Genetic diversity of Histoplasma capsulatum strains in Brazil

This study establishes the genetic relatedness among Brazilian Histoplasma capsulatum samples obtained from different sources. A PCR-based random amplified polymorphic DNA (RAPD) assay was used to delineate polymorphisms among isolates in geographically diverse regions in Brazil. RAPD fingerprints revealed distinct DNA profiles and provided a high level of discrimination among H. capsulatum strains from different locations. Cluster I was composed of H. capsulatum isolates from the northeast region. The majority of strains from southeast and south were categorized as major cluster II. The strain 84564 from Rio de Janeiro State showed no genetic correlation to any of the isolates from the same state. The RAPD patterns of H. capsulatum isolates from Goias (Cluster III) were unrelated to DNA fingerprints observed among the other H. capsulatum strains (48% similarity). This study is the first report that stratifies the clusters of H. capsulatum strains from Brazil by molecular typing and associates them with the geographical origin.     

Screening for acetylcholinesterase inhibitory activity in cyanobacteria of the genus Nostoc

Fifty-four cyanobacterial strains of the genus Nostoc from different habitats were screened for acetylcholinesterase inhibitory activity. Water-methanolic extracts from freeze-dried biomasses were tested for inhibitory activity using Ellman's spectrophotometric method. Acetylcholinesterase inhibitory activity higher than 90% was found in the crude extracts of Nostoc sp. str. Luke?ová 27/97 and Nostoc ellipsosporum Rabenh. str. Luke?ová 51/91. Extracts from Nostoc ellipsosporum str. Luke?ová 52/91 and Nostoc linckia f. muscorum (Ag.) Elenk. str. Gromov, 1988, CALU-980 inhibited AChE activity by 84.9% and 65.3% respectively. Moderate AChE inhibitory activity (29.1–37.5%) was found in extracts of Nostoc linckia Roth. str. Gromov, 1962/10, CALU-129, Nostoc muscorum Ag. str. Luke?ová 127/97, Nostoc sp. str. Lhotsky, CALU-327 and Nostoc sp. str. Gromov, CALU-998. Extracts from another seven strains showed weak anti-AChE activities.

The active component responsible for acetylcholinesterase inhibition was identified in a crude extract of Nostoc sp. str. Luke?ová 27/97 using HPLC and found to occur in one single peak.     

Strain-Specific Identification of Probiotic Lactobacillus rhamnosus with Randomly Amplified Polymorphic DNA-Derived PCR Primers

In the present work, strain-specific PCR primers for Lactobacillus rhamnosus Lc 1/3 are described. The randomly amplified polymorphic DNA (RAPD) technique was used to produce potential strain-specific markers. They were screened for specificity by hybridization with DNA from 11 L. rhamnosus strains. A 613-bp RAPD marker found to be strain-specific was sequenced, and a primer pair specific to L. rhamnosus Lc 1/3 was constructed based on the sequence. The primer pair was tested with 11 Lactobacillus species and 11 L. rhamnosus strains and was found to be strain specific. The nucleotide sequence of the specific RAPD marker was found to contain part of a protein encoding region which showed significant similarity to several transposases for insertion sequence elements of various bacteria, including other lactic acid bacterium species.     

One Group of Genetically Similar Listeria monocytogenes Strains Frequently Dominates and Persists in Several Fish Slaughter- and Smokehouses

Contamination of foods with the human pathogen Listeria monocytogenes may occur during processing, and the purpose of this study was to determine whether genetically similar strains colonize different processing plants or whether specific persistent strains are unique to each processing plant. We hypothesized that specific L. monocytogenes strains may be better adapted to specific environmental niches in the processing environment. L. monocytogenes contamination patterns were identified by the collection of 686 and 267 samples from the processing environments: raw fish and products of four fish smokehouses and four fish slaughterhouses, respectively. Samples were collected both during production and after cleaning and disinfection. Typically, these samplings were separated by 1 to 3 months. Sampling sites were targeted toward areas likely to harbor the bacterium. L. monocytogenes was isolated from 213 samples, and one strain from each positive sample was typed by RAPD (random amplified polymorphic DNA) analysis with four different primers. The 213 strains were divided into 37 RAPD types. One RAPD type was predominant; 86 of 213 strains belonged to this type. This type was found in three smokehouses and two slaughterhouses and was predominant in three of these plants. A subset of 35 strains was also analyzed by amplified fragment length polymorphism typing, which confirmed the genetic similarity of the groups. Moreover, strains of the dominant RAPD type were indistinguishable from strains isolated frequently from smoked fish products 10 years ago. One smokehouse was surveyed for a year and a half, and the dominant RAPD type persisted throughout the survey period and accounted for 94 of 118 isolates. Our study indicates that strains of L. monocytogenes that are genetically very closely related may be especially adapted to colonizing the processing equipment or especially resistant to cleaning and disinfection.     

Use of Conserved Randomly Amplified Polymorphic DNA (RAPD) Fragments and RAPD Pattern for Characterization of Lactobacillus fermentum in Ghanaian Fermented Maize Dough

The present work describes the use of randomly amplified polymorphic DNA (RAPD) for the characterization of 172 dominant Lactobacillus isolates from present and previous studies of Ghanaian maize fermentation. Heterofermentative lactobacilli dominate the fermentation flora, since approximately 85% of the isolates belong to this group. Cluster analysis of the RAPD profiles obtained showed the presence of two main clusters. Cluster 1 included Lactobacillus fermentum, whereas cluster 2 comprised the remaining Lactobacillus spp. The two distinct clusters emerged at the similarity level of <50%. All isolates in cluster 1 showed similarity in their RAPD profile to the reference strains of L. fermentum included in the study. These isolates, yielding two distinct bands of approximately 695 and 773 bp with the primers used, were divided into four subclusters, indicating that several strains are involved in the fermentation and remain dominant throughout the process. The two distinct RAPD fragments were cloned, sequenced, and used as probes in Southern hybridization experiments. With one exception, Lactobacillus reuteri LMG 13045, the probes hybridized only to fragments of different sizes in EcoRI-digested chromosomal DNA of L. fermentum strains, thus indicating the specificity of the probes and variation within the L. fermentum isolates.     

A RAPD Assay for Strain Typing of the Biotrophic Grape Powdery Mildew Fungus Uncinula necator Using DNA Extracted from the Mycelium

Délye, C., Corio-Costet, M.-F., and Laigret, F. 1995. A RAPD assay for strain typing of the biotrophic grape powdery mildew fungus Uncinula necator using DNA extracted from the mycelium. Experimental Mycology 19, 234-237. We describe, for the first time, a RAPD assay using DNA extracted from the mycelium of a powdery mildew fungus, Uncinula necator, a pathogen of grape. No contamination by plant DNA was observed, and the resulting patterns were fully repetitive. RAPD profiles were unchanged when using two different DNA polymerases or three different thermocyclers. Thirteen strains were tested for amplification, using 95 primers. Only 4% of the amplified fragments were polymorphic. Cluster analysis revealed that the strains from the same geographical origin had the higher genetic similarity, suggesting a short-range dissemination of U. necator. This RAPD assay was also successfully applied to the grape downy mildew fungus, Plasmopara viticola, indicating that it can be used for other fungi which cannot be grown on artificial media.     

DNA amplification fingerprinting as a tool for checking genetic purity of strains in the cyanobacterial inoculum

Summary The electrophoretic patterns for 17 different cyanobacterial cultures derived from 6 different decamer primers were analysed to provide diagnostic fingerprints for each culture and their genetic distances based on RAPD markers.The primer OPB 09 produced a maximum of 24 amplified products. The primers OPB 09, OPG 04 and OPAH 02 generated markers specific for Nostoc cultures. Westiellopsis was found to be distinct from other cyanobacterial cultures in the RAPD profile obtained with the primer OPAH 02. The primer OPF 03 generated specific markers for Tolypothrix tenuis. Fischerella cultures could be identified with the primers OPB 09, OPAG 03 and OPF 05. The study revealed that these RAPD markers could be further used to identify and establish the genetic purity of the strains in the cyanobacterial inoculum. There was a similarity of 60–90% within Westiellopsis cultures. Nostoc cultures shared 50–80% similarity with Westiellopsis cultures. Anabaenacultures were similar to Westiellopsiscultures by 60–70. The markers produced for each culture were also applied to phylogenetic analysis to infer genetic relatedness in this group of prokaryotes. The dendrogram analysis clearly revealed that free-living cyanobacterial cultures are closely related to each other and are distinct from the symbiotic forms.     

Genotyping of Ganoderma species by improved random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis

Species of Ganoderma are used in traditional medicines. An improved random amplified polymorphic DNA (RAPD) analysis, where the RAMP time is prolonged, has been used to characterize the genetic variation in some well known species of Ganoderma. The DNA materials were collected from ten Ganoderma strains, amplified with randomly selected 24 RAPD primers and evaluated by agarose gel electrophoresis. A cluster dendrogram was constructed for genetic analysis on the basis of amplification results. The improved RAPD amplified DNA with consistent and clear banding patterns. A total of 316 bands were found with 93% polymorphism. There was a significant genetic distance between the different strains of Ganoderma, with an index of similarity coefficient in the range of 0.52–0.74. The inter-simple sequence repeat (ISSR) analysis of the Ganoderma DNA samples showed similar trend results to the RAPD analysis with 0.49–0.81 similarity coefficients. This study reports the high level of genetic differences between different species or strains of a single species of Ganoderma and confirms the significance of the improved RAPD method in genetic characterization of organisms. Therefore, the improved RAPD combined with ISSR techniques might be used for the genetic characterization of organisms.     

Genomic fingerprinting of Bradyrhizobium japonicum isolates by RAPD and rep-PCR

Genetic diversity of indigenous Bradyrhizobium japonicum population in Croatia was studied by using different PCR-based fingerprinting methods. Characteristic DNA profiles for 20 B. japonicum field isolates and two reference strains were obtained using random primers (RAPD) and two sets of repetitive primers (REP- and ERIC-PCR). In comparison with the REP, the ERIC primer set generates fingerprints of lower complexity, but still several strain-specific bands were detected. Different B. japonicum isolates could be more efficiently distinguished by using combined results from REP- and ERIC-PCR. The most polymorphic bands were observed after amplification with four different RAPD primers. Both methods, RAPD and rep-PCR, resulted in identical grouping of the strains. Cluster analysis, irrespective of the fingerprinting method used, revealed that all the isolates could be divided into three major groups. Within the major groups, the degree of relative similarity between B. japonicum isolates was dependent upon the method used. Our results indicate that both RAPD and rep-PCR fingerprinting can effectively distinguish different B. japonicum strains. RAPD fingerprinting proved to be slightly more discriminatory than rep-PCR.     

Phylogenetic comparison among the heterocystous cyanobacteria based on a polyphasic approach

Phylogenetic comparison has been done among the selected heterocystous cyanobacteria belonging to the sections IV and V. The hierarchical cluster analysis based on antibiotics sensitivity showed a distant relationship between the members of Nostocales and Stigonematales. Thus, multiple antibiotic resistance pattern used as marker provide easy, fast, and reliable method for strain discrimination and genetic variability. However, morphological, physiological (both based on principal component analysis) and biochemical analysis grouped true branching cyanobacteria along with the members of section IV. Molecular analysis based on 16S rRNA gene sequences revealed that Hapalosiphon welwitschii and Westiellopsis sp. were grouped in cluster I whereas Scytonema bohnerii, a false branching genera showed a close proximity with Calothrix brevissima in cluster II. Cluster III of clade 2 included Nostoc calcicola and Anabaena oryzae which proved the heterogeneity at the generic level. Cluster IV the largest group of clade 2 based on 16S rRNA gene sequences includes six strains of the genera Nostoc, Anabaena, and Cylindrospermum showing ambiguous evolutionary relationship. In cluster IV, Anabaena sp. and Anabaena doliolum were phylogenetically linked by sharing 99% sequence similarity. Probably, they were of the same genetic makeup but appear differently under the diverse physiological conditions. Section IV showed polyphyletic origin whereas section V showed monophyletic origin. Results suggested that either morphological or physiological or biochemical or molecular attribute is not sufficient to provide true diversity and phylogeny of the cyanobacteria at the generic level and thus, a polyphasic approach would be more appropriate and reliable.     

Molecular genetic analysis of new Anabaena strains isolated from a plant-cyanobacterial community

Ecosystems of rice paddies are good sources of new strains of heterocyst-forming cyanobacteria that can be used in biotechnological systems for production of photohydrogen. The morphological and physiological properties of two novel epiphytic strains of cyanobacteria, Anabaena sp. 182 and Anabaena sp. 281, were studied. DNA typing of these strains based on PCR amplification of hydrogenase-encoding genes and DNA analysis using RAPD and Rep primers was carried out. The properties of the genome of strain Anabaena sp. 281 differed considerably from those of two reference strains (Anabaena variabilis ATCC 29413 and Nostoc sp. PCC 7120) with sequenced genomes, whereas strain Anabaena sp. 182 was found to be a close relative of A. variabilis ATCC 29413. Due to a number of physiological and biochemical advantages, Anabaena sp. 182 may be considered a new promising model for molecular and genetic engineering studies aimed at the development of H₂ producers.     

Genetic Analysis of Indian Lentil (Lens culinaris Medikus) Cultivars and Landraces Using RAPD and STMS Markers

Molecular analysis of 29 lentil (Lens culinaris) cultivars and landraces of Indian origin was carried out using twenty RAPD and ten cross-species STMS primers. A total of 97 markers (72 RAPD and 25 STMS) were amplified of which 42.3% were polymorphic. Genetic similarity among the cultivars and landraces was 89.7%. The observed results indicated low level of genetic diversity in the studied material. UPGMA cluster analysis for the combined data of RAPD and STMS revealed two broad clusters — Cluster I with three landraces and Cluster II containing all remaining landraces and cultivars except Precoz. Germplasm line Precoz was found to be the most distinct in individual as well as combined analyses. All cultivars and landraces except K-75 and L4076 could be discriminated from one another using combined data for the two techniques. Germplasm lines Precoz, L830 and cultivars L4147 and JL3 were quite distinct and could be potential germplasm resource.     

Physiological evidence indicates microcystin-LR to be a part of quantitative chemical defense system

The functional role of microcystins (MC) is poorly understood. Here, we investigated the effect of pure MC-LR recovered from the freshwater planktonic cyanobacterium Nostoc sp. BHU001 on five closely related cyanobacteria (Nostoc muscorum, Nostoc commune, Anabaena fertilissima, Anabaena doliolum, and Cylinderospermum majus) isolated from different habitats as well as on the producer itself (Nostoc sp. BHU001). MC-LR was found to be a general growth inhibitor active at nanomolar range (25–100 μg L^?1). It inhibited the growth of all cyanobacterial strains in a concentration-dependent manner, except the producer. A. fertilissima was the most sensitive species. MC-LR affected vital metabolic processes such as photosynthesis, respiration, and nitrogen fixation. Nitrogenase activity showed maximum sensitivity, followed by respiration, photosynthesis, and general growth. The photosynthetic electron transport activity was maximally inhibited at PSI, followed by whole chain and PSII activities. Thus, MC-LR is active at multiple sites causing energy constraint to the vital metabolic processes of the target organisms. However, its requirement at high concentration, which is environmentally irrelevant, and lack of quantitative information on the extracellular release of MC-LR suggest that MC-LR has no allelopathic function and could be a part of a quantitative chemical defense system.     

Pure culture and reconstitution of the Anthoceros-Nostoc symbiotic association

The partners of the symbiotic association between Anthoceros punctatus L. and Nostoc spp. have been cultured separately in a pure state. The symbiotic association was reconstituted following dual culture in liquid Anthoceros growth medium with a variety of axenic Nostoc isolates and mutant strains. The heterocyst frequency of competent Nostoc strains increased four- to fivefold when in symbiotic association relative to free-living N₂-grown cultures. Dinitrogen fixation by symbiotic Nostoc supported the growth of Anthoceros tissue, although this growth was nitrogen-limited relative to that supported by exogenous ammonium. When the association was reconstituted in the presence of two or three wild-type and mutant Nostoc strains some of these strains were found to compete in infection of Anthoceros tissue and a fraction of the symbiotic Nostoc colonies contained more than one strain. Exogenous ammonium did not affect infection, but repressed development of the symbiotic Nostoc colonies in Anthoceros tissue, and symbiotic Nostoc in N₂-grown Anthoceros tissue appeared to regress from the symbiotic state in the presence of exogenous ammonium. The results show that the Anthoceros-Nostoc symbiotic association is amenable to specific experimental manipulations; their implications are discussed with respect to infection of Anthoceros tissue and control of the development of symbiotic Nostoc.     

Phylogeny of symbiotic cyanobacteria within the genus <Emphasis Type="Italic">Nostoc</Emphasis> based on 16S rDNA sequence analyses

A phylogenetic analysis of selected symbiotic Nostoc strain sequences and available database 16S rDNA sequences of both symbiotic and free-living cyanobacteria was carried out using maximum likelihood and Bayesian inference techniques. Most of the symbiotic strains fell into well separated clades. One clade consisted of a mixture of symbiotic and free-living isolates. This clade includes Nostoc sp. strain PCC 73102, the reference strain proposed for Nostoc punctiforme. A separate symbiotic clade with isolates exclusively from Gunnera species was also obtained, suggesting that not all symbiotic Nostoc species can be assigned to N. punctiforme. Moreover, isolates from Azolla filiculoides and one from Gunnera dentata were well nested within a clade comprising most of the Anabaena sequences. This result supports the affiliation of the Azolla isolates with the genus Anabaena and shows that strains within this genus can form symbioses with additional hosts. Furthermore, these symbiotic strains produced hormogonia, thereby verifying that hormogonia formation is not absent in Anabaena and cannot be used as a criterion to distinguish it from Nostoc.The GenBank accession numbers for the cyanobacterial 16S rRNA gene sequences determined in this study are AY742447-AY742454.     

Genomic diversity and CRISPR-Cas systems in the cyanobacterium Nostoc in the High Arctic

Nostoc (Nostocales, Cyanobacteria) has a global distribution in the Polar Regions. However, the genomic diversity of Nostoc is little known and there are no genomes available for polar Nostoc. Here we carried out the first genomic analysis of the Nostoc commune morphotype with a recent sample from the High Arctic and a herbarium specimen collected during the British Arctic Expedition (1875–76). Comparisons of the polar genomes with 26 present-day non-polar members of the Nostocales family highlighted that there are pronounced genetic variations among Nostoc strains and species. Osmoprotection and other stress genes were found in all Nostoc strains, but the two Arctic strains had markedly higher numbers of biosynthetic gene clusters for uncharacterised non-ribosomal peptide synthetases, suggesting a high diversity of secondary metabolites. Since viral–host interactions contribute to microbial diversity, we analysed the CRISPR-Cas systems in the Arctic and two temperate Nostoc species. There were a large number of unique repeat-spacer arrays in each genome, indicating diverse histories of viral attack. All Nostoc strains had a subtype I-D system, but the polar specimens also showed evidence of a subtype I-B system that has not been previously reported in cyanobacteria, suggesting diverse cyanobacteria–virus interactions in the Arctic.     

Analysis of genetic diversity in cowpea (Vigna unguiculata L.Walp.) cultivars with random amplified polymorphic DNA markers

The genetic diversity among ten Indian cultivars of cowpea was analyzed using 18 sets of RAPD markers. A total of 181 bands with an average of 15 bands per primer were obtained. Out of 181 bands, 148 showed polymorphism (81.7%). The variation in genetic diversity among these cultivars ranged from 0.1742 to 0.4054. Cluster analysis based on Jaccard’s similarity coefficient using UPGMA with high bootstrap values revealed two distinct clusters I and II comprised of two and seven cultivars, respectively. Cluster II was further differentiated into various subclusters. Cultivar IC-9883 was found to be unique based on its altogether distinct position in the dendrogram and two-dimensional space projections.     

Genetic analysis of protoplast fusant Xhhh constructed for pharmaceutical wastewater treatment

In order to analyse genetic relationships between functional strain Xhhh previously constructed through protoplast fusion for pharmaceutical wastewater treatment and its parents, random amplification polymorphic DNA (RAPD) and polymerase chain reaction (PCR) were used to investigate genetic similarities among the strains based on genome and functional genes analyses. A total of 739 clear and consistent bands were produced in the RAPD fingerprint analysis with 40 primers. The genetic similarity indices between Xhhh and parental strains PC (Phanerochaete chrysosporium), SC (Saccharomyces cerevisiae) and XZ (native bacterium Bacillus sp.) were 36.21%, 37.73% and 37.48%, respectively. With PCR amplification and DNA sequencing, Xhhh was found containing functional genes of mnp and lip from PC, FLO1 from SC and 16S rDNA fragments from XZ. Experimental results of genetic analyses were in accordance with Xhhh biochemical and phenotypic characteristics, and protoplast fusion technique is considered as a promising technique in environmental pollution control.     

Molecular Assessment of Diversity among Pathotypes of Colletotrichum falcatum Prevalent in Sub-Tropical Indian Sugarcane

Summary Six pathotypes of Colletotrichum falcatum, responsible for Red-rot in sugarcane, prevalent in subtropical India were examined for genetic relationships using RAPD markers. A high degree of polymorphism (78.6%) was observed using 40 RAPD markers. More than 50% genetic divergence was found among the pathotypes and UPGMA cluster analysis of genetic similarity indices grouped the six pathotypes into two clusters. Cluster I comprised pathotypes Cf01 and Cf09, while cluster II comprised the remaining four pathotypes. Cf02 and Cf08 were the most closely related among all the pathotypes. Pathotype-specific unique bands generated in RAPD profiling are being used for developing markers for pathotype identification in diseased cane samples.     

Genetic diversity analysis of Zingiber Officinale Roscoe by RAPD collected from subcontinent of India

The present investigation was undertaken for the assessment of 12 accessions of Zingiber officinale Rosc. collected from subcontinent of India by RAPD markers. DNA was isolated using CTAB method. Thirteen out of twenty primers screened were informative and produced 275 amplification products, among which 261 products (94.90%) were found to be polymorphic. The percentage polymorphism of all 12 accessions ranged from 88.23% to 100%. Most of the RAPD markers studied showed different levels of genetic polymorphism. The data of 275 RAPD bands were used to generate Jaccard’s similarity coefficients and to construct a dendrogram by means of UPGMA. Results showed that ginger undergoes genetic variation due to a wide range of ecological conditions. This investigation was an understanding of genetic variation within the accessions. It will also provide an important input into determining resourceful management strategies and help to breeders for ginger improvement program.