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.     

Characterization of gibberellin producing strains of Fusarium moniliforme based on DNA polymorphism

The gibberellins are one of the major groups of growth promoting hormones and are secondary metabolites of the fungus Fusarium moniliforme (Perfect stage: Gibberella fujikuroi). Sixteen strains of Fusarium from different geographical regions and different hosts were analysed for their ability to produce gibberellins (GA) and for genetic relatedness by random amplified polymorphic DNA (RAPD). Range of gibberellin production varied between 28.9 to 600.0 mg g^-1 dry weight of mycelium in different strains of Fusarium. RAPD analysis showed completely different pattern between high, moderate and low producing strains. High producers formed nearly identical RAPD patterns, whereas the low and moderate producers gave heterologous amplification patterns. Since Fusarium pallidoroseum was in another group, it was possible to distinguish between different species of the genus Fusarium by RAPD. These investigations may find an application in the diagnosis of unknown Fusarium species and in distinguishing isolates of Gibberella fujikuroi within the section of Liseola. This revised version was published online in June 2006 with corrections to the Cover Date.     

Application of RAPD analysis for identification of Lactococcus lactis subsp. cremoris strains isolated from artisanal cultures

Randomly amplified polymorphic DNA (RAPD) was used for identification of Lactococcus lactis subsp. cremoris strains isolated 40 years ago from various dairy homemade products. Total genomic DNAs from six randomly chosen isolates and the reference strain Lactococcus lactis subsp. cremoris NIZO B64 were amplified using four different 10-mer primers. Although most RAPD fragments were common to all six isolates, a sufficient number of polymorphic fragments were also detected that allowed clear distinction of the isolates and the reference strain. The results indicate that RAPD analysis could be a useful and efficient method to distinguish Lactococcus lactis subsp. cremoris at the strain level and to detect genetic diversity.     

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.     

Genetic variation in cultivated strains of <Emphasis Type="Italic">Agaricus blazei</Emphasis>

Genetic differences among Agaricus blazei strains were investigated using somatic incompatibility testing, isozyme analysis, restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA), and random amplified polymorphic DNA (RAPD) analysis. Eight strains, one cultivated strain from Brazil and seven from Japan, were used in this study. Somatic incompatibility interactions were observed between the Brazilian cultivated strain and the Japanese strains. The Brazilian cultivated strain had its own distinct patterns of esterase isozyme and mtDNA RFLP, but all seven Japanese cultivated strains showed identical patterns. When the RAPD patterns, obtained using eight primers, were compared the eight strains had their own distinct RAPD profiles. Distance values were calculated between all pairs of the strains based on presence or absence of individual RAPD bands, and a dendrogram was constructed by unweighted pair-group method with arithmetic clustering (UPGMA) analysis. Seven Japanese cultivated strains were grouped to each other, and this group was finally linked to the Brazilian cultivated strain. Based on these results, the degree of genetic variation among the A. blazei strains used is discussed.     

Strain variation in Campylobacter pylori detected by numerical analysis of one-dimensional electrophoretic protein patterns

A total of 21 clinical isolates of Campylobacter pylori from Peru and the United Kingdom and two reference strains (from Australia), including the type strain (NCTC 11637^T), were characterized by high resolution one-dimensional SDS-polyacrylamide gel electrophoresis of cellular proteins. The protein patterns contained more than 40 discrete bands and the approximate molecular weights of the major bands were 22, 27, 46, 57, 60, 65 and 93 kD. The total patterns were used as the basis of numerical analysis. Most strains were clustered in four phenons at 91% similarity with the exception of six ungrouped strains. Overall similarity was high with all strains linked in the phenogram at 81%. Variation among strains was attributable principally to qualitative and quantitative band differences in the 47 to 56 kD (hypervariable) region of the C. pylori protein profile. From the analysis, ten different electropherotypes (EP-types) were identified. We demonstrated that differences were detectable among isolates from widely separated geographical locations as well as from the same location, although multiple isolates from two Peruvian patients had the same electropherotype. Our results indicate that determination of protein profiles provides the basis of a reproducible method for characterization of C. pylori isolates.     

Taxonomy of obligately homofermentative and facultatively heterofermentative lactobacilli in pig faeces

AIMS: Identification and characterization of obligately homofermentative and facultatively heterofermentative strains of Lactobacillus spp. isolated from the faeces of pigs that had been raised under different conditions. METHODS AND RESULTS: The phenotypic relatedness of the isolated strains and reference strains were determined by numerical analysis of total soluble cell protein patterns and simple physiological and biochemical tests. Of the 23 strains isolated from faeces, nine were obligately homofermentative and 14 facultatively heterofermentative. The strains clustered at r > or = 0.61 with Lactobacillus amylovorus (seven strains), Lactobacillus crispatus (one strain), Lactobacillus plantarum (14 strains) and Lactobacillus intestinalis (one strain). CONCLUSIONS: Results obtained from the physiological and biochemical tests confirmed the identity of the isolates as determined by numerical analysis of total soluble cell protein profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the association of Lact. crispatus and Lact. intestinalis with the gastro-intestinal tract of pigs.     

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.     

Differentiation of <Emphasis Type="Italic">Aspergillus niger</Emphasis> by random amplification of polymorphic DNA

The randomly amplified polymorphic DNA (RAPD) patterns of whole-cell lysates from five Aspergillus niger isolates, including one reference strain, two isolated from deep freeze, and two environmental strains from soil and plant infections, were investigated. PCR-RAPD analysis of genomic DNA was performed using eight primers (Tube-A1, Tube-A6, Tube-A17, Tube-B8, Tube-B11, Tube-B15, Tube-C5, Tube-C6). The RAPD assay discriminated between all strains. Comparison of deep freeze isolates showed identical RAPD patterns in some of the reference and environmental isolates. The data indicates that the RAPD technique is useful for fingerprinting A. niger.     

Discrimination of species and strains of basidiomycete genusCoprinus by random amplified polymorphic DNA (RAPD) analysis

All five examined strains ofCoprinus cinereus could be clearly discriminated from the strains of five otherCoprinus species by RAPD patterns with 12 of 13 primers. Also one specimen of unknownCoprinus strain was identified to beC. cinereus by this method. The RAPD patterns were similar among the strains in the same species; many common DNA fragments were recognized as well as some strain-specific DNA fragments. Thus all seven strains ofC. cinereus and all four strains ofC. angulatus examined could be distinguished individually. Diakryotic strains showed the combined RAPD patterns of the two monokaryotic strains constituting the dikaryon. The combined RAPD markers observed in the dikaryons were segregated in their basidiospore progeny. All 18 randomly picked progeny showed different combinations of RAPD markers from the parental strains.     

Molecular polymorphism and phenotypic variation in Aspergillus carbonarius

Thirteen collection strains and field isolates of Aspergillus carbonarius were examined by using various genotypic and phenotypic approaches. Restriction fragment length polymorphism analysis of the ribosomal RNA gene cluster and the mitochondrial DNA of the strains revealed only slight variations, except for one field isolate (IN7), which exhibited completely different ribosomal RNA gene cluster and mitochondrial DNA patterns. The mitochondrial DNAs of these strains were found to be much larger (45 to 57 kb) than those found earlier in the A. niger aggregate. Strain-specific characters could be detected by the random amplified polymorphic DNA technique. Isoenzyme analysis and examination of carbon source utilisation patterns of the strains also revealed some intraspecific variability, though much smaller than that observed by using DNA-based techniques. The dendrograms constructed based on genotypic and phenotypic data suggest that strain IN7 might represent a new subspecies of A. carbonarius.Abbreviations kb kilobase pair - mtDNA mitochondrial DNA - RAPD random amplified polymorphic DNA - rDNA ribosomal RNA gene cluster - RFLP restriction fragment length polymorphisms     

Molecular assessment of diversity among endophytic diazotrophs isolated from subtropical Indian sugarcane

Twenty-two endophytic bacterial isolates from the roots of sugarcane were compared morphologically, biochemically and genetically. Gram staining, colony pigment, texture and other cultural characteristics were taken for morphological characterization. Oxidation-fermentation tests for D-glucose and D-sucrose, production of acid and hydrogen from different carbon source, oxidase activity, antibiotic and drug resistance patterns were chosen as the biochemical and physiological criteria. Twelve random decamer primers were used to analyze and compare these isolates through RAPD among themselves as well as with known standard diazotrophic strains. The isolates were compared through dendrograms constructed on the basis of similarity patterns obtained from biochemical and RAPD analysis. The estimated diversity through RAPD analysis was more evident than the diversity on the basis of morphological and biochemical characters. Within Acetobacter group, the isolates showed substantial genetic diversity for future exploitation as PGPRs and diazotrophic associative endophytes.     

Variation revealed by RAPD‐PCR profiles of microsymbiont Anabaena azollae strains from four N2 fixing Azolla cultures

Nitrogen fixing Anabaena azollae strains isolated from four different Azolla cultures were characterized based on their total protein profile and RAPD profile to study the existing variation among them. As expected, the isolates showed almost similar protein banding patterns, but exhibited differences in 40–70 KDa protein subunits. Polymerase chain reaction of the DNA of the isolates, using four different primers, amplified specific sequences of DNA and showed clear polymorphism among the isolates. The RAPD profile generated the fingerprinting pattern characteristic of each strain based on the sequence of the primers used. Common band sharing observed between the strains A. azollae‐RS‐KK‐SK‐AM and A. azollae‐RS‐KK‐SK‐RP probably represents maternal inheritance of DNA to the progeny. The polymorphic bands were generated specifically for the isolates A. azollae‐RS‐KK‐SK‐RP and A. azollae‐RS‐KK‐SK‐AM with primers numbered 2 and 4, respectively, which could be developed as possible markers for these isolates.     

Molecular characterization of endophytic strains of Fusarium verticillioides (= Fusarium moniliforme) from maize (Zea mays. L)

The species Fusarium verticillioides (= F. moniliforme) is often found in maize seeds, constituting an important source of inoculum in the field. Fusarium spp., associated with symptomatic and asymptomatic plants, may be a primary causal agent of disease, a secondary invader or an endophyte. In the present work, endophytic fungi were isolated from two populations of Zea mays (BR-105 and BR-106) and their respective inbred lines. Within different inbred lines of maize, Fusarium was found at a frequency of 0 to 100% relative to the number of total isolated fungi. The frequency with which the genus occurred was practically the same in the two field sites (around 60%). Twenty-one F. verticillioides strains were analysed using the random amplified polymorphic DNA (RAPD) technique, employing 10 random primers. Variability analysis of endophytic isolates via RAPD showed genome polymorphism taxa of species around 60%. Endophytic isolates were clustered by their sites of origin. RAPD analysis clustered the endophytic isolates by their maize inbred lines hosts (Mil-01 to Mil-06), whereas at site A they clustered into two major groups related to the maize gene pool (BR-105 or BR-106 population). All strains isolated from seeds collected in Site A, except strains L9 and L10, were sub-grouped according to maize inbred lines. The analysis showed a discrete sub-grouping at site B. Results obtained here could be explained by a co-evolution process involving endophytic isolates of F. verticillioides and maize inbred lines.     

Molecular and phenotypic characterisation of Bacillus thuringiensis isolated during epizootics in Cydia pomonella L

Twelve Bacillus thuringiensis strains were isolated from intestinal tracts of Cydia pomonella larvae during epizootics in different laboratory insect culture lines. Phenotypic and genetic similarity of these isolates, together with two cultured from Leucoma salicis larvae and 14 reference B. thuringiensis strains were determined. The epizootic bacteria did not form a single group based on numerical analysis of biochemical properties. Simple RAPD method with only one primer does not allow estimating the genetic similarity of B. thuringiensis strains. We propose a novel strategy based on combining several DNA patterns obtained by RAPD technique with different primers for B. thuringiensis typing. Majority of infections in the C. pomonella culture lines were caused by bacteria with different genotypes. However, two isolates cultured from infected insects at different time (one strain was isolated in 1990 and the other in 1992) had identical DNA fingerprint that suggested a possibility of these bacteria to survive in the laboratory and to cause infections in different years. The results of SDS-PAGE of whole-cell proteins revealed a possibility to apply protein profile analysis in epidemiological investigations of infections caused by B. thuringiensis. Strains with identical DNA patterns had very similar whole-cell protein profiles.     

New available SCAR markers: potentially useful in distinguishing a commercial strain of the superior type from other strains of <Emphasis Type="Italic">Lentinula edodes</Emphasis> in China

At present, more than 100 strains of Lentinula edodes are cultivated on a commercial scale in China. A simple, reliable, and effective method to distinguish some commercial strains of the superior type from other commercial strains is very important for the Lentinula industry. In this study, 23 commercial strains of L. edodes cultivated widely in China at present were collected and analyzed with randomly amplified polymorphic DNA (RAPD) technique. Three informative dominant sequence characterized amplified region (SCAR) markers were developed by designing three pairs of specific SCAR primers from three sequenced differential RAPD bands, respectively. Based on the three SCAR markers, three different multiplex polymerase chain reaction (PCR) phenotypes were detected among the 23 studied commercial strains and in which a multilocus phenotype characterizing a commercial strain Cr02 of the superior type could potentially be used to distinguish this strain from the other 22 studied commercial strains. To our knowledge, this study is the first to describe the development of a multiplex PCR technique based on SCAR markers for detecting the molecular phenotypes among commercial strains of L. edodes in China.     

Genome Stability of Bacillus thuringiensis subsp. israelensis Isolates

Swedish soil isolates biochemically classified as Bacillus thuringiensis subsp. israelensis were further examined for genetic diversity by multilocus enzyme electrophoresis (MLEE), random amplified polymorphic DNA analysis (RAPD), pulse field gel electrophoresis (PFGE), and Southern blotting, and were compared with reference strains. All the tested strains belonging to the Bt. israelensis serotype H14 were found to be identical, as judged from the RAPD analysis. MLEE analysis gave a similar result; only one H14 strain was found to differ from the remaining H14 strains by one null allele. PFGE analysis confirmed a very close relationship between the H14 strains but revealed an SfiI restriction fragment of variable size. Southern blot analyses were carried out with probes for the chromosomally encoded flagellin gene(s) and the plasmid-encoded mosquitocidal toxins. All probes gave similar hybridization patterns in the H14 strains. The mosquito toxin probes hybridized only to the H14 strains, except for one probe hybridizing to strain 6:3, which was originally isolated from the same soil sample as strains 6:11 and 6:12. Because the RAPD, MLEE, and PFGE analyses showed that strain 6:3 appears to be unrelated to strains 6:11 and 6:12, the presence of a mosquito toxin sequence in strain 6:3 may suggest that gene transfer has occurred. Received: 8 July 1999 / Accepted: 9 August 1999     

Novel Endophytes of Rice form a Taxonomically Distinct Subgroup of Serratia marcescens

Six endophytic strains isolated from surface-sterilized rice roots and stems of different rice varieties grown in the Philippines were characterized. They were analyzed by physiological and biochemical tests, SDS-PAGE of whole-cell protein patterns, DNA-DNA hybridization and 16S rDNA sequencing. SDS-PAGE of whole-cell patterns showed that the six isolates fell into two subgroups which were similar but not identical in protein patterns to S. marcescens. The phylogenetic analysis of 16S rDNA sequences of two representative strains IRBG 500 and IRBG 501 indicated that they were closely related to S. marcescens(more than 99% identity). Physiological and biochemical tests corroborated that the isolates were highly related to each other and to S. marcescens. In cluster analysis, all six isolates were clustered together at 93% similarity level and grouped closely with Serratia marcescens at 86% similarity level. DNA-DNA hybridization studies revealed that the isolates shared high similarity levels with S. marcescens(≥86% DNA-DNA binding), indicating they belong to the same species. However, the isolates differed in several biochemical characteristics from the type strain. They produce urease and utilize urea and L(+) sorbose as a substrate, which is different from all known Serratia reference strains. These results suggest that the six endophytic isolates represent a novel, non-pigmented subgroup of S. marcescens.     

Candida parapsilosis fungemia in neonates: genotyping results suggest healthcare workers hands as source, and review of published studies

An outbreak of Candida parapsilosis fungemia involving 17 neonatal intensive care unit (NICU) patients was studied. There were 14 blood culture and nine colonizing isolates from other sites available. The hands of NICU healthcare workers (HCW) yielded eight isolates. Screening of the isolates by random amplified polymorphic DNA (RAPD) method showed only three profiles. Typing by restriction fragment length polymorphism (RFLP) revealed all blood isolates were RFLP subtype VII-1. Among the nine infant colonizing isolates, there were four different RFLP subtypes; four of the isolates were subtype VII-1. Seven of the eight isolates from HCW were RFLP subtype VII-1. The majority of infant colonizers were not found in the blood, suggesting a possible direct spread of the epidemic subtype VII-1 strain from HCW hands to infant blood. The source of the infant colonizing strains is unclear, but non-VII-1 strains may be largely of maternal origin and VII-1 strains from HCW. These findings reinforce prior studies that have implicated HCW hands as the source of nosocomial, including neonatal, fungemia.     

Characterization and infectivity of a spontaneous variant isolated fromFrankia sp. WEY 0131391

Summary A spontaneous variant, obtained from aFrankia isolate fromAlnus rubra nodules, was compared with the parent strain with regard to infectivity, nitrogenase activity, and electrophoretic and immunological profiles. Both the parent and the variant strain were equally effective in inducing nodulation in seedlings ofA. rubra. All inoculated plants had an active nitrogenase system as measured by the acetylene reduction assay. Electrophoresis of whole cell homogenates on SDS-polyacrylamide slab gels showed similar electrophoretic profiles; however, the variant strain also exhibited striking differences in protein patterns that distinguish it from the parent strain. Immunological analysis of the originalFrankia strain and its variant revealed shared antigens as well as immunologically distinct antigenic determinants in the two strains. The variant strain exhibits a distinct morphology and growth patterns which remain stable after many passages through culture.