Comparison of pulsed‐field gel electrophoresis and three rep‐PCR methods for evaluating the genetic relatedness of Stenotrophomonas maltophilia isolates

Aims: In this study, three facile repetitive‐sequence PCR (rep‐PCR) techniques have been compared with the pulsed‐field gel electrophoresis (PFGE) method for differentiating the genetic relatedness of clinical Stenotrophomonas maltophilia isolates. Methods and Results: The dendrograms of 20 S. maltophilia isolates were constructed based on the data obtained from PFGE and three PCR‐based methods, i.e. enterobacterial repetitive intergenic consensus‐PCR (ERIC‐PCR), BOX‐PCR and repetitive extragenic palindromic‐PCR (REP‐PCR). When compared with PFGE, ERIC‐PCR displayed a much lower discriminatory power, whereas BOX‐PCR and REP‐PCR had a comparable discriminatory power for close genetic‐related isolates. Conclusion: BOX‐PCR and REP‐PCR can be convenient and effective methods for evaluating the close genetic relatedness of clinical S. maltophilia isolates. Significance and Impact of the Study: A rapid method for determining S. maltophilia’s close genetic relatedness provides a convenient tool for understanding the epidemiology of S. maltophilia.     

Uneven Distribution of Mating‐Type Alleles Among Togninia minima Isolates,One of the Causal Agents of Leaf Stripe Disease on Grapevines in Northwest Iran

Togninia minima is the main fungal species associated with grapevine leaf stripe disease worldwide. This species is mainly known from its asexual state in nature; nevertheless, a biallelic heterothallic mating strategy has been confirmed for this species based on in vitro crossing studies. There are no data available on the incidence of an active sexual cycle within the populations of this species in many grapevine‐producing countries as well as Iran. The possibility of a clandestine sexual cycle within the Iranian isolates of T. minima was evaluated by analysing the distribution and frequency of the mating‐type alleles on a microspatial and a macrogeographical scales. Towards this aim, a total of 90 T. minima isolates were recovered from grapevines with esca disease from the vineyards in north and north‐western Iran. A multiplex PCR method previously designed by authors was applied for simultaneous identification and determination of the mating‐type alleles in T. minima populations. The results on the screening of mating‐type alleles using multiplex PCR method revealed the mating‐type identity of 77 isolates as Mat1‐2 and 23 isolates as Mat1‐1. Our results showed that both Mat1‐1 and Mat1‐2 isolates are present in a single vineyard and even on single vines. The distribution of mating‐type alleles in the sampled area skewed from the 1 : 1 ratio (77 : 23); however, co‐occurrence of both mating types in a single vineyard and even on single vines is suggestive for the presence of an active sexual cycle for T. minima in north‐western Iran.     

Genetic diversity among isolates of Paenibacillus larvae from Austria

Genetic diversity of 214 Paenibacillus larvae strains from Austria was studied. Genotyping of isolates was performed by polymerase chain reaction (PCR) with primers corresponding to enterobacterial repetitive intergenic consensus (ERIC), BOX repetitive and extragenic palindromic (REP) elements (collectively known as rep-PCR) using ERIC primers, BOX A1R and MBO REP1 primers. Using ERIC-PCR technique two genotypes could be differentiated (ERIC I and II), whereas using combined typing by BOX- and REP-PCR, five different genotypes were detected (ab, aB, Ab, AB and αb). Genotypes aB and αb are new and have not been reported in other studies using the same techniques.     

Differentiation of lineages within “Colletotrichum gloeosporioides s.l.” associated with cassava anthracnose disease by BOX‐ and ERIC‐PCRs

Several molecular techniques have been used to differentiate species or genetic lineages of microorganisms prior to sequencing. Among them, BOX‐ and ERIC‐PCRs may provide specific banding patterns for different species, allowing its differentiation. Therefore, the objective of this study was to evaluate these techniques as a tool for differentiation of phylogenetic lineages belonging to the Colletotrichum gloeosporioides species complex associated with cassava anthracnose disease. Sets of BOX‐ and ERIC‐PCR primers were used to assess the differentiation of lineages belonging to the complex with 81 C. gloeosporioides sensu lato (s.l.) isolates from different cassava producing regions. Some were identified by sequencing, such as Colletotrichum fructicola, Colletotrichum tropicale, C. gloeosporioides s.s, Colletotrichum theobromicola, Colletotrichum siamense, Colletotrichum brevisporum and Colletotrichum sichuanensis. The primers were able to amplify DNA fragments from all isolates. The ERIC‐PCR presented a wider range of banding patterns in comparison to BOX‐PCR, providing better differentiation of the individuals, as well as a higher correlation with the phylogenetic data was obtained by ERIC‐PCR and the combined data set for “BOX‐/ERIC‐PCRs,” inferred by Mantel test. However, the use of concatenated data (BOX‐/ERIC‐PCRs) reduced the discriminatory capacity presented by ERIC‐PCR alone, probably due to the lowest resolution of BOX‐PCR. Therefore, ERIC‐PCR technique enabled efficient differentiation of isolates belonging to the C. gloeosporioides complex and can be used to analyse multiple isolates in a collection and also being an important tool as a guide in the decision‐making process prior to sequencing. Based on this methodology, it was possible to identify two new species associated with cassava anthracnose disease, C. brevisporum and C. sichuanensis, being the first report of these two species associated with cassava anthracnose disease in Brazil.     

Rapid identification of pseudomonas avellanae field isolates,causing hazelnut decline in central italy,by repetitive PCR genomic fingerprinting

Pseudomonas avellanae is the main cause of hazelnut (Corylus avellana L.) decline, the so called ‘moria’, in central Italy where it has already killed more than 30 000 trees. Its current identification is very long requiring biochemical, physiological and nutritional tests as well as pathogenicity tests and takes not less than 6 months for its completion. In the present study the reliability of the repetitive polymerase chain reaction (rep‐PCR) technique for a rapid and accurate identification of such a pathogen was compared with the traditional identification method. In order to assess the variability of the pathogen, REP, BOX and ERIC primer sets were used in preliminary work to generate genomic fingerprints of 60 P. avellanae reference strains previously isolated from different areas of hazelnut cultivation. ERIC primers yielded the most discriminative clustering of strains that were grouped according to their geographic origin. Sixty field isolates collected from hazelnut orchards of central Italy, planted with different cultivars, during the years 1996–98 were submitted to either the traditional identification methods or to rep‐PCR by using ERIC primers. The latter technique accurately identified all the isolates that were also identified by the traditional methods. Whole‐cell protein analysis by means of sodium dodecyl sulphate‐polyacrylamide gel electrophoresis confirmed this achievement. Rep‐PCR can be successfully adopted for the rapid and accurate identification of P. avellanae in central Italy and it constitutes a very useful tool for the sanitation of the area.     

Characterization and genetic diversity of Pseudomonas syringae isolates from stone fruits in north‐western Iran

From 33 Iranian fluorescent Pseudomonas isolates originating from symptomatic tissues of peach (Prunus persica), plum (Prunus domestica), sweet (Prunus avium) and sour cherry (Prunus cerasus), 27 were identified as Pseudomonas syringae using LOPAT tests. Further characterization of those isolates by GATTa and L‐lactate utilization tests and the detection of syringomycin and coronatine and yersiniabactin coding genes showed that five of them belonged to race 1 and four to race 2 of P. syringae pv. morsprunorum (Psm) and eighteen other isolates were identified as P. syringae pv. syringae (Pss). Based on the analysis of the fingerprint patterns generated by REP, ERIC and BOX‐PCR, the strains were differentiated into three main groups at the 67% similarity level. Strains of the groups 1, 2 and 3 belong to Psm race 1, Psm race 2 and Pss, respectively. Rep‐PCR analysis showed high intra‐pathovar variation within the Pss isolates, which grouped into four distinct clusters. Using the REP primers, the percentage of polymorphic loci was 74.61%, whereas with BOX and ERIC primers, it was 60.5 and 55.21%, respectively. Finally, this study is the first report of the isolation of P. syringae pv. morsprunorum race 1 and 2 strains from stone fruit trees in Iran.     

Use of Rep‐PCR for Genetic Diversity Analyses in Fusarium culmorum

Fusarium culmorum is a pathogen of economically important grain crops. In this work, Rep‐PCR was used to identify genetic diversity in F. culmorum isolates which have been collected from wheat fields in Turkey. Reproducible genomic fingerprints were amplified in each strain by PCRs of prokaryotic repetitive extragenic palindromic (REP), enterobacterial repetitive intergenic consensus (ERIC) and BOX sequences. Totally 104 molecular markers were evaluated and similarity comparisons were shown as a dendrogram. The average genetic diversity was 52.3% ranging from 15.8% to 88.7% according to the Rep‐PCR data. Cluster analysis showed agreement with the distance of sampling locations. The highest genetic similarity (84.2%) was determined between two F. culmorum isolates (F1 and F2) originated from the same agro‐ecological region. Our results showed that Rep‐PCR is convenient and rapid for genetic diversity analyses and strain differentiation in F. culmorum.     

Characterization of a Chromobacterium haemolyticum population from a natural tropical lake

Aim: To study genetic diversity of Chromobacterium haemolyticum isolates recovered from a natural tropical lake. Methods and Results: A set of 31 isolates were recovered from a bacterial freshwater community by conventional plating methods and subjected to genetic and phenotypic characterization. The 16S ribosomal RNA (rRNA) gene phylogeny revealed that the isolates were related most closely with C. haemolyticum. In addition to the molecular data, our isolates exhibited strong β‐haemolytic activity, were nonviolacein producers and utilized i‐inositol, d ‐mannitol and d ‐sorbitol in contrast with the other known chromobacteria. Evaluation of the genetic diversity in the 16S rRNA gene, tRNA intergenic spacers (tDNA) and 16S‐23S internal transcribed spacers (ITS) unveiled different levels of genetic heterogeneity in the population, which were also observed with repetitive extragenic palindromic (rep)‐PCR genomic fingerprinting using the BOX‐AR1 primer. tDNA‐ and ITS‐PCR analyses were partially congruent with the 16S rRNA gene phylogeny. The isolates exhibited high resistance to β‐lactamic antibiotics. Conclusion: The population genetic heterogeneity was revealed by 16S rRNA gene sequence, ITS and BOX‐PCR analysis. Significance and Impact of the Study: This study provides for the first time an insight into the genetic diversity of phylogenetically close isolates to C. haemolyticum species.     

新疆土著大豆根瘤菌种群遗传结构的初步分析

应用重复序列REP（repetitive extragenic palindromic,重复基因外回）和ERIC（ente-robaterial repetitive intergenic consensus,肠细菌重复基因间共有序列）结合聚合酶链式反应（ERP－PCR和ERIC－PCR）对从新疆有集的27株土大豆根瘤菌染色体进行指纹分析,发现在相似水平0.5时可基本分为两大聚类群,一个类群主要包括慢生型根瘤菌,另一类群为快生型根瘤菌,来自同一地区的根瘤菌具有较高的遗传相似性,以上结果表明该技术中对大豆根瘤菌进行种群结构和遗传多样性分析的有效手段。     

Genetic diversity of Burkholderia solanacearum (synonym Pseudomonas solanacearum) race 3 in Kenya.

Genetic diversity among isolates of the bacterial plant pathogen Burkholderia solanacearum (synonym Pseudomonas solanacearum) race 3 biovar II of Kenya was determined by PCR with repetitive sequences (ERIC and BOX repetitive primer sets) and pulsed-field gel electrophoresis of genomic DNA digested by rare-cutting restriction endonucleases (RC-PFGE). The study comprised 46 isolates collected during 1992 from the major potato-growing regions of Kenya (45 were identified as race 3 biovar II, and 1 belonged to race 3 biovar N2) and 39 reference isolates from 19 other countries. RC-PFGE identified 10 distinct profile types among the Kenyan race 3 biovar II isolates (29 of the isolates exhibited identical profiles) and a further 27 distinct profile types among the reference isolates. ERIC and BOX primer sets were unable to differentiate race 3 biovar II isolates within the Kenyan population but differentiated a further two distinct profile types among the reference isolates. The race 3 biovar N2 isolate had a highly distinct RC-PFGE and repetitive sequence PCR profile. Statistical analysis of the data identified biogeographic trends consistent with conclusions drawn from previous studies on the origin and worldwide dissemination of race 3 biovar II isolates; however, genomic fingerprinting by RC-PFGE revealed a level of genetic diversity previously unrealized.     

Molecular intraspecific characterization of Photobacterium damselae ssp. damselae strains affecting cultured marine fish

Aims: The aim of this study was to analyse the intraspecific variability of Photobacterium damselae ssp. damselae strains isolated from different cultured marine fish species using molecular typing methods. Methods and Results: Twenty P. damselae ssp. damselae strains isolated from marine fish species were used in this study. Phenotypic characterization of the strains was carried out using standard microbiological methods. Genetic characterization was conducted using three PCR‐based methods [random amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus‐PCR (ERIC‐PCR) and repetitive extragenic palindromic‐PCR (REP‐PCR)]. Dice coefficient and the unweighted pair group method with average linkage were used for numerical analyses of banding patterns. At phenotypic level, the strains analysed showed seven different profiles, which could not be related to the host fish species, geographic area or outbreak of disease. Isolates were grouped into nine and eight clusters using the RAPD technique with primers 5 and 4, respectively. In both cases, the main cluster grouped 45% of strains. The techniques ERIC‐PCR and REP‐PCR were more discriminatory, both resulting in 14 different clusters, which grouped 15–20% of the isolates. Conclusions: In this study, the techniques tested are confirmed as good tools for molecular typing, because they allow discrimination between P. damselae ssp. damselae strains isolated within the same outbreak. In addition, ERIC‐PCR and REP‐PCR methods were more adequate for rapid typing of P. damselae ssp. damselae than RAPD, allowing the discrimination at strain level. Significance and Impact of the Study: The results, in agreement with previous studies, confirmed the high intraspecific variability among isolated P. damselae ssp. damselae strains at both phenotypic and genetic levels. This suggests the existence of different clonal lineages that coexist in the same geographic area, within a short period of time (2–3 years). The discrimination at strain level can be useful to study the traceability of infections.     

Characterization of Pectobacterium carotovorum subsp. carotovorum Causing Watermelon Soft Rot Disease in Iran

The aim of this study was characterized Pectobacterium carotovorum subsp. carotovorum (Pcc) the causal pathogen of watermelon soft rot disease in Iran. Of fifty bacterial isolates with white grey and convex colonies on nutrient agar obtained from symptomatic watermelon, ten isolates were selected for further tests. Pathogenicity tests results showed that all test isolates developed typical water‐soak symptoms after 2 days and signs of soft rot began 4 days after inoculation on watermelon fruits. Based on the phenotypic properties, the isolates were identified as Pectobacterium carotovorum subsp. carotovorum. The 16S rDNA sequences of isolates were 99% similar to the corresponding 16S rDNA sequence of the reference Pcc isolate. BOX and ERIC‐PCR analysis indicated that genetic diversity was present among the isolated Pcc isolates did not relate to the geographic location isolated from. To the best of our knowledge, this is the first study of biochemical and genotypic characterization of Pcc isolates the causal agents of soft rot disease on watermelon, in Iran.     

Variation in extragenic repetitive DNA sequences in Pseudomonas syringae and potential use of modified REP primers in the identification of closely related isolates

In this study, Pseudomonas syringe pathovars isolated from olive, tomato and bean were identified by species-specific PCR and their genetic diversity was assessed by repetitive extragenic palindromic (REP)-PCR. Reverse universal primers for REP-PCR were designed by using the bases of A, T, G or C at the positions of 1, 4 and 11 to identify additional polymorphism in the banding patterns. Binding of the primers to different annealing sites in the genome revealed additional fingerprint patterns in eight isolates of P. savastanoi pv. savastanoi and two isolates of P. syringae pv. tomato. The use of four different bases in the primer sequences did not affect the PCR reproducibility and was very efficient in revealing intra-pathovar diversity, particularly in P. savastanoi pv. savastanoi. At the pathovar level, the primer BOX1AR yielded shared fragments, in addition to five bands that discriminated among the pathovars P. syringae pv. phaseolicola, P. savastanoi pv. savastanoi and P. syringae pv. tomato. REP-PCR with a modified primer containing C produced identical bands among the isolates in a pathovar but separated three pathovars more distinctly than four other primers. Although REP- and BOX-PCRs have been successfully used in the molecular identification of Pseudomonas isolates from Turkish flora, a PCR based on inter-enterobacterial repetitive intergenic concensus (ERIC) sequences failed to produce clear banding patterns in this study.     

Assessment of genetic relatedness among commercial and wild strains of Agaricus bisporus using repetitive extragenic palindromic sequences and polymerase chain reaction

The genetic relatedness among 18 strains of Agaricus bisporus was assessed based on the fragment pattern analysis obtained by the amplification of genomic DNA by BOX, ERIC (ERICIR-I/ERIC2) and REP (REP1RI/REP2I) gene sequences. Based on the banding patterns of PCR-amplified products, eight putative groups among the 18 commercial and wild strains were recognized. REP-PCR generated multiple distinct products showing considerable variability among the strains with ERIC and REP elements successfully enabled detection of wild and commercial A. bisporus. Strains originating from the same geographical location were not always genetically related. To our knowledge, this was the first relevance study of biodiversity in commercial and native populations of A. bisporus by using the REP-PCR technique. The results confirmed the usefulness REP-PCR typing in intraspecific genetic variation assessments of the button mushroom. High level of Iranian wild strains distance with the commercial cultivars approves their importance as a promising new source of diversity in A. bisporus breeding program.     

Genotypic and phenotypic diversity of PGPR fluorescent pseudomonads isolated from the rhizosphere of sugarcane (Saccharum officinarum L.)

The genetic diversity of plant growth-promoting rhizobacterial (PGPR) fluorescent pseudomonads associated with the sugarcane (Saccharum officinarum L.) rhizosphere was analyzed. Selected isolates were screened for plant growthpromoting properties including production of indole acetic acid, phosphate solubilization, denitrification ability, and production of antifungal metabolites. Furthermore, 16S rDNA sequence analysis was performed to identify and differentiate these isolates. Based on 16S rDNA sequence similarity, the isolates were designated as Pseudomonas plecoglossicida, P. fluorescens, P. libaniensis, and P. aeruginosa. Differentiation of isolates belonging to the same group was achieved through different genomic DNA fingerprinting techniques, including randomly amplified polymorphic DNA (RAPD), amplified ribosomal DNA restriction analysis (ARDRA), repetitive extragenic palindromic (REP), enterobacterial repetitive intergenic consensus (ERIC), and bacterial repetitive BOX elements (BOX) analyses. The genetic diversity observed among the isolates and rep-PCR-generated fingerprinting patterns revealed that PGPR fluorescent pseudomonads are associated with the rhizosphere of sugarcane and that P. plecoglossicida is a dominant species. The knowledge obtained herein regarding the genetic and functional diversity of fluorescent pseudomonads associated with the sugarcane rhizosphere is useful for understanding their ecological role and potential utilization in sustainable agriculture.     

Genetic and phenotypic microdiversity of Ochrobactrum spp

The diversity of Ochrobactrum anthropi, Ochrobactrum intermedium, Ochrobactrum tritici and Ochrobactrum grignonense in agricultural soil and on the wheat rhizoplane was investigated. O. anthropi was isolated both from soil and from the rhizoplane, O. intermedium and grignonense only from bulk soil, and O. tritici only from the wheat rhizoplane. On the genetic level, the immunotrapped isolates and a number of strains from culture collection mainly of clinical origin were compared with rep-PCR profiling using BOX primers, and a subset of these isolates and strains using REP primers. The isolates clustered according to their species affiliation. There was no correlation between rep clusters of O. anthropi isolates and habitat (place of isolation). The genetic diversity of Ochrobactrum at the species level as well as microdiversity of O. anthropi (number of BOX groups) was higher in soil than on the rhizoplane. Similarity values from genetic rep-PCR profiles correlated positively with DNA-DNA reassociation percentages. Isolates with >80.7% similarity in BOX profile and >86.4% in rep profile clustered within the same species. Similarity analysis of rep-PCR profiles is hence an alternative to DNA-DNA hybridization as a genomic criterion for species delineation within the genus Ochrobactrum. We used the substrate utilization system BIOLOG-GN to compare the immunotrapped isolates on the phenetic level. For the isolates from bulk soil, substrate utilization versatility (number of utilized substrates) and substrate utilization capacity (mean conversion rate of substrates) were slightly but significantly higher than for the isolates from the rhizoplane. This trend was also seen using API 20E and 20NE systems. Plate counts of total bacteria and the number of immunotrapped Ochrobactrum isolates per gram dry weight were higher for the rhizoplane than for the soil samples. The results of genetic and phenotypic analyses indicated a 'rhizosphere effect'; the diversity and metabolic capacity of Ochrobactrum isolates were higher in bulk soil, and the population density was higher on the wheat rhizoplane.     

Genotypic characterization of Brochothrix spp. isolated from meat,poultry and fish

Aim: To study genotypic diversity of isolates of Brochothrix thermosphacta recovered from meat, poultry and fish. Methods and Results: A total of 27 bacteria isolated from 19 samples of meat, poultry and fish were identified phenotypically and genotypically using PCR amplification of 16S‐23S rDNA intergenic transcribed spacer (ITS‐PCR), repetitive sequence‐based PCR (rep‐PCR) and 16S rDNA sequencing. Using ITS‐PCR, all bacteria showed the same DNA profile as the reference strains of Br. thermosphacta, allowing typing of the isolates at species level. Using 16S rDNA sequencing, all isolates were identified, at genus and species level, as Br. thermosphacta. Identification as Br. campestris was observed with a lower, but very close, level of similarity. Rep‐PCR was more discriminatory than ITS‐PCR and allowed differentiation of four subgroups among the isolates. Conclusion: Minor genotypic differences among Br. thermosphacta strains from meat, poultry and fish were observed. Significance and Impact of the Study: A rudimentary exploration of genotypic differences of Br. thermosphacta from meat, poultry and fish resulted in preliminary confirmation of the suitability of ITS‐PCR for typing Br. thermosphacta and confirmed the value of rep‐PCR fingerprinting to discriminate between Br. thermosphacta strains.     

Determination of genetic differences between fluid and nonfluid variants of<Emphasis Type="Italic">Clavibacter michiganensis</Emphasis> subsp.<Emphasis Type="Italic">sepedonicus</Emphasis> using rep-PCR technique

Testing of 23 isolates of Clavibacter michiganensis subsp. sepedonicus for analysis by rep-PCR (using BOX, ERIC, REP primer sets) was used for the purpose of localization of genetic markers for fluid and/or nonfluid strains. None of the primer sets was successful in detecting genetic differences between the isolates and no polymorphism was generated.     

Development of Pathogenicity and AFLP to Characterize Fusarium oxysporum f.sp. momordicae Isolates from Bitter Gourd in China

Bitter gourd (Momordica charantia L.) cultivated in China is regarded as an important vegetable crop and is of considerable economic importance. However, it is susceptible to fusarium wilt, which causes heavy economic losses. Forty‐eight isolates were isolated from diseased bitter gourd plants that displayed typical fusarium wilt symptoms. Based on the morphological features, the rDNA internal transcribed space (ITS) sequences, pathogenicity and host biotypes, all of the isolates tested were pathogenic to the susceptible bitter gourd plants species (cv. ‘Guinongke No. 2’) and were identified as Fusarium oxysporum f. sp. momordicae (FOM). Our results classified different isolates as slightly, moderately or highly virulent. Among the isolates tested, 43 isolates slightly infected bottle gourd (Lagenaria siceraria var. clavata), whereas they did not infect other species from the family Cucurbitaceae. Genetic diversity among 48 isolates was characterized using amplified fragment length polymorphism (AFLP) analysis. The number of bands amplified by each primer pairs ranged from 41 to 66, with sizes ranging from 200 to 500 bp. A total of 366 bands were observed, out of which 363 were polymorphic (99.14%). The Nei's genetic identity of the six geographical populations varied from 0.7362 to 0.9707. The mean Nei's gene diversity index (H = 0.2644) and the mean Shannon's information index (I = 0.4071) at species level were higher than ones at populations level, indicated that the variation within populations was greater than that among populations. The Nei's GST (0.5103) and gene flow (Nm = 0.4923) revealed that genetic differentiation was mainly among populations and few gene exchanges. The dendrogram obtained from AFLP marker showed that there was a good correlation between isolates from different geographical locations and their pathogenicity. The AFLP marker effectively distinguished the high virulent isolates from the less virulent isolates. The highly virulent isolates were distinctly separated in different clusters, which indicated a significantly high correlation with the geographical origin in the AFLP dendrogram. The pathogenicity and molecular marker analysis confirmed the presence of variation in virulence as well as genetic diversity among the FOM isolates studied.     

First evidence for a restriction-modification system in Leptospira sp

Anaplasma marginale genomic DNA was tested for the presence of repetitive extragenic palindromic (REP) and enterobacterial repetitive intergenic consensus (ERIC)-like sequences in order to evaluate the genetic diversity of multiple A. marginale isolates. A. marginale isolates were obtained from cattle of six different states of Brazil, from the US and an Anaplasma centrale strain was obtained from Uruguay. Patterns obtained from A. marginale isolates varied from 14 to 17 fragments by REP-polymerase chain reaction (PCR) and 6 to 14 fragments by ERIC-PCR. All A. marginale isolates presented a 0.75-kb fragment by REP and two common fragments (0.38 and 1.0 kb) by ERIC-PCR. These two fragments were not detectable in A. centrale. Both methods produced similar patterns (80%) among A. marginale isolates obtained from the same region, although some isolates within regions shared less similarity. Isolates from Parana and Pernambuco, were differentiated by these methods. The study demonstrates the presence of ERIC and REP-like elements in A. marginale isolates and shows that A. marginale isolates and strains can be differentiated by these methods.