Genetic Diversity of Fusarium oxysporum Populations Isolated from Tomato Plants in Tunisia

Fusarium crown and root rot of tomato (Lycopersicon esculentum) caused by Fusarium oxysporum f. sp. radicis‐lycopersici is a new devastative disease of tomato greenhouse crops in Tunisia. Nothing is known neither about the population of this pathogen in this region, nor about the population of F. oxysporum f. sp. lycopersici the causal agent of Fusarium wilt of tomato. In order to examine the genetic relatedness among the F. oxysporum isolates by intergenic spacer restriction fragment length polymorphism (IGS‐RFLP) analysis and to elucidate the origin of the formae specialesradicis‐lycopersici in Tunisia by looking for genetic similarity of Tunisians isolates with isolates from a foreign source, the genetic diversity among F. oxysporum f. sp. radicis‐lycopersici and F. oxysporum f. sp. lycopersici populations was investigated. A total of 62 isolates of F. oxysporum, obtained from symptomless tomato plants, were characterized using IGS typing and pathogenicity tests on tomato plants. All Fusarium isolates were highly pathogenic on tomato. Fusarium oxysporum f. sp. radicis‐lycopersici isolates were separated into five IGS types. From the 53 F. oxysporum f. sp. radicis‐lycopersici isolates, 34 isolates have the same IGS types (IGS type 25), and the remaining 19 isolates were distributed into four IGS types. However, the only nine isolates of F. oxysporum f. sp. lycopersici have six different IGS types. This difference of diversity between the two formae speciales suggests that F. oxysporum f. sp. radicis‐lycopersici isolates have a foreign origin and may have been accidentally introduced into Tunisia.     

Restriction analysis of PCR amplified nrDNA regions revealed intraspecific variation within populations of Fusarium culmorum

Seventy-five isolates of Fusarium culmorum with diverse geographical origin and host were analyzed using restriction digestion of polymerase chain reaction amplified nuclear ribosomal DNA intergenic spacer (IGS) and 28S gene regions. The 28S gene was conserved and has produced identical restriction patterns, however, the IGS region was substantially variable. The isolates were divided into 29 unique IGS haplotypes. There was limited resolution between clustering of isolates and their origin and/or host. The variability was distributed largely equally at both macro- and micro-geographical scale. The phylogeographic distribution pattern suggests a seed-borne dispersal of F. culmorum.     

Genetic variability in intergenic spacers of ribosomal DNA in Pisolithus isolates associated with pine, eucalyptus and Afzelia in lowland Kenyan forests

Basidiocarps of Pisolithus associated with indigenous ( Afzelia quanzensis Welw.) and introduced ( Pinus caribaea Mor. and Eucalyptus camaldulensis Dehnh.) hosts in the lowland forests of the Coast Province of Kenya are morphologically distinct. Genetic variability among 52 Pisolithus basidiocarps, collected beneath the various host plants, was examined based on sequence polymorphism within the internal transcribed spacer (ITS) and intergenic spacer (IGS1) of ribosomal DNA genes. Variability in ITS and IGS1 sequences indicated that the three host-associated morphotypes were genetically different. Consensus trees generated by bootstrap analysis of sequence data of Pisolithus isolates from Australia and Kenya are polyphyletic and strongly suggest that the three different morphotypes/genotypes present in Kenya represent separate biological species. In addition, our data indicate that little genetic exchange occurs in silva between these species.     

Mycoplasma hominis in Cuban Trichomonas vaginalis isolates: association with parasite genetic polymorphism

Trichomonas vaginalis can be naturally infected with intracellular Mycoplasma hominis. This bacterial infection may have implications for trichomonal virulence and disease pathogenesis. The objective of the study was to report the presence of M. hominis in Cuban T. vaginalis isolates and to describe the association between the phenotype M. hominis infected with RAPD genetic polymorphism of T. vaginalis. The Random Amplified Polymorphic DNA (RAPD) technique was used to determine genetic differences among 40 isolates of T. vaginalis using a panel of 30 random primers and these genetic data were correlated with the infection of isolates with M. hominis. The trees drawn based on RAPD data showed no relations with metronidazole susceptibility and significantly association with the presence of M. hominis (P=0.043), which demonstrates the existence of concordance between the genetic relatedness and the presence of M. hominis in T. vaginalis isolates. This result could point to a predisposition of T. vaginalis for the bacterial enters and/or survival.     

Cysteine proteinase 30 in clinical isolates of T. vaginalis from symptomatic and asymptomatic infected women

A cysteine proteinase of 30 kDa (CP30) of Trichomonas vaginalis, is known to play a role in cytoadherence of the parasite to host cells. However, the CP30 activity in clinical isolates from symptomatic and asymptomatic patients has not been analyzed. In the present study, CP30 was detected in 20 fresh and long-term culture maintained T. vaginalis isolates each from symptomatic and asymptomatic women by substrate gel electrophoresis and immunoblotting. Though CP30 was detected in all the fresh isolates from 20 symptomatic and 20 asymptomatic women, the intensity of CP30 band was significantly higher in isolates from symptomatic as compared to asymptomatic women indicating higher expression in former. CP30 was found in all the 20 long-term cultured isolates from symptomatic whereas only in 70% of asymptomatic women indicating that CP30 expression is a more stable characteristic of symptomatic isolates. The isolates from symptomatic women, demonstrated significantly higher cytoadherence to VECs as compared to asymptomatic women. In both the types of isolates, this cytoadherence was inhibited significantly by CP30 specific hyperimmune serum. These results confirm that CP30 is an important virulence factor of T. vaginalis and has an important role in cytoadherence to VECs and thus has a role in pathogenesis of trichomoniasis.     

Molecular and physiological evaluation of subtropical environmental isolates of Acanthamoeba spp., causal agent of Acanthamoeba keratitis

Previous molecular examination of Acanthamoeba spp. has resulted in the determination of distinct genotypes in this genus (designated T1-T12, T14). Genotype T4 has been responsible for the majority of cases of Acanthamoeba keratitis. Here we examine the relative abundance of environmental T4 isolates on beaches and ask whether they have temperature and salinity tolerances that could enhance pathogenicity. Twenty-four Acanthamoeba strains were isolated from beach sand (n = 20), soil (n = 3), and tap water (n = 1) in south Florida. Phylogenetic analysis identified 19 of 24 isolates as T4, the Acanthamoeba keratitis-associated genotype. The remaining isolates were genotype T5 (4) and T11 (1). Nearly all beach isolates were genotype T4, whereas the tap water and soil isolates were mostly T5. All amoebae grew at 0, 1.0, and 2.0% salt and 19 of 20 beach isolates also grew at 3.2%. No soil or tap-water acanthamoebae reproduced at 3.2%. All isolates grew at 37 degrees C and two (T5) at 42 degrees C. Little correlation existed between beach location, salt-tolerance, and genetic relatedness. Overall, the large majority of environmental isolates obtained were genotype T4, suggesting it may be the most common genotype in this environment and could be a potential source of Acanthamoeba keratitis infections.     

Double-stranded RNA virus in Korean isolate IH-2 of Trichomonas vaginalis

In this study, we describe Korean isolates of Trichomonas vaginalis infected with double-stranded (ds) RNA virus (TVV). One T. vaginalis isolate infected with TVV IH-2 evidenced weak pathogenicity in the mouse assay coupled with the persistent presence of a dsRNA, thereby indicating a hypovirulence effect of dsRNA in T. vaginalis. Cloning and sequence analysis results revealed that the genomic dsRNA of TVV IH-2 was 4,647 bp in length and evidenced a sequence identity of 80% with the previously-described TVV 1-1 and 1-5, but only a 42% identity with TVV 2-1 and 3 isolates. It harbored 2 overlapping open reading frames of the putative capsid protein and dsRNA-dependent RNA polymerase (RdRp). As previously observed in the TVV isolates 1-1 and 1-5, a conserved ribosomal slippage heptamer (CCUUUUU) and its surrounding sequence context within the consensus 14-nt overlap implied the gene expression of a capsid protein-RdRp fusion protein, occurring as the result of a potential ribosomal frameshift event. The phylogenetic analysis of RdRp showed that the Korean TVV IH-2 isolate formed a compact group with TVV 1-1 and 1-5 isolates, which was divergent from TVV 2-1, 3 and other viral isolates classified as members of the Giardiavirus genus.     

Polymorphism in the internal transcribed spacer (ITS) region of the ribosomal DNA among different Fusarium species

Fusarium species causing wilt diseases in different plants were characterised by comparing nonpathogenic and different pathogenic species using rDNA RFLP analysis. The ITS (internal transcribed spacer) region of 12 isolates belonging to the section Elegans, Laseola, Mortiella, Discolor, Gibbosum, Lateritium and Sporotrichiella were amplified by universal ITS primers (ITS-1 and ITS-4) using polymerase chain reaction (PCR). Amplified products, which ranged from 522 to 565 bp were obtained from all 12 Fusarium isolates. The amplified products were digested with seven restriction enzymes, and restriction fragment length polymorphism (RFLP) patterns were analysed. A dendrogram derived from PCR-RFLP analysis of the rDNA region divided the Fusarium isolates into three major groups. Assessment of molecular variability based on rDNA RFLP clearly indicated that Fusarium species are heterogeneous and most of the forma speciales have close evolutionary relationships.     

Trichomonas vaginalis: random amplified polymorphic DNA analysis of isolates from symptomatic and asymptomatic women in India

Trichomonas vaginalis, the causative agent for human trichomoniasis, is a protozoan parasite. Trichomoniasis is the most common non-viral sexually transmitted disease. The infection in women may be asymptomatic or may lead to severe vaginitis, cervicitis and severe sequelae. Despite its high prevalence, the genetic variability and factors leading to symptomatic infection have been poorly understood. One thousand women in childbearing age group were screened for the presence of T vaginalis. Thirty-eight women were found positive for T vaginalis and out of these 22 (57.9%) were having symptomatic infection and 16 (42%) were asymptomatic. Fresh isolates from 15 symptomatic and 15 asymptomatic women were axenised and subjected to random amplified polymorphic DNA (RAPD) analysis with the use of five different random primers (OPD 1-OPD 5). The isolates with similar banding pattern were assigned as a single type. OPD 3 indicated least (nine types) while OPD 4 indicated highest typing (18 types) ability. Phylogenetic analysis using RAPD distance software indicated two distinct lineages; upper branch consisting of only seven symptomatic isolates while lower branch consisting of all the 15 asymptomatic isolates the other eight symptomatic isolates were recorded in separate cluster. The study indicated that RAPD technique might be helpful to delineate the pathogenic mechanism(s) for its virulence; however, further studies on large number of isolates are desired to elucidate the findings.     

Characterization of natural populations of Nitrobacter spp. using PCR/RFLP analysis of the ribosomal intergenic spacer

DNA sequences from the intergenic spacer (IGS) region of the ribosomal operon were amplified by the polymerase chain reaction (PCR) technique using two primers derived from 16S and 23S rRNA conserved sequences. The PCR products, cleaved by 4 base cutting restriction enzymes, were used to differentiate Nitrobacter strains. This method offered a convenient alternative to serological testing for characterization of Nitrobacter isolates and enabled a large number of strains to be genotypically characterized easily and rapidly. This method was successfully used to characterize natural populations of Nitrobacter from various soils and a lake. A diversity was demonstrated in various soils, and in a lake both in freshwater and in sediments. Strains closely related to both WL and LL were found in these eco-systems. It seems that the diversity of Nitrobacter populations was not associated with global environments but may be related to the presence of locally coexisting niches.Non-commun abbreviations PCR polymerase chain reaction - RFLP restriction fragment length polymorphism - IGS intergenic spacer     

Genetic,morphological, and virulence characterization of the entomopathogenic fungus Verticillium lecanii

In order to clarify relationships among genetic diversity, virulence, and other characteristics of conidia, 46 isolates of Verticillium lecanii from various hosts and geographical locations were examined. The internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of ribosomal DNA (rDNA), mitochondrial small subunit rDNA (mt-SrDNA) and beta-tubulin were analyzed by PCR-RFLP. PCR-single stranded conformational polymorphism (SSCP) was performed on regions of the mitochondrial large subunit rDNA, mt-SrDNA, beta-tubulin and histone 4. There were no relationships among the results of RFLP, SSCP, isolation source, and location. However, amplified product size of IGS did have relationships with conidia size and sporulation. Six isolates with 4.0-kb IGS products had large conidia dimensions, and yielded low numbers of conidia compared with other isolates. Three out of the six isolates were high virulence (over 90%) against green peach aphids. Furthermore, double-stranded RNA (dsRNA) was detected in 22 out of 35 V. lecanii isolates and related with the amplicon sizes of IGS, though not with virulence or isolation location. Isolates containing dsRNA were divided into six distinct types based on banding pattern. These data demonstrate the level of genetic diversity of V. lecanii, and suggest relations among the genetic properties and conidial morphology.     

Race Differentiation in Fusarium oxysporum f.sp. chrysanthemi

The pathogenicity of different isolates of Fusarium oxysporum obtained from plants of Gerbera (Gerbera jamesonii), Chrysanthemum (Chrysanthemum morifolium), Paris daisy (Argyranthemum frutescens) and African daisy (Osteospermum sp.), all in the family Asteraceae, was tested on different cultivars of these hosts, to assess their pathogenicity. The reactions were compared with those of isolates of F. oxysporum f. sp. chrysanthemi and of f.sp. tracheiphilum obtained from the American Type Culture Collection. We found that isolates of F. oxysporum f. sp. chrysanthemi can be distinguished as three physiological races on the basis of their pathogenicity to the panel of differential cultivars. Sequencing of the intergenic spacer (IGS) region of ribosomal DNA (rDNA) and phylogenetic analysis showed that the Fusarium races fell into three phylogenetic groups, which coincided with those observed in pathogenicity tests. Analysis of the IGS sequences revealed a high degree of similarity among strains from Italy and Spain from different host species, suggesting that recent outbreaks in these ornamentals were probably caused by introduction of infected nursery material from a common origin.     

Multiple polymorphic sites at the ITS and ATAN loci in cultured isolates of Perkinsus marinus

Sequence analysis of genomic DNA from the protozoan parasite Perkinsus marinus at two loci revealed genetic polymorphisms within and among different cultured isolates. Genomic DNA from 12 Perkinsus marinus isolates was amplified at the internal transcribed spacer region and at an anonymous locus previously identified to contain polymorphisms by restriction fragment length polymorphism analysis. Fourteen polymorphic nucleotide positions were identified at the internal transcribed spacer region; eight in internal transcribed spacer 1 and six in internal transcribed spacer 2. Thirteen polymorphic nucleotide sites were identified within the anonymous locus. In some instances, more than three different sequences were observed at both the internal transcribed spacer region and at the anonymous locus from a single clonal isolate, suggesting the possibility of recombination in cultured cells and/or strand jumping during the polymerase chain reaction. Intra-isolate sequence variation (3.46% for the anonymous locus and 3.08% for internal transcribed spacer 1) was in several cases as high as inter-isolate sequence variation, even in one isolate where recombination was not evident. High intra- and inter-isolate variation detected at both loci demonstrates the importance of determining the genetic variation of each locus prior to development of sequence-based molecular diagnostics.     

Trypanosoma evansi: molecular homogeneity as inferred by phenetical analysis of ribosomal internal transcribed spacers DNA of an eclectic parasite

The protozoan Trypanosoma evansi is described as presenting high morphological and genetic similarities among the isolates despite its biological heterogeneity and wide geographical distribution. PCR amplification of the internal transcribed spacers of the ribosomal gene in combination with the coding region of the 5.8S ribosomal subunit further submitted to restriction enzymes digestion were carried out in DNAs extracted from 41 T. evansi strains isolated from horses, dogs, coatis and capybaras from two distinct regions of the Brazilian Pantanal. We also used one T. evansi isolate from Africa, one from Asia and one isolate of T. b. brucei from Africa. Analysis of the RFLP profiles yielded a unique "riboprinting" that does not vary intraspecifically. These results provide insights on the ribosomal gene organization of T. evansi and showed that ITS analysis by RFLP show high genetic similarity of this locus among isolates of this protozoan parasite.     

PCR-RFLP patterns of four isolates of Trichinella for rDNA ITS1 region

We have studied the genetic differences among four isolates of Trichinella including a new strain of Trichinella spiralis (ISS 623) recently found from a human case who took a badger in Korea. Because they have a different host origin and came from geographically separated regions, we supposed the genetic pattern of the isolates might be different as had been previously reported. It was analysed by PCR-RFLP analysis of the rDNA repeat that can readily distinguish a species or strain from others. Isolated genomic DNA of each isolate of Trichinella larvae was amplified with ITS1 specific primers and digested with restriction endonucleases. The PCR product of ITS1 was confirmed using Southern blot analysis to be a 910 bp fragment. The restriction fragments of each isolate had variable patterns when it was digested with Rsa 1 only. According to the RFLP patterns, the estimated genetic divergence between each isolate was different. In conclusion, four isolates of Trichinella including a new strain of T. spiralis obtained from a Korean patient may have genetic differences in the ITS1 region and the Shanghai isolate was genetically more similar to the Japanese unknown isolate than others in the ITS1 region.     

Genetic diversity of carbofuran-degrading soil bacteria

The genetic diversity of 128 carbofuran-degrading bacteria was determined by ARDRA (amplified ribosomal DNA restriction analysis) of 16S rDNA and restriction fragment length polymorphism analysis of the 16S-23S rDNA spacer region (IGS) using five endonucleases. The isolates were distributed in 26 distinct ARDRA groups and 45 IGS types revealing a high level of microbial diversity confirmed by ARDRA clustering and sequencing of 16S rDNA. The occurrence of a methylcarbamate-degrading gene (mcd) was monitored by polymerase chain reaction amplification using specific primers. The mcd gene was detected only in 58 bacteria and there was no clear relationship between the presence of this gene and the phylogenetic position of the strain.     

Genetic diversity of Fusarium oxysporum populations isolated from different soils in France

The genetic diversity of soil-borne populations of Fusarium oxysporum was assessed using 350 isolates collected from six different French soils. All isolates were characterised by restriction fragment analysis of the PCR-amplified ribosomal intergenic spacer (IGS). Twenty-six IGS types were identified among the 350 isolates analysed. Five to nine different IGS types were detected in each soil. None of the IGS types was common to all of the soils. An analysis of the molecular variance based on IGS type relationships and frequency revealed that the genetic structure of the populations of F. oxysporum varied widely among the soils. Some populations were both highly diverse within the soils and differentiated between the soils. A possible relationship between the intrapopulation or interpopulation level of diversity and some external factors such as the soil type or the crop history was evaluated. A subsample representative of the diversity of the six populations was further characterised by analysing the genomic distribution of two transposable elements, impala and Fot1. One to 10 copies of the impala element were present in most of the isolates, irrespective of their soil of origin. The Fot1 element was only detected in 40% of the isolates originating from the three populations less diverse in terms of IGS types, but in 82.6% of the isolates originating from the three more diverse populations.     

Monitoring the persistence of Laccaria bicolor as an ectomycorrhizal symbiont of nursery-grown Douglas fir by PCR of the rDNA intergenic spacer

The large-scale inoculation of selected beneficial ectomycorrhizal fungi in forest nurseries has generated renewed interest in the ecology of these symbiotic fungi. However, information on the dissemination and persistence of introduced symbionts is scarce due to the limitation of the current identification methods. To identify ectomycorrhizal fungi on single root tips, we investigated the polymorphism of the PCR-amplified ribosomal DNA intergenic spacer (IGS) from a wide range of ectomycorrhizal fungi. To investigate the reliability of this molecular approach in large-scale surveys, the dissemination and persistence on Douglas fir seedlings of the introduced Laccaria bicolor S238N were assessed in a forest nursery in the Massif Central (France). Several hundred ectomycorrhizas and fruiting bodies were sampled from plots where control and L. bicolor inoculated-Douglas fir seedlings were grown for 1.5 years. PCR typing of mycorrhizas indicated that trees inoculated with L. bicolor S238N remained exclusively colonized by that isolate (or sexually derived isolates) for the entire test period. In contrast, control seedlings were infected by indigenous isolates of Laccaria laccata and Thelephora terrestris. The molecular evidence for the persistence of the introduced mycobiont despite the competition from indigenous isolates of the same species provides further illustration of the potential of exotic species for large-scale microbial application.     

Intraspecific variation and population structure of the German cockroach, Blattella germanica, revealed with RFLP analysis of the non-transcribed spacer region of ribosomal DNA

Little information is available on genetic variation within and between populations of pest cockroaches. In this study, intraspecific HindIII polymorphism was investigated in the German cockroach, Blattella germanica (Linnaeus) (Dictyoptera, Blattaria: Blattellidae), using restriction fragment length polymorphisms (RFLP) of the non-transcribed spacer (NTS) region of ribosomal DNA (rDNA). Individual male insects were collected from infestations at three different pig farms. Each population was characterized by HindIII restriction fragment frequencies and haplotype (a particular X-chromosome pattern) frequencies. The inheritance of the X-chromosome HindIII rDNA patterns over 12 generations (3 years) follows Mendelian patterns, and the stability of this polymorphic marker indicates infrequent genetic recombination of variable sites. Although pairwise genetic distance measures were uncorrelated with geographical distance, the pattern of genetic differentiation of the three cockroach populations suggests that human-mediated transport of cockroaches is an important force in shaping the population genetic structure of cockroach infestations, at least at the regional scale of 10-100 km. Sequence variation in the ribosomal NTS is a useful marker, and RFLP of rDNA is a simple, robust and reproducible technique for differentiating recently diverged cockroach populations.     

Structure of the intergenic spacer region from the ribosomal RNA gene family of white spruce (Picea glauca)

Five genomic clones containing ribosomal DNA repeats from the gymnosperm white spruce (Picea glauca) have been isolated and characterized by restriction enzyme analysis. No nucleotide variation or length variation was detected within the region encoding the ribosomal RNAs. Four clones which contained the intergenic spacer (IGS) region from different rDNA repeats were further characterized to reveal the sub-repeat structure within the IGS. The sub-repeats were unusually long, ranging from 540 to 990 bp but in all other respects the structure of the IGS was very similar to the organization of the IGS from wheat, Drosophila and Xenopus.