Genetic diversity and phylogenetic relationships among microsporidia infecting the silkworm,Bombyx mori,using random amplification of polymorphic DNA: Morphological and ultrastructural characterization

Random amplification of polymorphic DNA polymerase chain reaction (RAPD-PCR) and pathological, morphological and ultrastructural characterization were used to differentiate seven new microsporidian isolates infecting the mulberry silkworm, Bombyx mori. The pathogenicity observed was dose-dependent and differed from each of the microsporidian isolates; the NIK-4m was found to be more virulent than other isolates. However, all the isolates, except NIK-4m, showed heavy gonadal infection and vertical transmission in the infected silkworms. Differences in the spore shape ranging from oval to elongate were observed, and the polar filament has 8–16 coils arranged in one or two rows. Of the 80 decamer random primers tested, 50 generated reproducible RAPD profiles and yielded a total of 600 fragments, of which 594 were polymorphic (99%). Forty nine RAPD primers produced 179 unique genetic markers, whose presence or absence differed among the microsporidians, albeit with varied efficiency of polymorphism detection. The degree of band sharing was used to evaluate genetic distances between different microsporidian isolates and to construct a phylogenetic tree using Dice coefficients. Cluster analysis based on Dice coefficients resulted in the formation of one major cluster consisting of NIK-1s, NIAP-7g, NIK-2r and NIK-5d and NIK-4m in the other; while NIAP-6p was intermediate between these two. NIK-8b and NITN-9n were found to be entirely different from others. Reproducible RAPD patterns of all microsporidian isolates enabled us to differentiate the microsporidian isolates. The results demonstrate that besides ultrastructural studies, RAPD-PCR can be a useful and reliable tool to detect polymorphism, genetic relationships, and for the identification of the microsporidians. In addition, DNA fingerprints generated in this process have potential applications as diagnostic tools for identification of different microsporidia with considerable accuracy.     

Phenotypic and molecular analysis of dominant occurring antibiotic active-producing Streptomyces soil flora in Northern Jordan

This investigation aimed to determine the relatedness of dominant occurring soil Streptomyces spp. in Northern Jordan based on their RAPD-PCR fingerprints, and to compare RAPD technique with the conventional phenotypic characterization of Streptomyces isolates. Fifty-eight white and gray color-bearing aerial mycelia antibiotic active-producing Streptomyces soil isolates along with three reference strains were genetically analyzed by RAPD-PCR. Polymorphisms between the isolates showed 1 to 10 bands per isolate and ranged from 200 to 3200 bp in size. Results revealed one common band of ~600 bp shared by ~85% of the isolates, and the observation of bands specific to some reference strains and some soil isolates. When RAPD patterns were analyzed with the UPGMA, results revealed clustering the tested isolates into two equal main super clusters (50% each). Super cluster I appeared to be homogenous and include the three reference strains. However, super cluster II was heterogeneous and but not including any of the reference strains. The association of the antibiotic activity of the dominant white and gray aerial mycelium-bearing Streptomyces isolates to RAPD clustering is reported for the first time, and the RAPD-PCR fingerprints generated here deserve to be cloned, characterized and sequenced in future as Streptomyces species-specific DNA markers. The more random primers used in the analysis may add to RAPD technique a cost-effective, fast, precise result, and less labor work solution for analyzing the similarities and differences among the Streptomyces isolates.     

RAPD-PCR and 16S rDNA phylogenetic analysis of alkaline protease producing bacteria isolated from soil of India: Identification and detection of genetic variability

178 bacterial strains were isolated from the soil samples collected from different regions of India out of which, 20 bacterial isolates were selected for alkaline protease production. The alkaline protease production efficiency of organisms was monitored at regular intervals (24 h) upto 7 days at 37 °C, pH 10. The 16S rDNA sequencing and RAPD-PCR based technique were used to identify the genetic variability among the 20 isolates of alkaline protease producing bacteria. The phylogenetic analysis indicated that the isolates can be separated into two clusters which could be further subdivided into five groups. Group 1 and 5 represented the family Bacillaceae, Groups 2 represented the Micrococcaceae family while Group 3 included the Arthrobacter bacterial group (family Micrococcaceae) from different geographical locations, respectively. Group 4 was identified as Pseudomonadaceae which was gram (−) bacteria. 21 different oligonucleotide primers were used to amplify approximately 261 fragments from each DNA sample. The bands were scored on the basis of their presence and absence and similarity between DNA samples was checked using Jaccard’s coefficient. Isolates were distinguished into distinct groups based on RAPD profiles from different geographical locations, morphological features and enzyme production efficiency. For cluster analysis the dendrogram was constructed using the unweighted pair group method with arithmetic averages (UPGMA). The results indicated that 16S rDNA and RAPD-PCR are suitable methods for rapid identification and differentiation of alkaline protease producing bacteria.     

Polyphasic study of the genetic diversity of lactobacilli associated with 'Almagro' eggplants spontaneous fermentation, based on combined numerical analysis of randomly amplified polymorphic DNA and pulsed-field gel electrophoresis patterns

AIMS: The goal of this study was to assess the genetic diversity of lactic acid bacteria (LAB) from the complex natural ecosystem present in the spontaneous fermentation of 'Almagro' eggplants by a polyphasic approach based on molecular techniques. METHODS AND RESULTS: Randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) were applied to 149 Lactobacillus isolates obtained from that fermentation process. Two random primers, OPL-05 and ArgDei-For, and two rare-cutting enzymes, SfiI and SmaI, chosen after preliminary testing on the basis of band intensity and distribution, were used. RAPD and PFGE generated electrophoretic patterns suitable for strain discrimination, but further discrimination was achieved when combined numerical analysis of the results from both methods and the results previously obtained by SDS-PAGE whole cell protein analysis, was carried out. The findings indicated a considerable degree of genomic diversity in the LAB microbiota studied and especially in the Lactobacillus plantarum isolates. In terms of species assignment, the polyphasic study allowed a definite and well-founded identification of 98.7% of the isolates. CONCLUSIONS: The combined numerical analysis of RAPD and PFGE patterns represented a useful tool to discriminate the diversity of the Lactobacillus strains responsible for the spontaneous fermentation of this pickle. The species identification and strain typing results from the polyphasic study were regarded as the most exact compromise yielding the fewest contradictions based on the available data. SIGNIFICANCE AND IMPACT OF THE STUDY: Combined numerical analysis of RAPD-PCR and PFGE patterns has not yet been employed to study the genetic diversity of LAB from an ecosystem like that found in fermenting vegetables.     

Evaluation of random amplified polymorphic DNA (RAPD)-PCR as a method to differentiate Lactobacillus acidophilus,Lactobacillus crispatus,Lactobacillus amylovorus,Lactobacillus gallinarum,Lactobacillus gasseri,and Lactobacillus johnsonii

The technique random amplified polymorphic DNA (RAPD)-PCR was evaluated as a method to differentiate Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus gasseri, and Lactobacillus johnsonii. Representative strains, including the type of each species, were selected from different clusters obtained by numerical analysis of total soluble cell protein patterns. Results obtained by RAPD-PCR corresponded well with results obtained by numerical analysis of total soluble cell protein patterns. The type strains of each species displayed different RAPD profiles. Strains with identical L(+)- nicotinamide adenine dinucleotide-dependent lactic dehydrogenase (nLDH) electrophoretic profiles could be distinguished on the basis of their RAPD profiles.     

Genetic diversity and phylogenetic relationships among microsporidia infecting the silkworm, Bombyx mori, using random amplification of polymorphic DNA: morphological and ultrastructural characterization

Random amplification of polymorphic DNA polymerase chain reaction (RAPD-PCR) and pathological, morphological and ultrastructural characterization were used to differentiate seven new microsporidian isolates infecting the mulberry silkworm, Bombyx mori. The pathogenicity observed was dose-dependent and differed from each of the microsporidian isolates; the NIK-4m was found to be more virulent than other isolates. However, all the isolates, except NIK-4m, showed heavy gonadal infection and vertical transmission in the infected silkworms. Differences in the spore shape ranging from oval to elongate were observed, and the polar filament has 8-16 coils arranged in one or two rows. Of the 80 decamer random primers tested, 50 generated reproducible RAPD profiles and yielded a total of 600 fragments, of which 594 were polymorphic (99%). Forty nine RAPD primers produced 179 unique genetic markers, whose presence or absence differed among the microsporidians, albeit with varied efficiency of polymorphism detection. The degree of band sharing was used to evaluate genetic distances between different microsporidian isolates and to construct a phylogenetic tree using Dice coefficients. Cluster analysis based on Dice coefficients resulted in the formation of one major cluster consisting of NIK-1s, NIAP-7g, NIK-2r and NIK-5d and NIK-4m in the other; while NIAP-6p was intermediate between these two. NIK-8b and NITN-9n were found to be entirely different from others. Reproducible RAPD patterns of all microsporidian isolates enabled us to differentiate the microsporidian isolates. The results demonstrate that besides ultrastructural studies, RAPD-PCR can be a useful and reliable tool to detect polymorphism, genetic relationships, and for the identification of the microsporidians. In addition, DNA fingerprints generated in this process have potential applications as diagnostic tools for identification of different microsporidia with considerable accuracy.     

Morphological, Biochemical and Molecular Characterization of Trichoderma harzianum Isolates for their Efficacy as Biocontrol Agents

The random amplified polymorphic DNA (RAPD) procedure was used to examine the genetic variability among 8 isolates of Trichoderma harzianum , and their ability to antagonize Sclerotium rolfsii using a dual culture assay was correlated with RAPD profiles. Eight oligodeoxynucleotide primers were selected for the RAPD assays, which resulted in 86 bands for 8 isolates of T . harzianum . The data were entered into a binary matrix and a similarity matrix was constructed using the DICE similarity (SD) index. An unweighted pair grouping mathematical averaging (UPGMA) cluster based on SD values was generated using the NTSYS computer program. A mean coefficient of similarity obtained for pairwise comparisons was c. 30% and it showed that the variability among the isolates of T. harzianum was very high. Using the dual culture method in antagonism experiments, the T. harzianum isolates were classified in to antagonism classes. Further, T. harzianum isolates were screened for chitinase and β-1,3-glucanase activity. RAPD was efficient in demonstrating the high intraspecific genetic variation among isolates. The dendrogram did not show the grouping of isolates by their level of antagonism. Relationship among polymorphism existent, the aggressiveness and the origin of isolates were not found.     

A survey on distribution and toxigenicity of <Emphasis Type="Italic">Aspergillus flavus</Emphasis> from indoor and outdoor hospital environments

In the present study, genetic diversity and mycotoxin profiles of Aspergillus flavus isolated from air (indoors and outdoors), levels (surfaces), and soils of five hospitals in Southwest Iran were examined. From a total of 146 Aspergillus colonies, 63 isolates were finally identified as A. flavus by a combination of colony morphology, microscopic criteria, and mycotoxin profiles. No Aspergillus parasiticus was isolated from examined samples. Chromatographic analyses of A. flavus isolates cultured on yeast extract–sucrose broth by tip culture method showed that approximately 10% and 45% of the isolates were able to produce aflatoxin B₁ (AFB₁) and cyclopiazonic acid (CPA), respectively. Around 40% of the isolates produced sclerotia on Czapek–Dox agar. The isolates were classified into four chemotypes based on the ability to produce AF and CPA that majority of them (55.5%) belonged to chemotype IV comprising non-mycotoxigenic isolates. Random amplified polymorphic DNA (RAPD) profiles generated by a combination of four selected primers were used to assess genetic relatedness of 16 selected toxigenic and non-toxigenic isolates. The resulting dendrogram demonstrated the formation of two separate clusters for the A. flavus comprised both mycotoxigenic and non-toxigenic isolates in a random distribution. The obtained results in this study showed that RAPD profiling is a promising and efficient tool to determine intra-specific genetic variation among A. flavus populations from hospital environments. A. flavus isolates, either toxigenic or non-toxigenic, should be considered as potential threats for hospitalized patients due to their obvious role in the etiology of nosocomial aspergillosis.     

Evaluation of RAPD-PCR and protein profile analysis to differentiate <Emphasis Type="Italic">Vibrio harveyi</Emphasis> strains prevalent along the southwest coast of India

Sixty five isolates of Vibrio harveyi were subjected to random amplified polymorphic DNA (RAPD)-PCR analysis and protein profiling to investigate the genetic variability among V. harveyi prevalent along the coast and also assess the discriminating ability of these two molecular methods. A total of 10 RAPD primers were assayed for their specificity in detecting V. harveyi, of which only two primers: PM3 and CRA25 were highly reproducible and found suitable for use in RAPD-PCR. The genetic diversity among V. harveyi isolates assessed by RAPD-PCR using PM3 primer yielded 35 different RAPD patterns which clustered the isolates into 15 groups at 72% similarity level. Similarly, RAPD-PCR with CRA25 clustered the 38 patterns into 10 groups at 74% similarity. The discriminatory index (D) value calculated for RAPD fingerprints generated with PM3 and CRA25 were 0.90 and 0.85, respectively. On the other hand, molecular typing of V. harveyi using whole cell proteins generated profiles that showed no major difference indicating the technique to be not useful in typing strains of this bacterium. However, a few of the isolates showed the presence of unique band of 28 kDa that needs to be further investigated to understand the role of the protein in disease process if any.     

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.     

Lack of Genetic Variation of Bursaphelenchus xylophilus in Portugal Revealed by RAPD-PCR Analyses

Random Amplified Polymorphic DNA (RAPD-PCR) technique was used to assess the level of genetic variability and genetic relationships among 24 Portuguese isolates of pinewood nematode, Bursaphelenchus xylophilus. The isolates represent the main infested areas of Portugal. Two additional isolates of B. xylophilus representing North America and East Asia were included, and B. mucronatus was used as out-group. Twenty-eight random primers generated a total of 640 DNA fragments. The Nei and Li similarity index revealed a high genetic similarity among the Portuguese isolates (above 90%). Hierarchical cluster analysis was performed to illustrate the relatedness among the isolates. No indication for separate groups among the Portuguese isolates was obtained, and the low level of genetic diversity strongly suggests that they were dispersed recently from a single introduction. The lack of apparent relationship between the genetic and the geographic matrices of the Portuguese isolates limits the use of this technique for following recent pathways of distribution. Genetic distance of the Portuguese isolates towards an isolate from China was much lower as compared to an isolate from the USA. This confirmed previous results suggesting an East Asian origin of the Portuguese B. xylophilus.     

Genetic characterization of antibiotic-resistant Staphylococcus aureus from milk in the North-West Province,South Africa

Food borne diseases are a major public health concern worldwide. Staphylococcus aureus is one of the potential food borne pathogens which causes nosocomial and community acquired infections. In the present study, 74 representative strains of S. aureus isolated and characterized in previous study from different milk samples were subjected to random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) and enterobacterial repetitive intergenic consensus (ERIC)-PCR to generate fingerprints to determine the genetic relationships of the isolated strains. A total of 20 RAPD patterns were generated and the number of amplified fragments obtained ranged from 0 to 8 with molecular weight ranging from 250 to 2000 bp. A dendrogram based on fingerprinting pattern grouped isolates into twelve major clusters (I–XII). In the case of ERIC-PCR 9 banding patterns were obtained with amplicons ranging from 1 to 8 and band sizes ranging from 250 to 2000 bp. A total of four major clusters (I–IV) were observed in the dendrogram based on ERIC fingerprints. The discrete banding patterns obtained both from ERIC-PCR and RAPD-PCR showed remarkably the genetic diversity of S. aureus. The findings of this study indicate that raw, bulk and pasteurized milk in the North-West Province was contaminated with toxigenic and multi-drug resistant S. aureus strains. This emphasizes the need to implement appropriate control measures to reduce contamination as well as the spread of virulent S. aureus strains to reduce the burden of disease in humans.     

Detection of genetic variability in various isolates of cattle tick, <Emphasis Type="Italic">Boophilus microplus</Emphasis> from Tamil Nadu,India using PCR-RAPD analysis

The genomic DNA from ten isolates of the cattle tick, Boophilus microplus collected in and around Chennai, India, was analyzed by random amplified polymorphic DNA (RAPD) using PCR. Selected five random primers were used for the study of genetic variability among different isolates of B. microplus. A high degree of genetic polymorphism with a different pattern of RAPD profiles for each tick isolate was detected with all these random primers. This variability was also confirmed by similarity coefficient values and dendrogram which were performed using mean RAPD profiles for all the primers between various isolates of ticks. The findings suggest the existence of a complex genotypic diversity of the tick B. microplus in an endemic region such as Chennai.     

Analysis of intraspecific and interspecific variation in the genus Alternaria by the use of RAPD-PCR

The RAPD-PCR profiles of 13 phytopathogenic Alternaria species and two closely related outgroups were examined using six different primers. Each species produced a distinct pattern of DNA fragments which were used as a measure of the degree of relatedness between species. A. brassicae isolates of diverse origin showed high levels of similarity but little similarity was noted between other species. The closest interspecific genetic distances were recorded between A. citri, A. alternata and A. longipes. The outgroup genera Embellisia and Stemphylium, which are recognised as distinct, could not be clearly separated using RAPD banding criteria, suggesting a high level of genetic diversity amongst these groups of fungi.     

Morphological and genetic differentiation among four pigment producing Indian species of <Emphasis Type="Italic">Phoma</Emphasis> (Saccardo, 1899)

A PCR-based technique, involving the random amplification of polymorphic DNA (RAPD), was used for assessing genetic relatedness among isolates of the genus Phoma. Randomly Amplified Polymorphic DNA (RAPD) revealed the presence of interspecific genetic variation among the pigment producing isolates of Phoma and has shown distinct phylogenetic cluster. The major objective of the study was to study the genetic variation, if any. Study was aimed to differentiate four pigment producing species of Phoma based on morphological studies and molecular markers in general and RAPD in particular. We found that the test species of Phoma can be very well differentiated using molecular markers. Phoma sorghina was differentiated from P. exigua, P. fimeti and P. herbarum. RAPD profiles of P. herbarum and P. fimeti has shown the maximum similarity, which indicates the genetic relatedness among these two species which were considered earlier as distinct species based on morphological observation.     

Evaluation of Numerical Analysis of Random Amplified Polymorphic DNA (RAPD)-PCR as a Method to Differentiate Lactobacillus plantarum and Lactobacillus pentosus

Lactobacillus plantarum and Lactobacillus pentosus grouped into one protein profile cluster at r ≥ 0.70, separate from Lactobacillus casei, Lactobacillus sake, and Lactobacillus curvatus. Similar sugar fermentation reactions were recorded for representative strains of L. plantarum and L. pentosus. Representative strains, including the type of each species, were selected from the different protein profile clusters and their genetic relatedness determined by using numerical analysis of random amplified polymorphic DNA (RAPD)-PCR. The type strains of L. plantarum (ATCC 14917^T) and L. pentosus (NCFB 363^T) displayed different RAPD profiles and grouped into two independent clusters, well separated from L. casei, L. curvatus, and L. sake. Numerical analysis of RAPD-PCR proved a reliable and accurate method to distinguish between strains of L. plantarum and L. pentosus.     

Assessment of genetic diversity in Colletotrichum falcatum Went accessions based on RAPD and ISSR markers

Sugarcane is susceptible to red rot disease caused by phytopathogenic fungus Colletotrichum falcatum Went which ultimately affect the economy of farmers as well as sugar based industry. One of the various ways to control this devastating disease is to develop disease resistance sugarcane cultivar and this requires the complete understanding of genetic makeup of pathogen. Although South Gujarat is well known sugarcane cultivating area, less published data can be found about PCR-based genetic diversity in prevalent C. falcatum accessions. So, present investigation aims at finding molecular variation among the ten accessions of C. falcatum using RAPD and ISSR molecular markers. A total of 35 RAPD and 39 ISSR primers were screened across 10 C. falcatum accessions, of which 15 RAPD and 21 ISSR primers have showed consistent amplification. Statistics related to genetic variation were estimated using NTSYS-PC by means of Dice’s coefficient. The results revealed 80.6% and 68.07% polymorphism and similarity coefficient ranged from 0.43 to 0.91 and 0.73 to 0.93 in RPAD and ISSR analysis respectively. The dendrogram generated using RAPD, ISSR and combined RAPD-ISSR grouped accessions into different clusters which reveal considerable level molecular variation among the C. falcatum accessions. It is also evident from PCA plots that accessions are rather dispersed with tested marker systems indicating good genetic base. So, in nut shell, we found considerable genetic variation and relatedness within C. falcatum accessions collected from different areas of south Gujarat, India using RAPD and ISSR markers.     

Variations in random amplified polymorphic DNA profile and toxin production among isolates of Colletotrichum falcatum Went from sugarcane in Tamil Nadu,India

Red rot, caused by Colletotrichum falcatum Went, is one of the most important diseases of sugarcane (Saccharum officinarum L.). The pathogen shows a great diversity in virulence as a number of pathotypes are known to occur in nature. In the present study, the toxin producing ability and genetic variability among isolates of C. falcatum collected from major sugarcane growing areas of Tamil Nadu, India were analysed. The C. falcatum isolates differed significantly in their ability to produce toxin in vitro. The toxin from C. falcatum isolate Cf 671a induced the maximum electrolyte leakage (300 μS) from sugarcane leaf tissues. The genetic relatedness of the isolates of C. falcatum differing in toxin production potential was investigated by using RAPD analysis. Analysis of the genetic coefficient matrix derived from the scores of RAPD profiles showed that minimum and maximum percent similarities among the tested C. falcatum isolates were in the range of 19 to 95% respectively. The phylogenetic analysis by the UPGMA identified two main clusters. Cluster A contains only one isolate (Cf 98061) and all the other isolates were placed in Cluster B confirming high genetic diversity among the isolates. No correlation was observed between clustering of the C. falcatum isolates in the dendrogram and their toxin producing abilities.     

Molecular and genetic analyses of geographic variation in isolates of Phoma macrostoma used for biological weed control

Molecular and genetic approaches were used to evaluate the genetic relatedness among isolates of the fungus Phoma macrostoma Montagne originating from Canada and Europe and to other species in the genus Phoma. Distinct differences were observed in genetic variation among nine species of the genus Phoma. Randomly amplified polymorphic DNA (RAPD) revealed the presence of intraspecific genetic variation among the isolates of P. macrostoma, with the isolates being used for biological weed control being distributed in a distinct phylogenetic cluster. Additional variation within the biocontrol isolate cluster in P. macrostoma was revealed by pulsed field gel electrophoresis (PFGE), which showed that biocontrol isolates generated two different chromosomal profiles, however the profiles did not relate to their Canadian ecozone origin. Mating studies showed that biocontrol isolates of P. macrostoma from Canada did not produce sexual reproductive structures and were incapable of crossing. These studies also confirmed that no obvious differentiation exists among the biocontrol isolates of P. macrostoma from Canadian Ecozones 3 and 4.