Molecular and phenotypic characterization of strains nodulating Anthyllis vulneraria in mine tailings, and proposal of Aminobacter anthyllidis sp. nov., the first definition of Aminobacter as legume-nodulating bacteria

Bacterial strains from Zn-Pb mine tailings were isolated by trapping with Anthyllis vulneraria, a legume-host suitable for mine substratum phytostabilisation. Sequence analysis of the 16S rRNA gene and three housekeeping genes (atpD, dnaK and recA) showed that they were related to those of the genus Aminobacter. DNA-DNA relatedness of representative isolates supported the placement of novel strains in Aminobacter as a new species. Phenotypic data emphasize their differentiation from the other related species of Aminobacter and Mesorhizobium. Aminobacter isolates exhibited nodA sequences tightly related with M. loti as the closest nodA relative. By contrast, their nodA sequences were highly divergent from those of M. metallidurans, another species associated with A. vulneraria that carries two complete copies of nodA. Therefore, the novel bacterial strains efficient on A. vulneraria represented the first occurrence of legume symbionts in the genus Aminobacter. They represent a new species for which the name Aminobacter anthyllidis sp. nov. is proposed (type strain STM4645(T)=LMG26462(T)=CFBP7437(T)).     

Molecular systematics of rhizobia based on maximum likelihood and Bayesian phylogenies inferred from rrs, atpD, recA and nifH sequences, and their use in the classification of Sesbania microsymbionts from Venezuelan wetlands

A well-resolved rhizobial species phylogeny with 51 haplotypes was inferred from a combined atpD + recA data set using Bayesian inference with best-fit, gene-specific substitution models. Relatively dense taxon sampling for the genera Rhizobium and Mesorhizobium was achieved by generating atpD and recA sequences for six type and 24 reference strains not previously available in GenBank. This phylogeny was used to classify nine nodule isolates from Sesbania exasperata, S. punicea and S. sericea plants native to seasonally flooded areas of Venezuela, and compared with a PCR-RFLP analysis of rrs plus rrl genes and large maximum likelihood rrs and nifH phylogenies. We show that rrs phylogenies are particularly sensitive to strain choice due to the high levels of sequence mosaicism found at this locus. All analyses consistently identified the Sesbania isolates as Mesorhizobium plurifarium or Rhizobium huautlense. Host range experiments on ten legume species coupled with plasmid profiling uncovered potential novel biovarieties of both species. This study demonstrates the wide geographic and environmental distribution of M. plurifarium, that R. galegae and R. huautlense are sister lineages, and the synonymy of R. gallicum, R. mongolense and R. yanglingense. Complex and diverse phylogeographic, inheritance and host-association patterns were found for the symbiotic nifH locus. The results and the analytical approaches used herein are discussed in the context of rhizobial taxonomy and molecular systematics.     

Multilocus sequence analysis reveals multiple symbiovars within Mesorhizobium species

The genus Mesorhizobium includes species nodulating several legumes, such as chickpea, which has a high agronomic importance. Chickpea rhizobia were originally described as either Mesorhizobium ciceri or M. mediterraneum. However, rhizobia able to nodulate chickpea have been shown to belong to several different species within the genus Mesorhizobium. The present study used a multilocus sequence analysis approach to infer a high resolution phylogeny of the genus Mesorhizobium and to confirm the existence of a new chickpea nodulating genospecies. The phylogenetic structure of the Mesorhizobium clade was evaluated by sequence analysis of the 16S rRNA gene, ITS region and the five core genes atpD, dnaJ, glnA, gyrB, and recA. Phylogenies obtained with the different genes are in overall good agreement and a well-supported, almost fully resolved, phylogenetic tree was obtained using the combined data. Our phylogenetic analyses of core genes sequences and their comparison with the symbiosis gene nodC, corroborate the existence of one new chickpea Mesorhizobium genospecies and one new symbiovar, M. opportunistum sv. ciceri. Furthermore, our results show that symbiovar ciceri spreads over six species of mesorhizobia. To our knowledge this study shows the most complete Mesorhizobium multilocus phylogeny to date and contributes to the understanding of how a symbiovar may be present in different species.     

Ensifer mexicanus sp. nov. a new species nodulating Acacia angustissima (Mill.) Kuntze in Mexico

A new lineage of Ensifer nodulating the American legume Acacia angustissima in the tropical forest of Chiapas and Morelos, Mexico is described. Bacteria were identified as Ensifer with ssb or nolR specific primers. Phylogenetic analysis with partial sequences of the five chromosomal genes gyrA, nolR, recA, rpoB and rrs revealed that this new lineage is related to African Ensifer terangae. The results of total DNA-DNA hybridization and selected phenotypic tests among the A. angustissima strains and E. terangae indicated that they belong to different species. The phylogeny with the symbiotic nifH gene also separates this group as a different clade but with close affinities to bacteria belonging to the genus Ensifer isolated from American hosts. ITTG R7(T) (=CFN ER1001, HAMBI 2910, CIP 109033, ATCC BAA-1312, DSM18446) is the type strain of a new species for which the name Ensifer mexicanus sp. nov. is proposed.     

Horizontal gene transfer and recombination shape mesorhizobial populations in the gene center of the host plants Astragalus luteolus and Astragalus ernestii in Sichuan, China

Thirty-three rhizobial strains isolated from the root nodules of Astragalus luteolus and Astragalus ernestii growing on the west plateau at two different altitudes in Sichuan province, China, were characterized by amplified rDNA restriction analysis (ARDRA), amplified fragment length polymorphism (AFLP), and by sequencing of rrs, glnA, glnII and nifH . The ARDRA analysis revealed considerable genomic diversity. In AFLP analysis, 20 of 33 Astragalus rhizobia formed three distinct clades, with others dispersed into different groups with the reference strains. Phylogenetic analysis of the rrs gene of six representative strains showed that the isolates were members of the genus Mesorhizobium . Three of the isolates formed a sister clade to Mesorhizobium loti and Mesorhizobium ciceri , whereas the other three formed a sister clade to a clade harboring the species Mesorhizobium huakuii, Mesorhizobium plurifarum, Mesorhizobium septentrionale and Mesorhizobium amorphae , indicating the existence of two new species. Phylogenetic analysis of glnA and glnII confirmed the rrs phylogenies for four strains, but the trees were incongruent. The nifH sequences of the strains formed a monophyletic clade and were typical of those of mesorhizobia forming symbioses with inverted repeat lacking clade legume species. The incongruent phylogenies of the genes studied suggest that horizontal gene transfer and recombination shape mesorhizobial populations in the gene center of the host plants.     

Sinorhizobium americanum symbiovar mediterranense is a predominant symbiont that nodulates and fixes nitrogen with common bean (Phaseolus vulgaris L.) in a Northern Tunisian field

A total of 40 symbiotic bacterial strains isolated from root nodules of common bean grown in a soil located in the north of Tunisia were characterized by PCR-RFLP of the 16S rRNA genes. Six different ribotypes were revealed. Nine representative isolates were submitted to phylogenetic analyses of rrs, recA, atpD, dnaK, nifH and nodA genes. The strains 23C40 and 23C95 representing the most abundant ribotype were closely related to Sinorhizobium americanum CFNEI 156(T). S. americanum was isolated from Acacia spp. in Mexico, but this is the first time that this species is reported among natural populations of rhizobia nodulating common bean. These isolates nodulated and fixed nitrogen with this crop and harbored the symbiotic genes of the symbiovar mediterranense. The strains 23C2 and 23C55 were close to Rhizobium gallicum R602sp(T) but formed a well separated clade and may probably constitute a new species. The sequence similarities with R. gallicum type strain were 98.7% (rrs), 96.6% (recA), 95.8% (atpD) and 93.4% (dnaK). The remaining isolates were, respectively, affiliated to R. gallicum, E. meliloti, Rhizobium giardinii and Rhizobium radiobacter. However, some of them failed to re-nodulate their original host but promoted root growth.     

The phylogenetic significance of peptidoglycan types: Molecular analysis of the genera Microbacterium and Aureobacterium based upon sequence comparison of gyrB, rpoB, recA and ppk and 16SrRNA genes

The type strains of 27 species of the genus Microbacterium, family Microbacteriaceae, were analyzed with respect to the phylogeny of the housekeeping genes coding for DNA gyrase subunit B (gyrB), RNA-polymerase subunit B (rpoB), recombinase A (recA) and polyphosphate kinase (ppk). The resulting gene trees were compared to the 16S rRNA gene phylogeny of the same species. The topology of neighbour-joining and maximum parsimony phylogenetic trees based upon nucleic acid sequences and protein sequences of housekeeping genes differed among each other and no gene tree was identical to that of the 16S rRNA gene tree. Only some species showed consistent clustering by all genes analyzed, but the majority of species branched with different neighbours in most gene trees. The failure to phylogenetically cluster type strains into two groups based upon differences in the amino acid composition of peptidoglycan on the basis of 16S rRNA gene sequence similarity, once leading to the union of the genera Microbacterium and Aureobacterium, was also seen in the analysis of recA, rpoB and gyrB gene and protein phylogenies. Analysis of the pkk gene and protein as well as of a concatenate tree, combining sequences of all five genes (total of 3.700 nucleotides), sees members of the former genus Aureobacterium and other type strains with lysine as diagnostic diamino acid to form a coherent cluster that branches within the radiation of Microbacterium species with ornithine in the peptidoglycan.     

Rhizobia with different symbiotic efficiencies nodulate Acaciella angustissima in Mexico, including Sinorhizobium chiapanecum sp. nov. which has common symbiotic genes with Sinorhizobium mexicanum

Bacteria from nodules of the legume Acaciella angustissima native to the south of Mexico were characterized genetically and their nodulation and competitiveness were evaluated. Phylogenetic studies derived from rpoB gene sequences indicated that A. angustissima is nodulated by Sinorhizobium mexicanum, Rhizobium tropici, Mesorhizobium plurifarium and Agrobacterium tumefaciens and by bacteria related to Sinorhizobium americanum, Sinorhizobium terangae, Rhizobium etli and Rhizobium gallicum . A new lineage related to S. terangae is recognized based on the sequences of gyrA, nolR, recA, rpoB and rrs genes, DNA–DNA hybridization and phenotypic characteristics. The name for this new species is Sinorhizobium chiapanecum and its type strain is ITTG S70^T. The symbiotic genes nodA and nifH were similar to those from S. mexicanum strains, which are Acaciella symbionts as well, with nodA gene sequences grouped within a cluster of nod genes from strains that nodulate plants from the Mimosoideae subfamily of the Leguminosae. Sinorhizobium isolates were the most frequently obtained from A. angustissima nodules and were among the best strains to promote plant growth in A. angustissima and to compete in interstrain nodule competition assays. Lateral transfer of symbiotic genes is not evident among the genera that nodulate A. angustissima ( Rhizobium, Sinorhizobium and Mesorhizobium ) but may occur among the sympatric and closely related sinorhizobia that nodulate Acaciella .     

Genetic diversity, community structure and distribution of rhizobia in the root nodules of Caragana spp. from arid and semi-arid alkaline deserts, in the north of China

The genetic diversity of 88 Caragana nodule rhizobial isolates, collected from arid and semi-arid alkaline sandy soils in the north of China, was assessed by PCR-RFLP of the 16S rRNA gene and the 16S-23S IGS, as well as the phylogenies of housekeeping genes (atpD, glnII and recA) and symbiotic genes (nodC and nifH). Of the 88 strains, 69 were placed in the genus Mesorhizobium, 16 in Rhizobium and 3 in Bradyrhizobium. Mesorhizobium amorphae, Mesorhizobium septentrionale, Mesorhizobium temperatum and Rhizobium yanglingense were the four predominant microsymbionts associated with Caragana spp. in the surveyed regions, and M. septentrionale was widely distributed among the sampling sites. Phylogenies of nodC and nifH genes showed that two kinds of symbiotic genes existed, corresponding to Mesorhizobium and Rhizobium, respectively. Available phosphorous (P) and potassium (K) contents were the main soil factors correlated with the distribution of these rhizobia in the sampling regions. Positive correlations between the available higher P content/lower K content and the dominance of Mesorhizobium species (M. temperatum, M. amorphae and M. septentrionale), and between the lower P content/higher K content and the dominance of R. yanglingense were found.     

Genetic diversity of root nodule bacteria nodulating Lotus corniculatus and Anthyllis vulneraria in Sweden

Very little is known about the genetic diversity and phylogeny of rhizobia nodulating Lotus species in northern temperate regions. We have therefore studied the genetic diversity among a total of 61 root nodule bacteria isolated from Lotus corniculatus and Anthyllis vulneraria from different geographic sites and habitats in Sweden by restriction fragment length polymorphism (RFLP) of the internal transcribed spacer between their 16S rRNA and 23S rRNA (IGS) region. A high diversity consisting of 26 IGS types from 54 L. corniculatus isolates and five IGS types from seven A. vulneraria isolates was found. The 16S rRNA sequences and phylogeny of representatives of the different IGS types showed four interesting exceptions from the majority of the isolates belonging to the genus Mesorhizobium: Two isolates were both found to be closely related to Rhodococcus spp., and two other isolates showed close relationship with Geobacillus spp. and Paenibacillus spp., respectively. The nodA sequences and phylogeny showed that all the isolates, including those not belonging to the traditional rhizobia genera, harbored nodA sequences which were typical of Mesorhizobium loti. Generally, the 16S rRNA and nodA phylogenetic trees were not congruent in that isolates with similar 16S rRNA sequences were associated with isolates harboring different nodA sequences. All the isolates were confirmed to nodulate L. corniculatus in an inoculation test. This is the first report of members of these non-rhizobia genera being able to nodulate legumes, and we suggest that they may have acquired their nodulating properties through lateral gene transfer.     

Multilocus sequence analysis provides basis for fast and reliable identification of Vibrio harveyi-related species and reveals previous misidentification of important marine pathogens

Vibrio harveyi and related bacteria are important pathogens responsible for severe economic losses in the aquaculture industry worldwide. Phenotypic tests and 16S rRNA gene analysis fail to discriminate species within the V. harveyi group because these are phenotypically and genetically nearly identical. This study used multilocus sequence analysis to identify 36 V. harveyi-like isolates obtained from a wide range of sources in Australia and to re-evaluate the identity of important pathogens. Phylogenies inferred from the 16S rRNA gene and five concatenated protein-coding genes (rpoA-pyrH-topA-ftsZ-mreB) revealed four well-supported clusters identified as V. harveyi, V. campbellii, V. rotiferianus and V. owensii. Results revealed that important V. campbellii and V. owensii prawn pathogens were previously misidentified as V. harveyi and also that the recently described V. communis sp. nov. is likely a junior synonym of V. owensii. Although the MLSA topologies corroborated the 16S rRNA gene phylogeny, the latter was less informative than each of the protein-coding genes taken singularly or the concatenated dataset. A two-locus phylogeny based on topA-mreB concatenated sequences was consistent with the five-locus MLSA phylogeny. Global Bayesian phylogenies inferred from topA-mreB suggested that this gene combination provides a practical yet still accurate approach for routine identification of V. harveyi-related species.     

Pseudomonas asturiensis sp. nov., isolated from soybean and weeds

Five strains of gram negative bacteria, isolated from soybean (LPPA 221^T, 222 and 223) and weeds (LPPA 816 and 1442), were analyzed by a polyphasic approach. The isolates showed variation in their phenotypic traits and were placed in the Pseudomonas fluorescens lineage, based on 16S rRNA gene sequence phylogeny, as a single but well separated cluster. MLSA analysis based on gyrB and rpoD sequences clustered the strains in a single branch in the Pseudomonas syringae group, and revealed P. viridiflava as closest relative. DNA–DNA hybridizations showed medium levels of DNA–DNA relatedness with the type strain of P. viridiflava (50%) and lower levels (<32%) with other type strains of the P. syringae group, supporting classification within a novel species of the genus Pseudomonas. The strains can be distinguished from species of the P. syringae group by the fatty acid C_17:0 cyclo that is present in a low amount (2.5%) and from P. viridiflava by their inability to assimilate d-tartrate and d-sorbitol, and by the formation of red colonies on TTC medium. For this new species, the name Pseudomonas asturiensis sp. nov. is proposed. The type strain is LPPA 221^T (=LMG 26898^T = CECT 8095^T).     

Core and symbiotic genes reveal nine Mesorhizobium genospecies and three symbiotic lineages among the rhizobia nodulating Cicer canariense in its natural habitat (La Palma,Canary Islands)

Cicer canariense is a threatened perennial wild chickpea endemic to the Canary Islands. In this study, rhizobia that nodulate this species in its natural habitats on La Palma (Canary Islands) were characterised. The genetic diversity and phylogeny were estimated by RAPD profiles, 16S-RFLP analysis and sequencing of the rrs, recA, glnII and nodC genes. 16S-RFLP grouped the isolates within the Mesorhizobium genus and distinguished nine different ribotypes. Four branches included minority ribotypes (3–5 isolates), whereas another five contained the predominant ribotypes that clustered with reference strains of M. tianshanense/M. gobiense/M. metallidurans, M. caraganae, M. opportunistum, M. ciceri and M. tamadayense. The sequences confirmed the RFLP groupings but resolved additional internal divergence within the M. caraganae group and outlined several potential novel species. The RAPD profiles showed a high diversity at the infraspecific level, except in the M. ciceri group. The nodC phylogeny resolved three symbiotic lineages. A small group of isolates had sequences identical to those of symbiovar ciceri and were only detected in M. ciceri isolates. Another group of sequences represented a novel symbiotic lineage that was associated with two particular chromosomal backgrounds. However, nodC sequences closely related to symbiovar loti predominated in most isolates, and they were detected in several chromosomal backgrounds corresponding to up to nine Mesorhizobium lineages. The results indicated that C. canariense is a promiscuous legume that can be nodulated by several rhizobial species and symbiotypes, which means it will be important to determine the combination of core and symbiotic genes that produce the most effective symbiosis.     

Mycobacterium species identification--a new approach via dnaJ gene sequencing

The availability of the dnaJ1 gene for identifying Mycobacterium species was examined by analyzing the complete dnaJ1 sequences (approximately 1200 bp) of 56 species (54 of them were type strains) and comparing sequence homologies with those of the 16S rRNA gene and other housekeeping genes (rpoB, hsp65). Among the 56 Mycobacterium species, the mean sequence similarity of the dnaJ1 gene (80.4%) was significantly less than that of the 16S rRNA, rpoB and hsp65 genes (96.6%, 91.3% and 91.1%, respectively), indicating a high discriminatory power of the dnaJ1 gene. Seventy-one clinical isolates were correctly clustered to the corresponding type strains, showing isolates belonging to the same species. In order to propose a method for strain identification, we identified an area with a high degree of polymorphism, bordered by conserved sequences, that can be used as universal primers for PCR amplification and sequencing. The sequence of this fragment (approximately 350 bp) allows accurate species identification and may be used as a new tool for the identification of Mycobacterium species.     

Phylogenetic relationships of three new microsporidian isolates from the silkworm, Bombyx mori

The pathogenicity, mode of transmission, tissue specificity of infection and the small subunit rRNA (SSU-rRNA) gene sequences of the three new microsporidian isolates from the silkworm Bombyx mori were studied. Out of the three, NIK-2r revealed life cycle features and SSU-rRNA gene sequence similar to Nosema bombycis, suggesting that it is N. bombycis. The other two, NIK-4m and NIK-3h, differed from each other as well as from N. bombycis. NIK-4m was highly pathogenic and did not show any vertical transmission, in accordance with the apparent lack of gonadal infection, whereas NIK-3h was less pathogenic and vertical transmission was not detected but could not be excluded. Phylogenetic analysis based on SSU-rRNA gene sequence placed NIK-3h and NIK-4m in a distinct clade that included almost all the Vairimorpha species and Nosema species that infect lepidopteran and non-lepidopteran hosts, while NIK-2r was included in a clade containing almost all the Nosema isolates that infect only lepidopteran hosts. Thus, we have presented molecular evidence that one of the three isolates is in fact the type species N. bombycis, while the other two isolates are Vairimorpha spp. There was distinct separation of microsporidian isolates infecting only lepidopteran hosts and those infecting lepidopteran and non-lepidopteran hosts, reflecting possible co-evolution of hosts and microsporidian isolates.     

Diverse rhizobia associated with woody legumes Wisteria sinensis, Cercis racemosa and Amorpha fruticosa grown in the temperate zone of China

Fifty-nine bacterial isolates from root nodules of the woody legumes Wisteria sinensis, Cercis racemosa and Amorpha fruticosa grown in the central and eastern regions of China were characterized with phenotypic analysis, PCR-based 16S and 23S rRNA gene RFLP, Box PCR and 16S rRNA gene sequencing. Seven main phena were defined in numerical taxonomy, which corresponded to distinct groups within the genera Agrobacterium, Bradyrhizobium, Mesorhizobium and Rhizobium in 16S and 23S rRNA gene PCR-RFLP. The phylogenetic relationships of the 16S rRNA genes supported the grouping results of PCR-RFLP. Most of the isolates from Amorpha fruticosa were classified into two groups closely related to Mesorhizobium amorphae. Seventeen of the 21 isolates from Wisteria sinensis were identified as two groups related to Rhizobium and Agrobacterium. Six out of 10 isolates from Cercis racemosa were identified as a group related to Bradyrhizobium. Our results indicated that each of the investigated legumes nodulated mainly with one or two rhizobial groups, although isolates from different plants intermingled in some small bacterial groups. In addition, correlation between geographic origin and grouping results was found in the isolates from Amorpha fruticosa. These results revealed that the symbiotic bacteria might have been selected by both the legume hosts and the geographic factors.     

Both mating types in the heterothallic fungus Ophiostoma quercus contain MAT1-1 and MAT1-2 genes

In heterothallic Ascomycota, two opposite but distinct mating types control all sexual processes. Using mating crosses, mating types were assigned to ten isolates of the heterothallic fungal species Ophiostoma quercus. Primers were subsequently designed to target the MAT1-1-1, MAT1-1-3 (of the mating type 1 idiomorph), and MAT1-2-1 (of the mating type 2 idiomorph) genes in these isolates. Results showed that all isolates contained the full gene sequence for the MAT1-2-1 gene. In addition, fragments of the MAT1-1-1 and MAT1-1-3 genes were sequenced from all isolates. These results were unexpected, as each isolate from a heterothallic species would typically contain only one of the two possible MAT idiomorphs.     

Phylogenetic analyses of a combined data set suggest that the Attheya lineage is the closest living relative of the pennate diatoms (Bacillariophyceae)

A Bayesian analysis of a seven gene data set was conducted to reconstruct phylogenetic relationships among a sample of centric and pennate diatoms and to test alternative hypotheses about the closest living relative of Bacillariophyceae. A lineage, composed of two Attheya species, was inferred to share the most recent common ancestor with Bacillariophyceae--a relationship that was also corroborated by the combined parsimony analysis. All competing hypotheses about the closest living relative of Bacillariophyceae were rejected because 100% of the trees in the post-burn-in sample in the Bayesian analysis supported the Attheya-Bacillariophyceae clade. According to a partitioned Bremer support analysis, the majority of the genes in the combined data matrix supported the Attheya--Bacillariophyceae relationship. The global topology of the phylogenetic tree indicated that a monophyletic group consisting of Thalassiosirales and Toxarium undulatum formed the deepest branch followed by a node uniting a clade composed of Bacillariophyceae/Attheya species and a lineage made up of Eucampia zoodiacus, Chaetocerotales, Lithodesmiales, Triceratiales, Biddulphiales and Cymatosirales. Except for the phylogenetic positions of Lithodesmiales, Thalassiosira sp and Skeletonema costatum, the optimal tree obtained from the combined parsimony analysis showed the same branching order of taxa as those seen in the consensus tree inferred from three independent Markov chain Monte Carlo analyses. Noteworthy findings are that Toxarium undulatum shares a strongly supported node with Thalassiosirales and that the genus Attheya is not a member of the Chaetocerotales lineage.     

Phylogenetic relationships of Fusarium poae based on EF-1 alpha and mtSSU sequences

A molecular phylogenetic analysis of Fusarium poae isolates from South America (Argentina) and Europe (mainly England, Germany, Italy) was performed using 98 F. poae, four Fusarium culmorum, two Fusarium sporotrichioides and one Fusarium langsethiae isolates. Phylogenetic analyses were performed using nuclear (translation elongation factor 1-alpha, EF-1 alpha) and mitochondrial (mitochondrial small subunit rDNA, mtSSU) sequences. Partitioned (each dataset separately) and combined (EF-1 alpha+mtSSU) analyses did not reveal any clear correlations from the inferred branching topology, between the distribution of observed haplotypes and the geographic origin and/or host species. Results from the present study confirmed that isolates from F. poae form a monophyletic group, and the low variability within isolates from a broad geographic range suggests a common lineage history. Among F. poae isolates from Argentina, however, some were found to possess an insert within mtSSU with structural similarities to group IC2 introns. F. poae isolates differing by the presence/absence of a mtSSU insertion were characterized further by analysis of a portion of the Tri5 gene, but this sequence was unable to reveal variability. The presence of this insert only within isolates from Argentina suggests that evolutionary events (insertions/deletions) are probably taking place within the Argentinian F. poae isolates, and that the acquisition of this insert occurred after geographic isolation of the Argentinian and European populations.