Mycorrhizal Fungus Gigaspora Margarita Research Articles

Siderophores in plant root tissue: Tagetes patula nana colonized by the arbuscular mycorrhizal fungus Gigaspora margarita.

More than 70% of vascular plant species live in symbiosis with arbuscular mycorrhizal (AM) fungi. In addition to other effects this symbiosis is known for its significance for plant nutrition including iron. Fungal iron mobilization from soil is commonly dependent on siderophores. This study reports on a search for such iron-chelators in root tissue of Tagetes patula nana var. plena colonized by Gigaspora margarita. The AM colonized plants and uninoculated controls were grown under strictly axenic conditions. HPLC analyses of aqueous extracts from plant roots have provided clear evidence for the presence of a rhizoferrin type siderophore, named glomuferrin, in root tissue of mycorrhizal seedlings. Results from HPLC analytical work are seconded by molecular biological data: A BLASTp search revealed that the AM fungal species Gigaspora rosea, Rhizophagus irregularis (formerly Glomus intraradices), Glomus cerebriformis and Diversispora epigea encode a non-ribosomal peptide synthetase (NRPS)-independent siderophore synthase (NIS), which is homologous to the rhizoferrin synthetase of Rhizopus delemar. Thus this study indicates that the biosynthesis of rhizoferrin type siderophores such as glomuferrin (= bis-imidorhizoferrin) may be widespread in the AM symbiosis.

Steel slag and phosphate nutrition of corn inoculated with arbuscular mycorrhizal fungi

Abstract: The objective of this work was to evaluate the effect of the use of steel slag as a soil acidity corrective and of mycorrhizal fungi associated with phosphate fertilization on corn plants. The study was performed in a greenhouse, using 3-kg pots with a Typic Haplorthox, in a 4x5x2 factorial arrangement, with four acidity correction treatments (C1, correction with dolomitic limestone at a dose of 4 Mg ha-1; C2, correction with steel slag at a dose of 4 Mg ha-1; C3, correction with a 1:1 mixture of 2 Mg ha-1 dolomitic limestone and 2 Mg ha-1 steel slag; and C4, control, without pH correction), five phosphorus doses (0, 42, 95, 213, and 480 mg dm-3), and the presence or absence of two arbuscular mycorrhizal fungi (Rhizophagus clarus and Gigaspora margarita), with five replicates. Steel slag was efficient in correcting soil pH and providing Ca and Mg for the plants; therefore, it could replace limestone. The inoculation with R. clarus and G. margarita, associated with P doses of 42, 95, and 213 mg dm-3, improved the development of corn plants after 45 days, resulting in greater plant height, stem diameter, leaf area, and shoot and root dry matter.

Metabolome changes are induced in the arbuscular mycorrhizal fungus Gigaspora margarita by germination and by its bacterial endosymbiont.

Metabolomic profiling is becoming an increasingly important technique in the larger field of systems biology by allowing the simultaneous measurement of thousands of small molecules participating in and resulting from cellular reactions. In this way, metabolomics presents an opportunity to observe the physiological state of a system, which may provide the ability to monitor the whole of cellular metabolism as the technology progresses. The arbuscular mycorrhizal fungus Gigaspora margarita has not previously been explored with regard to metabolite composition. To develop a better understanding of G. margarita and the influences of its endosymbiont Candidatus Glomeribacter gigasporarum, a metabolomic analysis was applied to quiescent and germinated spores with and without endobacteria. Over 100 metabolites were identified and greater than 2600 unique unidentified spectral features were observed. Multivariate analysis of the metabolomes was performed, and a differentiation between all metabolic states of spores and spores hosting the endobacteria was observed. The known metabolites were recruited to many biochemical pathways, with many being involved in maintenance of the antioxidant potential, tyrosine metabolism, and melanin production. Each of the pathways had higher metabolite abundances in the presence of the endosymbiont. These metabolomics data also agree with previously reported transcriptomics results demonstrating the capability of this technique to confirm hypotheses and showing the feasibility of multi-omic approaches for the study of arbuscular mycorrhizal fungi and their endobacterial communities. Challenges still exist in metabolomic analysis, e.g., the identification of compounds is demanding due to incomplete libraries. A metabolomics technique to probe the effects of bacterial endosymbionts on fungal physiology is presented herein, and this method is useful for hypothesis generation as well as testing as noted above.

The virome of the arbuscular mycorrhizal fungus Gigaspora margarita reveals the first report of DNA fragments corresponding to replicating non-retroviral RNA viruses in fungi.

Arbuscular Mycorrhizal Fungi (AMF) are key components of the plant microbiota. AMF genetic complexity is increased by the presence of endobacteria, which live inside many species. A further component of such complexity is the virome associated to AMF, whose knowledge is still very limited. Here, by exploiting transcriptomic data we describe the virome of Gigaspora margarita. A BLAST search for viral RNA-dependent RNA polymerases sequences allowed the identification of four mitoviruses, one Ourmia-like narnavirus, one Giardia-like virus, and two sequences related to Fusarium graminearum mycoviruses. Northern blot and RT-PCR confirmed the authenticity of all the sequences with the exception of the F. graminearum-related ones. All the mitoviruses are replicative and functional since both positive strand and negative strand RNA are present. The abundance of the viral RNA molecules is not regulated by the presence or absence of Candidatus Glomeribacter gigasporarum, the endobacterium hosted by G. margarita, with the exception of the Ourmia-like sequence which is absent in bacteria-cured spores. In addition, we report, for the first time, DNA fragments corresponding to mitovirus sequences associated to the presence of viral RNA. These sequences are not integrated in the mitochondrial DNA and preliminary evidence seems to exclude integration in the nuclear genome.

Microbiological Assessment of Soil Planted with Cover Crops, and Soybean and Maize in Succession

This study aimed to assess the population density of nematodes and mycorrhizal soil fungi, in areas cultivated with oats, brachiaria, forage and white lupine, as well as in maize and soybean crops in succession, in order to generate a microbiological indicator of soil quality. In order to assess nematode and arbuscular mycorrhizal fungi (AMF) population densities, the experiment was performed in two stages: the first assessment was performed in the area where different cover crops were planted, in five seasons (0, 60, 90, 120, 150 days after sowing—DAS). In the second stage, soybean and maize crops in succession were assessed. According to the results, free-living nematodes and arbuscular mycorrhizal fungi population densities were not affected by the cover crop species used and, therefore, these can be grown prior to soybean and corn crops, without impact to free-living nematodes and AMF. The largest population of saprophyte nematodes and AMF occurred at 90 days. The cultivation of soybean and corn did not influence the number of free-living nematodes, but influenced the number of arbuscular mycorrhizal fungi. The highest numbers of mycorrhizal fungi Gigaspora margarita and Glomus macrocarpum were found in maize.

Carlactone-type strigolactones and their synthetic analogues as inducers of hyphal branching in arbuscular mycorrhizal fungi

Hyphal branching in the vicinity of host roots is a host recognition response of arbuscular mycorrhizal fungi. This morphological event is elicited by strigolactones. Strigolactones are carotenoid-derived terpenoids that are synthesized from carlactone and its oxidized derivatives. To test the possibility that carlactone and its oxidized derivatives might act as host-derived precolonization signals in arbuscular mycorrhizal symbiosis, carlactone, carlactonoic acid, and methyl carlactonoate as well as monohydroxycarlactones, 4-, 18-, and 19-hydroxycarlactones, were synthesized chemically and evaluated for hyphal branching-inducing activity in germinating spores of the arbuscular mycorrhizal fungus Gigaspora margarita. Hyphal branching activity was found to correlate with the degree of oxidation at C-19 methyl. Carlactone was only weakly active (100 ng/disc), whereas carlactonoic acid showed comparable activity to the natural canonical strigolactones such as strigol and sorgomol (100 pg/disc). Hydroxylation at either C-4 or C-18 did not significantly affect the activity. A series of carlactone analogues, named AD ester and AA’D diester, was synthesized by reacting formyl Meldrum’s acid with benzyl, cyclohexylmethyl, and cyclogeranyl alcohols (the A-ring part), followed by coupling of the potassium enolates of the resulting formylacetic esters with the D-ring butenolide. AD ester analogues exhibited moderate activity (1 ng–100 pg/disc), while AA’D diester analogues having cyclohexylmethyl and cyclogeranyl groups were highly active on the AM fungus (10 pg/disc). These results indicate that the oxidation of methyl to carboxyl at C-19 in carlactone is a prerequisite but BC-ring formation is not essential to show hyphal branching activity comparable to that of canonical strigolactones.

Crescimento e produção de capítulos florais de calêndula em resposta à inoculação micorrízica e fósforo

RESUMO: O objetivo foi avaliar o efeito da inoculação com fungos micorrízicos arbusculares e fósforo no crescimento e produção de capítulos florais da calêndula (Calendula officinalis). O experimento foi conduzido em casa de vegetação, em vasos. Foram feitas inoculações com os fungos micorrízicos arbusculares Glomus clarum, Gigaspora margarita e controle (sem fungo) e adubadas ou não com fósforo (50 mg/dm3). O arranjo foi em fatorial 3 x 2, no delineamento experimental blocos casualizados, com quatro repetições. Em solos sem adição de adubação fosfatada, plantas de lendula officinalis, inoculadas com o fungo micorrízico arbuscular Gigaspora margarita, apresentaram maior altura (27,9 cm), maior massa seca da parte aérea (4,22 g/planta) e da raiz, maior número (16,5) e maior massa seca de capítulos florais (0,51 g/capítulo) em comparação com plantas não micorrizadas (19,8 cm, 3,49 g/planta, 7,0 e 0,22 g/capítulo, respectivamente).

Mycoavidus cysteinexigens gen. nov., sp. nov., an endohyphal bacterium isolated from a soil isolate of the fungus Mortierella elongata.

An endohyphal bacterium (strain B1-EBT) living in association with the fungus Mortierella elongata FMR23-6 I-B1 was isolated from a fungal cell homogenate and studied for its taxonomic allocation. Cells were Gram-stain-negative, rod-shaped, non-spore-forming, non-motile, and negative for oxidase and catalase. Strain B1-EBT required cysteine for growth and grew at temperatures between 4 and 35 °C. A comparative analysis of 16S rRNA gene sequences revealed that strain B1-EBT forms a distinct clade in the family Burkholderiaceae, encompassing a group of endosymbionts associated with several soil isolates of M. elongata. The most closely related genus is 'Candidatus Glomeribacter gigasporarum', an endosymbiont of the arbuscular mycorrhizal fungus Gigaspora margarita. The major cellular fatty acids of strain B1-EBT were C16 : 0, summed feature 3 (C16 : 1ω7c and C16 : 1ω6c) and summed feature 8 (C18 : 1ω7c or C18 : 1ω6c). Ubiquinone Q-8 was the only quinone detected. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unknown aminophospholipid and two unknown aminolipids. The DNA G+C content was 49.8 mol%. On the basis of phenotypic, chemotaxonomic, and phylogenetic characteristics, strain B1-EBT represents a novel genus and novel species in the family Burkholderiaceae, for which the name Mycoavidus cysteinexigens gen. nov., sp. nov. is proposed. The type strain is B1-EBT ( = JCM 30646T = LMG 28693T = NBRC 110909T).

Medicaol, a strigolactone identified as a putative didehydro-orobanchol isomer, from Medicago truncatula

A major strigolactone produced by the model legume Medicago truncatula (barrel medic) has been tentatively identified as a didehydro-orobanchol isomer. In this study, a putative didehydro-orobanchol isomer was isolated from root exudates collected from barrel medic grown hydroponically under phosphate-starved conditions. The structure and absolute configurations of this strigolactone, named medicaol, were determined by a combination of spectroscopic analysis and spectral comparison with 4-deoxymedicaol which was synthesized using solvolysis and rearrangement of hydroxymethylhexahydroindenone to tetrahydroazulenone as a key step. Medicaol has a seven-membered cycloheptadiene in the A ring instead of a typical six-membered cyclohexene. Medicaol and 4-deoxymedicaol showed activity comparable to their corresponding six-membered A ring relatives, orobanchol and 4-deoxyorobanchol (ent-2′-epi-5-deoxystrigol), in inducing hyphal branching of germinating spores of an arbuscular mycorrhizal fungus Gigaspora margarita. Plausible biosynthetic pathways from 4-deoxyorobanchol to medicaol are also proposed.

PH measurement of tubular vacuoles of an arbuscular mycorrhizal fungus, Gigaspora margarita.

Arbuscular mycorrhizal fungi play an important role in phosphate supply to the host plants. The fungal hyphae contain tubular vacuoles where phosphate compounds such as polyphosphate are accumulated. Despite their importance for the phosphate storage, little is known about the physiological properties of the tubular vacuoles in arbuscular mycorrhizal fungi. As an indicator of the physiological state in vacuoles, we measured pH of tubular vacuoles in living hyphae of arbuscular mycorrhizal fungus Gigaspora margarita using ratio image analysis with pH-dependent fluorescent probe, 6-carboxyfluorescein. Fluorescent images of the fine tubular vacuoles were obtained using a laser scanning confocal microscope, which enabled calculation of vacuolar pH with high spatial resolution. The tubular vacuoles showed mean pH of 5.6 and a pH range of 5.1-6.3. These results suggest that the tubular vacuoles of arbuscular mycorrhizal fungi have a mildly acidic pH just like vacuoles of other fungal species including yeast and ectomycorrhizal fungi.

Interações entre fungos micorrízicos arbusculares, rizóbio e actinomicetos na rizosfera de soja

Isolados de actinomicetos e de fungos micorrízicos arbusculares foram avaliados em sistemas de inoculação conjunta in vivo, em plantas de soja inoculadas com rizóbios, com a finalidade de se observar interações microbianas. A pesquisa foi realizada no Centro Nacional de Pesquisa de Agrobiologia (Embrapa CNPAB). Foram utilizados, como substrato, solo da série Itaguaí (textura média) misturado com areia de rio na proporção 1:1 (v:v), com adição de isolados de actinomicetos (ACT-78 e ACT-370); estirpes de rizóbios (Bradyrhizobium elkanii - BR 29 e Bradyrhizobium japonicum - BR 33) e fungos micorrízicos arbusculares (Gigaspora margarita e Glomus clarum). Os resultados revelam que a inoculação com os fungos micorrízicos arbusculares influenciou de modo expressivo as variáveis de crescimento, nodulação e densidade de actinomicetos na rizosfera de soja, que apresentaram valores maiores do que as plantas sem o inóculo fúngico. Houve efeitos sinergísticos e antagônicos dos fungos micorrízicos arbusculares com os actinomicetos e com rizóbios.

The mitochondrial genome of the arbuscular mycorrhizal fungus Gigaspora margarita reveals two unsuspected trans-splicing events of group I introns.

• Arbuscular mycorrhizal fungi (AMF) are ubiquitous organisms that benefit ecosystems through the establishment of an association with the roots of most plants: the mycorrhizal symbiosis. Despite their ecological importance, however, these fungi have been poorly studied at the genome level. • In this study, total DNA from the AMF Gigaspora margarita was subjected to a combination of 454 and Illumina sequencing, and the resulting reads were used to assemble its mitochondrial genome de novo. This genome was annotated and compared with those of other relatives to better comprehend the evolution of the AMF lineage. • The mitochondrial genome of G. margarita is unique in many ways, exhibiting a large size (97 kbp) and elevated GC content (45%). This genome also harbors molecular events that were previously unknown to occur in fungal mitochondrial genomes, including trans-splicing of group I introns from two different genes coding for the first subunit of the cytochrome oxidase and for the small subunit of the rRNA. • This study reports the second published genome from an AMF organelle, resulting in relevant DNA sequence information from this poorly studied fungal group, and providing new insights into the frequency, origin and evolution of trans-spliced group I introns found across the mitochondrial genomes of distantly related organisms.

Endobacteria affect the metabolic profile of their host Gigaspora margarita, an arbuscular mycorrhizal fungus

The aim of this paper was to understand whether the endobacterium identified as Candidatus Glomeribacter gigasporarum has an effect on the biology of its host, the arbuscular mycorrhizal fungus Gigaspora margarita, through the study of the modifications induced on the fungal proteome and lipid profile. The availability of G. margarita cured spores (i.e. spores that do not contain bacteria), represented a crucial tool to enable the comparison between two fungal homogeneous populations in the presence and the absence of the bacterial components. Our results demonstrate that the endobacterial presence leads to a modulation of fungal protein expression in all the different conditions we tested (quiescent, germinating and strigolactone-elicited germinating spores), and in particular after treatment with a strigolactone analogue. The fungal fatty acid profile resulted to be modified both quantitatively and qualitatively in the absence of endobacteria, being fatty acids less abundant in the cured spores. The results offer one of the first comparative metabolic studies of an AM fungus investigated under different physiological conditions, reveal that endobacteria have an important impact on the host fungal activity, influencing both protein expression and lipid profile, and suggest that the bacterial absence is perceived by G. margarita as a stimulus which activates stress-responsive proteins.

Detection of a novel bacterium associated with spores of the arbuscular mycorrhizal fungusGigaspora margarita

With PCR-denaturing gradient gel electrophoresis analysis, two bacterial 16S rRNA gene V3 region sequences, 7A and 7B, were detected in association with the crushed spores of the arbuscular mycorrhizal fungus Gigaspora margarita W.N. Becker & I.R. Hall 1976 MAFF520054. DNA sequencing and phylogenetic analysis revealed that 7B was mostly related to the documented cytoplasm endosymbiotic bacterium Candidatus Glomeribacter gigasporarum of G. margarita, but 7A could not be confidently assigned to a known taxon. Further characterization of 7A was conducted by obtaining its almost complete 16S rRNA gene sequence via PCR amplification and sequencing. BLAST search indicates that the 16S rRNA gene sequence did not match any identified species sequences in the GenBank database. Further detection revealed that 7A was also associated with the clean G. margarita MAFF520054 spores that were obtained by the surface-sterilized method or dual culture with Ri T-DNA transformed carrot roots. Many ellipse-shaped or egg-shaped bacterium-like organisms were clustered in layer 3 of the fungal spore wall by transmission electron microscopy observation. Our results indicate that 7A represents a novel bacterial population associated with G. margarita MAFF520054 spores, and its doubtless location (wall or cytoplasm) remains unclear based on the present data.

The ftsZ Gene of the Endocellular Bacterium ‘Candidatus Glomeribacter gigasporarum’ Is Preferentially Expressed During the Symbiotic Phases of Its Host Mycorrhizal Fungus

The arbuscular mycorrhizal fungus (AM) Gigaspora margarita consistently hosts bacteria, named 'Candidatus Glomeribacter gigasporarum,' inside its cytoplasm. Endobacteria have a positive impact on fungal fitness during the presymbiotic phase, prior to plant roots colonization. We tested the hypothesis that the endobacterium and its cell divisions depend on fungal metabolism, mirroring also the events of the fungal life cycle which are influenced by plant signals. We first cloned a fragment of ftsZ, a marker gene for bacterial division, and then analyzed its expression along the different stages of fungus development. The bacterial gene transcripts showed the highest values when the fungus was associated to the plant, and peaked in the extraradical mycelium. Strigolactones, which are known to stimulate the AM fungal growth, caused a significant transcript increase in the germinated spores in the absence of the plant. The quantitative real-time reverse-transcription polymerase chain reaction data were strengthened by the quantification of the dividing bacteria, which were increasing in number in germinating spores after the strigolactone treatment. The bioactive molecule alone did not cause any change in the number of bacteria after their isolation from the fungus, thus showing that the strigolactone alone cannot confer free-living capacities to the bacterium.

Effect of peat on mycorrhizal colonization and effectiveness of the arbuscular mycorrhizal fungus Gigaspora margarita

The influence of the addition of Chinese peat and Canadian peat on arbuscular mycorrhizal colonization, mycorrhizal effectiveness and host-plant growth was investigated in a pot experiment. Chinese peat or Canadian peat was mixed with Masa soil (weathered granite soil) at different levels (0, 25, 50, 100, 150 or 200 g kg−1) into which an arbuscular mycorrhizal fungus (AMF) Gigaspora margarita Becker & Hall was inoculated, and seedlings of Miscanthus sinensis Anderess were planted. There was a significant increase in plant growth with increasing amounts of Chinese peat. The growth-promoting effect of the AMF on the host was enhanced when the addition of Chinese peat was increased from 25 to 100 g kg−1. Root colonization and the number of spores proliferating increased with increases at low levels of Chinese peat (from 25 to 100 g kg−1), and decreased gradually with higher Chinese peat increments. Although plant growth and root colonization with the addition of Canadian peat increased slightly, Canadian peat suppressed mycorrhizal effectiveness. In contrast to Canadian peat, the addition of Chinese peat improved considerably the physical and chemical properties of the soil, which might result in the promotion of AM formation and mycorrhizal effectiveness.

Formation of appressoria by the arbuscular mycorrhizal fungus Gigaspora margarita on roots of Allium cepa is linked with root age

Spores of the arbuscular mycorrhizal fungus, Gigaspora margarita, were placed near the root tip, the middle of the root (equal distance from root base and root tip), or the root base (close to the shoot) of the first primary root of 9-day-old onion. Two weeks later, the number and position of appressoria and the appressoria with penetrating hyphae were determined in the first and the newly formed second primary roots. The total number of appressoria was not significantly different among the treatments. Inoculation near the root tip of the first primary root resulted in the formation of a large number of appressoria on the first primary root and the formation of about three times fewer appressoria on the second primary root. Inoculation near the base of the first primary root resulted in the formation of no appressoria on the first primary root, whereas many appressoria were formed on the second primary root. Our results suggest that the root age is a determinant of the appressorium formation.

Enhanced activity of the GmarMT1 promoter from the mycorrhizal fungus Gigaspora margarita at limited carbon supply

Metallothioneins are low molecular weight polypeptides, present in most eukaryotic phyla, with role in metal homeostasis and detoxification. We previously reported the identification and the characterization of a metallothionein gene ( GmarMT1) from the arbuscular mycorrhizal fungus Gigaspora margarita. Here, we have used real-time quantitative RT-PCR to show that GmarMT1 expression was turned off during the symbiotic fungal growth in the hexose-rich mycorrhizal apoplast, whereas transcripts were abundant during all other fungal growth stages, when products of lipid breakdown and the glyoxylate cycle feed carbohydrate-consuming pathways. In support of a nutritional regulation of GmarMT1 expression, we show that transcriptional activity of GmarMT1 promoter in yeast was strongly induced by glucose starvation (up to 20 times the basal level). We speculate that GmarMT1-encoded protein, with its proved metal binding ability, could regulate the homeostasis of zinc, a fundamental cofactor involved in C metabolism regulation and glucose repression.

Presymbiotic growth and sporal morphology are affected in the arbuscular mycorrhizal fungus Gigaspora margarita cured of its endobacteria

Some arbuscular mycorrhizal fungi contain endocellular bacteria. In Gigaspora margarita BEG 34, a homogenous population of beta-Proteobacteria is hosted inside the fungal spore. The bacteria, named Candidatus Glomeribacter gigasporarum, are vertically transmitted through fungal spore generations. Here we report how a protocol based on repeated passages through single-spore inocula caused dilution of the initial bacterial population eventually leading to cured spores. Spores of this line had a distinct phenotype regarding cytoplasm organization, vacuole morphology, cell wall organization, lipid bodies and pigment granules. The absence of bacteria severely affected presymbiotic fungal growth such as hyphal elongation and branching after root exudate treatment, suggesting that Ca. Glomeribacter gigasporarum is important for optimal development of its fungal host. Under laboratory conditions, the cured fungus could be propagated, i.e. could form mycorrhizae and sporulate, and can therefore be considered as a stable variant of the wild type. The results demonstrated that - at least for the G. margarita BEG 34 isolate - the absence of endobacteria affects the spore phenotype of the fungal host, and causes delays in the growth of germinating mycelium, possibly affecting its ecological fitness. This cured line is the first manipulated and stable isolate of an arbuscular mycorrhizal fungus.

Repetitive DNA sequences include retrotransposons in genomes of the Glomeromycota

Twenty-five repetitive elements are first described in the genomes of the arbuscular mycorrhizal (AM) fungi Gigaspora margarita, Gig. rosea and Glomus mosseae. Nineteen repetitive DNA sequences isolated by genomic library screening and four by self-priming PCR had no homology to known DNA sequences, except for two Gig. margarita sequences and one Gig. rosea sequence which showed amino acid similarity to retrotransposons. Part of the Gig. rosea sequence was also similar to a DNA transposon. Two other retrotransposon sequences were isolated using PCR targeting of reverse transcriptase and ribonuclease H domains. Evidence is provided for three gypsy-like LTR retrotransposon and two non-LTR retrotransposon sequences in the AM fungal genomes. Four contain stop codons indicating that they cannot be active. Expression of three retrotransposons was not detected in germinating spores or intraradical hyphae of Gig. margarita. Southern blot analyses indicated that these three sequences are dispersed in the genome and that two are methylated. Sequence analysis of different GmarLTR1 copies showed they have undergone mutations by transitions, which may have been induced by cytosine methylation. Transposable elements may have played a major role in shaping genome structure and size during evolution of the Glomeromycota.