Phylogeny and distribution of Bradyrhizobium symbionts nodulating cowpea (Vigna unguiculata L. Walp) and their association with the physicochemical properties of acidic African soils

In the N₂-fixing symbiosis, the choice of a symbiotic partner is largely influenced by the host plant, the rhizobial symbiont, as well as soil factors. Understanding the soil environment conducive for the survival and multiplication of root-nodule bacteria is critical for microbial ecology. In this study, we collected cowpea-nodules from acidic soils in Ghana and South Africa, and nodule DNA isolates were characterized using 16S–23S rRNA-RFLP, phylogenetic analysis of housekeeping and symbiotic genes, and bradyrhizobial community structure through canonical correspondence analysis (CCA). The CCA ordination plot results showed that arrow of soil pH was overlapping on CCA2 axis and was the most important to the ordination. The test nodule DNA isolates from Ghana were positively influenced by soil Zn, Na and K while nodule DNA isolates from South Africa were influenced by P. The amplified 16S–23S rRNA region yielded single polymorphic bands of varying lengths (573–1298 bp) that were grouped into 28 ITS types. The constructed ITS-dendrogram placed all the nodule DNA isolates in five major clusters at low cut-off of approx. 0.1 Jaccard’s similarity coefficient. The phylogenetic analysis of 16S rRNA and housekeeping genes (glnII, gyrB, and atpD) formed distinct Bradyrhizobium groups in the phylogenetic trees. It revealed the presence of highly diverse bradyrhizobia (i.e. Bradyrhizobium vignae, Bradyrhizobium elkanii, Bradyrhizobium iriomotense, Bradyrhizobium pachyrhizi, and Bradyrhizobium yuanmingense) together with novel/unidentified bradyrhizobia in the acidic soils from Ghana and South Africa. Discrepancies noted in the phylogenies of some nodule DNA isolates could be attributed to horizontal gene transfer or recombination.     

Microsymbionts of Phaseolus vulgaris in acid and alkaline soils of Mexico

In order to investigate bean-nodulating rhizobia in different types of soil, 41 nodule isolates from acid and alkaline soils in Mexico were characterized. Based upon the phylogenetic studies of 16S rRNA, atpD, glnII, recA, rpoB, gyrB, nifH and nodC genes, the isolates originating from acid soils were identified as the phaseoli symbiovar of the Rhizobium leguminosarum-like group and Rhizobium grahamii, whereas the isolates from alkaline soils were defined as Ensifer americanum sv. mediterranense and Rhizobium radiobacter. The isolates of “R. leguminosarum” and E. americanum harbored nodC and nifH genes, but the symbiotic genes were not detected in the four isolates of the other two species. It was the first time that “R. leguminosarum” and E. americanum have been reported as bean-nodulating bacteria in Mexico. The high similarity of symbiotic genes in the Rhizobium and Ensifer populations showed that these genes had the same origin and have diversified recently in different rhizobial species. Phenotypic characterization revealed that the “R. leguminosarum” population was more adapted to the acid and low salinity conditions, while the E. americanum population preferred alkaline conditions. The findings of this study have improved the knowledge of the diversity, geographic distribution and evolution of bean-nodulating rhizobia in Mexico.     

Identification of a Saxitoxin Biosynthesis Gene with a History of Frequent Horizontal Gene Transfers

The paralytic shellfish poisoning (PSP) toxins, saxitoxin, and its derivatives, are produced by a complex and unique biosynthetic pathway. It involves reactions that are rare in other metabolic pathways, however, distantly related organisms, such as dinoflagellates and cyanobacteria, produce these toxins by an identical pathway. Speculative explanations for the unusual phylogenetic distribution of this metabolic pathway have been proposed, including a polyphyletic origin, the involvement of symbiotic bacteria, and horizontal gene transfer. This study describes for the first time the identity of one gene, sxt1, that is involved in the biosynthesis of saxitoxin in cyanobacteria. It encoded an O-carbamoyltransferase (OCTASE) that was proposed to carbamoylate the hydroxymethyl side chain of saxitoxin precursor. Orthologues of sxt1 were exclusively present in PSP-toxic strains of cyanobacteria and had a high sequence similarity to each other. L. wollei had a naturally mutated sxt1 gene that encoded an inactive enzyme, and was incapable of producing carbamoylated PSP-toxin analogues, supporting the proposed function of Sxt1. Phylogenetic analysis revealed that OCATSE genes were present exclusively in prokaryotic organisms and were characterized by a high rate of horizontal gene transfer. OCTASE has most likely evolved from an ancestral O-sialoglycoprotein endopeptidase from proteobacteria, whereas the most likely phylogenetic origin of sxt1 was an ancestral alpha-proteobacterium. The phylogeny of sxt1 suggested that the entire set of genes required for saxitoxin biosynthesis may spread by horizontal gene transfer.     

Genetic allelism and linkage tests of a soybean gene,Rfg1, controlling nodulation with Rhizobium fredii strain USDA 205

A soybean gene, Rfg1, controlling nodulation with strain USDA 205, the type strain for the fast-growing species Rhizobium fredii, was tested for allelism with the Rj4 gene. The Rj4 gene conditions ineffective nodulation primarily with certain strains of the slow-growing soybean microsymbiont, Bradyrhizobium elkanii. The F2 seeds of the cross of the cultivars Peking, carrying the alleles rfg1, Rj4, i (controlling inhibition of seed coat color) and W1 (controlling flower color), and Kent, carrying the alleles Rfg1, rj4, i-i and w1, were evaluated for nodulation response with strain USDA 205 by planting surface disinfested seeds in sterilized vermiculite in growth trays and inoculating with a stationary phase broth culture of strain USDA 205 at planting. Plants were classified for nodulation response visually after four weeks growth and transplanted to the field for F3 seed production. Flower color, purple (W1) vs white (w1), was determined in the field. The allele present at the i locus was determined by classification of F3 seed coat color. The F3 seeds were planted in growth trays and inoculated with strain USDA 61 of Bradyrhizobium elkanii to determine the genotype for the Rj4 locus. The Rfg1 and Rj4 genes were determined to be located at separate loci. Chi-square analysis for linkage indicated that Rfg1 segregated independently of the Rj4, I and W1 loci.     

Shifting abundances of communities associated with nitrogen cycling in soils promoted by sugarcane harvest systems

Sugarcane cultivation supports Brazil as one of the largest world sugar and ethanol producer. In order to understand the impact of changing sugarcane harvest from manual to mechanized harvest, we studied the effect of machinery traffic on soil and consequently soil compaction upon soil microbial communities involved in nitrogen cycling. The impact of sugarcane harvest was dependent on soil depth and texture. At deeper soil layers, mechanized harvesting increases the abundance of nitrogen fixers and denitrifying communities (specifically nosZ clade I and II) while manual harvesting increases the abundance of ammonia oxidizers (specifically AOA) and increases denitrifying communities (nosZ clade I and II) on top and at intermediate depth. The effect of change on the harvest system is more evident on sandy soil than on clay soil, where soil indicators of compaction (bulk density and penetration resistance) were negatively correlated with soil microorganisms associated with the nitrogen cycle. Our results point to connections between soil compaction and N transformations in sugarcane fields, besides naming biological variables to be used as proxies for alterations in soil structure.     

Dimensions and distribution of intercellular spaces in cryo-planed soybean nodules

The ability of legume nodules to regulate their permeability to gas diffusion has been attributed to physiological control over the size and distribution of gas-filed intercellular spaces within the nodule cortex. To examine the size and distribution of intercellular spaces and to determine whether they were filled with gas (high diffusion permeability) or liquid (low diffusion permeability), whole nodules were frozen in liquid nitrogen slush (-210°C), and then either cryo-fractured or cryo-planed before being examined by cold-stage scanning electron microscopy (SEM). The cryo-planed tissue was found to have many advantages over cryo-fractured nodules in providing images which were easier to interpret and quantify. Intercellular spaces throughout the nodule were examined in both tangential and medial planed faces. Since no differences were observed between views in either the size or shape of the open intercellular spaces, it was concluded that the intercellular spaces of nodules were not radially oriented as assumed in many mathematical models of gas diffusion. The inner cortex region in the nodules had the smallest intercellular spaces compared to other zones, and less than 10% of the intercellular spaces were occluded with any type of material in the central zone regions. Vacuum infiltration of nodules with salt solutions and subsequent cryo-planing for SEM examination showed that open and water-filled intercellular spaces could be differentiated. The potential is discussed for using this method to study the mechanism of diffusion barrier regulation in legume nodules.     

Soybeans inoculated with root zone soils of Canadian native legumes harbour diverse and novel Bradyrhizobium spp. that possess agricultural potential

An assessment was made of the evolutionary relationships of soybean nodulating bacteria associated with legumes native to eastern Canada to identify potential new sources of soybean inoculant strains.Short season soybeans were used to selectively trap bacteria from root zone soils of four native legume species. Screening of more than 800 bacterial isolates from soybean root nodules by analysis of recA gene sequences followed by analyses of selected genotypes using six core and two symbiosis (nodC and nifH) gene sequences permitted identification of diverse taxa that included eight novel and four named Bradyrhizobium species as well as lineages attributed to the genera Afipia and Tardiphaga.Plant tests showed that symbionts related to four named species as well as a novel Bradyrhizobium lineage were highly efficient with regard to nitrogen fixation on soybeans relative to an inoculant strain.A new symbiovar (sv. septentrionalis) is proposed based on a group of four novel Bradyrhizobium spp. that possess distinctive nodC and nifH gene sequences and symbiotic characteristics.Evidence is provided for horizontal transfer of sv. septentrionalis symbiosis genes between novel Bradyrhizobium spp., a process that rendered recipient bacteria ineffective on soybeans.Diverse lineages of non-symbiotic and symbiotic Bradyrhizobium spp. co-occured within monophyletic clusters in a phylogenetic tree of concatenated core genes, suggesting that loss and/or gain of symbiosis genes has occurred in the evolutionary history of the bacterial genus.Our data suggest that symbiont populations associated with legumes native to eastern Canada harbour elite strains of Bradyrhizobium for soybean inoculation.     

Diversity and geographical distribution of rhizobia associated with <Emphasis Type="Italic">Lespedeza</Emphasis> spp. in temperate and subtropical regions of China

Eighty-eight root-nodule isolates from Lespedeza spp. grown in temperate and subtropical regions of China were characterized by a polyphasic approach. Nine clusters were defined in numerical taxonomy and SDS-PAGE analysis of whole cell proteins. Based upon further characterizations of amplified 16S rDNA restriction analysis (ARDRA), PCR-based restriction fragment length polymorphism of ribosomal IGS, 16S rDNA sequence analysis and DNA-DNA hybridization, these isolates were identified as Bradyrhizobium japonicum, B. elkanii, B. yuanmingense, Mesorhizobium amorphae, M. huakuii, Sinorhizobium meliloti and three genomic species related to B. yuanmingense, Rhizobium gallicum and R. tropici. The Bradyrhizobium species and R. tropici-related rhizobia were mainly isolated from the subtropical region and the species of Mesorhizobium, S. meliloti and R. gallicum-related species were all isolated from the temperate region. Phylogenetic analyses of nifH and nodC indicated that the symbiotic genes of distinct rhizobial species associated with Lespedeza spp. might have different origins and there was no evidence for lateral gene transfer of symbiotic genes. The results obtained in the present study and in a previous report demonstrated that Lespedeza spp. are nodulated by rhizobia with diverse genomic backgrounds and these Lespedeza-nodulating rhizobia were not specific to the host species, but specific to their geographic origins. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. GenBank sequence accession numbers: The GenBank accession numbers were EF61095 through EF061114 and EF051240 for acquired 16S rDNA sequences; EF153395 through EF153402 for nifH sequences; and EF153403 through EF153410 for nodC sequences.     

Deep placement: A method of nitrogen fertilizer application compatible with algal nitrogen fixation in wetland rice soils

Summary The effect of different methods of nitrogen fertilizer application on the algal flora and biological nitrogen fixation (Acetylene-reducing activity) in a wetland rice soil was studied in pot and field experiments. Broadcast application of urea inhibited nitrogen fixation and favored the growth of green algae. In contrast, deep placement of urea supergranules (1–2 g urea granules) did not suppress the growth of N₂-fixing blue-green algae and permitted acetylene-reducing activity on the soil surface to continue virtually uninhibited.     

Application of genistein to inocula and soil to overcome low spring soil temperature inhibition of soybean nodulation and nitrogen fixation

In the soybean (Glycine max. (L.) Merr)– Bradyrhizobium japonicum symbiosis, suboptimal root zone temperatures (RZTs) slow nodule development by disruption of the interorganismal signal exchange between the host plant and bradyrhizobia. Two field experiments were conducted on two adjacent sites in 1994 to determine whether the incubation of B. japonicum with genistein prior to application as an inoculant, or genistein, without B. japonicum, applied onto seeds in the furrow at the time of planting, increased soybean nodulation, N fixation, and total N yield. The results of these experiments indicated that genistein application increased nodule number and nodule dry matter per plant and hastened the onset of N fixation during the early portion of the soybean growing season, when the soils were still cool. Because these variables were improved, total fixed. N, fixed N as a percentage of total plant N, and N yield increased due to genistein application. The interaction between genistein application and soybean cultivars indicated that genistein application was more effective on N-stressed plants.     

Selection and characterization of Spanish Trifolium-nodulating rhizobia for pasture inoculation

Identification of elite nitrogen-fixing rhizobia strains is a continuous and never ending effort, since new legume species can be cultivated in different agro systems or are introduced into new areas. This current study reports on the taxonomic affiliation and symbiotic proficiency of nine strains of Trifolium-nodulating rhizobia isolated from different pasture areas in Spain, as well as three Rhizobium leguminosarum bv. trifolii reference strains, on eleven Trifolium species. Based on 16S rRNA gene sequences the strains belonged to the R. leguminosarum species complex. Additional phylogenetic analyses of the housekeeping genes recA, atpD and rpoB showed the strains were closely related to the species R. leguminosarum, R. laguerreae, R. indicum, R. ruizarguesonis or R. acidisoli. In addition, three strains had no clear affiliation and could represent putative new species, although two of the reference strains were positioned close to R. ruizarguesonis. nodC gene phylogeny allowed the discrimination between strains isolated from annual or perennial Trifolium species and placed all of them in the symbiovar trifolii. Neither geographic origin nor host-plant species could be correlated with the taxonomic affiliation of the strains and a high degree of phenotypic diversity was found among this set of strains. The strong interaction of plant species with the rhizobial strains found for biological nitrogen fixation (BNF) was noteworthy, and allowed the identification of rhizobial strains with a maximum proficiency for certain trefoil species. Several strains showed high BNF potential with a wide range of clover species, which made them valuable strains for inoculant manufacturers and they would be particularly useful for inoculation of seed mixtures in natural or cultivated pastures.     

Relationship between autoregulation and nitrate inhibition of nodulation in soybeans

Ten of 11 supernodulating mutants of soybean [ Glycine max (L.) Merr.] cv. Bragg, in which nodulation was far in excess of that in the wild type, showed pronounced tolerance of nodulation to applied nitrate. Mutant nts (nitrate-tolerant symbiosis) 1116 had an intermediate nodulation response and also showed some inhibition by nitrate. Mutant 1029, a revertant of nts382 (an extreme supernodulator), showed a wild-type nodulation pattern and was equally sensitive to nitrate as cv. Bragg. Grafting experiments with cv. Bragg and nts382 indicated that both supernodulation and tolerance of nodulation to nitrate were dependent on shoot factors. Total leaf nitrate reductase (EC 1.6.6.1 and EC 1.6.6.2) activity of the supernodulating mutants was similar to that in cv. Bragg. We conclude from these results that the inhibitory effect of nitrate on nodule initiation and development in soybean depends on an interaction between nitrate and the autoregulation singal. In the supernodulating mutants, the autoregulation signal is either altered or absent and cosequently nodulation in these mutants is not sensitive to nitrate.     

Effect of Rhizobium sp. BARIRGm901 inoculation on nodulation,nitrogen fixation and yield of soybean (Glycine max) genotypes in gray terrace soil

Soybean plants require high amounts of nitrogen, which are mainly obtained from biological nitrogen fixation. A field experiment was conducted by soybean (Glycine max) genotypes, growing two varieties (Shohag and BARI Soybean6) and two advanced lines (MTD10 and BGM02026) of soybean with or without Rhizobium sp. BARIRGm901 inoculation. Soybean plants of all genotypes inoculated with Rhizobium sp. BARIRGm901 produced greater nodule numbers, nodule weight, shoot and root biomass, and plant height than non-inoculated plants. Similarly, inoculated plants showed enhanced activity of nitrogenase (NA) enzyme, contributing to higher nitrogen fixation and assimilation, compared to non-inoculated soybean plants in both years. Plants inoculated with Rhizobium sp. BARIRGm901 also showed higher pod, stover, and seed yield than non-inoculated plants. Therefore, Rhizobium sp. BARIRGm901 established an effective symbiotic relationship with a range of soybean genotypes and thus increased the nodulation, growth, and yield of soybean grown in gray terrace soils in Bangladesh.     

Haloalkaliphilic diazotrophs in soda solonchak soils

Nitrogen fixation (NF) potential was measured in more than 40 samples of soda solonchak soils with the pH of water extract between 9.5 and 11.0 collected in several locations of Central Asia and in Egypt, using the acetylene reduction method. NF was detected in most of the samples. Maximal rates were observed under microaerophilic-anaerobic conditions with glucose as a substrate. In most cases, the NF negatively correlated with salt content and alkalinity. Five enrichments at pH 10 under micro-oxic conditions with glucose resulted in stable haloalkaliphilic mixed cultures, with diazotrophic component(s) active up to 2.0-3.0 M total Na(+). The cultures were dominated by Gram-positive spore-forming bacteria. Molecular cloning of nifH genes demonstrated the presence of two phylogenetic lineages of diazotrophs in the enrichments affiliated with the low-GC Gram-positive bacteria (in rRNA groups 1 and 6 of bacilli and in Clostridiales). Isolation of pure cultures of haloalkaliphilic diazotrophs from micro-oxic enrichments yielded nine strains, comprising two phylogenetic lineages. Most of the isolates (eight) were affiliated with the aerotolerant fermentative haloalkaliphilic bacterium Amphibacillus tropicus and a single strain clustered with the obligately anaerobic haloalkaliphile Bacillus arseniciselenatis. Diazotrophy has never been recognized previously in these groups of Gram-positive bacteria. Overall, the results demonstrated the existence, in soda solonchak soils, of a novel group of free-living fermentative diazotrophic bacteria active at extremely haloalkaline conditions.     

Characterization of Pisum sativum and Vicia faba microsymbionts in Morocco and definition of symbiovar viciae in Rhizobium acidisoli

In this work, we analyzed the diversity of seventy-six bacteria isolated from Pea and faba bean nodules in two regions of Morocco. The molecular diversity was realized using the analysis of the sequences of 16S rRNA and six housekeeping genes (recA, glnII, atpD, dnaK, rpoB and gyrB) and two symbiotic genes (nodA and nodC).The phylogeny of the 16S rRNA gene sequences revealed that all strains belong to the genus Rhizobium, being related to the type strains of R. leguminosarum, R. laguerreae, R. indigoferae, R. anhuiense and R. acidisoli. The housekeeping genes phylogenies showed that some strains formed a subclade distinct from the rhizobial species that usually nodulate Vicia faba and Pisum sativum which are closely related to R. acidisoli FH23 with sequence similarity of 98.3%.Analysis of the PGPR activities of the different isolates showed that the strains related to R. laguerreae were able to solubilize phosphates and to produce siderophores and auxin phytohormone. However, R. acidisoli strain F40D2 was unable to solubilize phosphates although they produce siderophores and IAA.The phylogenetic analysis of the nodA and nodC sequences showed that all isolated strains were closely related with the strains of symbiovar viciae. The nodulation tests confirmed the ability to nodulate V. faba and P. sativum but not Cicer arietinum or Phaseolus vulgaris. Hence, in Morocco P. sativum is nodulated by R. laguerreae; whereas V. faba is nodulated by R. laguerreae and the symbiovar viciae of R. acidisoli which has been not previously described in this species.     

Comparative genomics of Bradyrhizobium japonicum CPAC 15 and Bradyrhizobium diazoefficiens CPAC 7: elite model strains for understanding symbiotic performance with soybean

Background

The soybean-Bradyrhizobium symbiosis can be highly efficient in fixing nitrogen, but few genomic sequences of elite inoculant strains are available. Here we contribute with information on the genomes of two commercial strains that are broadly applied to soybean crops in the tropics. B. japonicum CPAC 15 (=SEMIA 5079) is outstanding in its saprophytic capacity and competitiveness, whereas B. diazoefficiens CPAC 7 (=SEMIA 5080) is known for its high efficiency in fixing nitrogen. Both are well adapted to tropical soils. The genomes of CPAC 15 and CPAC 7 were compared to each other and also to those of B. japonicum USDA 6^T and B. diazoefficiens USDA 110^T.

Results

Differences in genome size were found between species, with B. japonicum having larger genomes than B. diazoefficiens. Although most of the four genomes were syntenic, genome rearrangements within and between species were observed, including events in the symbiosis island. In addition to the symbiotic region, several genomic islands were identified. Altogether, these features must confer high genomic plasticity that might explain adaptation and differences in symbiotic performance. It was not possible to attribute known functions to half of the predicted genes. About 10% of the genomes was composed of exclusive genes of each strain, but up to 98% of them were of unknown function or coded for mobile genetic elements. In CPAC 15, more genes were associated with secondary metabolites, nutrient transport, iron-acquisition and IAA metabolism, potentially correlated with higher saprophytic capacity and competitiveness than seen with CPAC 7. In CPAC 7, more genes were related to the metabolism of amino acids and hydrogen uptake, potentially correlated with higher efficiency of nitrogen fixation than seen with CPAC 15.

Conclusions

Several differences and similarities detected between the two elite soybean-inoculant strains and between the two species of Bradyrhizobium provide new insights into adaptation to tropical soils, efficiency of N₂ fixation, nodulation and competitiveness.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-420) contains supplementary material, which is available to authorized users.     

Identification and use of actinomycetes for enhanced nodulation of soybean co-inoculated with Bradyrhizobium japonicum

The utilization of actinomycetes as potential soybean (Glycine max (L.)) co-inoculants was evaluated. Soil samples from Carbondale and Belleville, Ill., were used to inoculate pre-germinated soybean plants to determine antibiotic sensitivity in the native Bradyrhizobium japonicum population. Sensitivity was in the order kanamycin > tetracycline > oxytetracycline > rifampicin > neomycin. Antagonism by five actinomycete cultures toward seven test strains of B. japonicum was also assessed. The ranking average inhibition (across all seven B. japonicum strains) by these actino mycetes was Streptomyces kanamyceticus = Streptomyces coeruleoprunus > Streptomyces rimosus > Streptomyces sp. > Amy colatopsis mediterranei. Ten antibiotic combinations were used to isolate antibiotic-resistant mutants of B. japonicum I-110 and 3I1B-110 via successive cycles of mutation. Eighty-one antibiotic-resistant strains were isolated and tested for symbiotic competency; nine of which were selected for further characterization in a greenhouse pot study. Few differences in nodule number were caused by these treatments. Nodule occupancy varied from 0% to 18.3% when antibiotic-resistant strains of B. japonicum were used as the sole inoculants. However, when three mutant strains of B. japonicum were co-inoculated with S. kanamyceticus, significant increases in nodule occupancy (up to 55%) occurred. Increases in shoot nitrogen composition (27.1%-40.9%) were also caused by co-inoculation with S. kanamyceticus.     

Identification and distribution of sequences having similarity to mitochondrial plasmids in mitochondrial genomes of filamentous fungi

Mitochondrial plasmids are autonomously replicating genetic elements commonly associated with fungal and plant species. Analysis of several plant and fungal mitochondrial genomes has revealed regions that show significant homology to mitochondrial plasmids, suggesting that plasmids have had a long-term association with their mitochondrial hosts. To assess the degree to which plasmids have invaded fungal mitochondrial genomes, BLAST search parameters were modified to identify plasmid sequences within highly AT-rich mtDNAs, and output data were parsed by E value, score, and sequence complexity. High scoring hits were evaluated for the presence of shared repetitive elements and location within plasmids and mtDNAs. Our searches revealed multiple sites of sequence similarity to four distinct plasmids in the wild-type mtDNA of Neurospora crassa, which collectively comprise more than 2% of the mitochondrial genome. Regions of plasmid similarity were not restricted to plasmids known to be associated with senescence, indicating that all mt plasmids can potentially integrate into mitochondrial DNA. Unexpectedly, plasmid-related sequences were found to be clustered in regions that have disproportionately low numbers of PstI palindromic sequences, suggesting that these repetitive elements may play a role in eliminating foreign DNA. A separate class of GC-rich palindromes was identified that appear to be mobile, as indicated by their occurrence within regions of plasmid homology. Sites of sequence similarity to mitochondrial plasmids were also detected in other filamentous fungi, but to a lesser degree. The tools developed here will be useful in assessing the contribution plasmids have made to mitochondrial function and in understanding the co-evolution of mitochondrial plasmids and their hosts.Electronic Supplementary Material Supplementary material is available for this article at