Arbuscular Mycorrhizal Fungus Gigaspora Margarita Research Articles

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.

Structural requirements of strigolactones for hyphal branching in AM fungi.

Strigolactones are a group of terpenoid lactones that act as a host-derived signal in the rhizosphere communication of plants with arbuscular mycorrhizal (AM) fungi and root parasitic weeds as well as an endogenous plant hormone regulating shoot branching in plants. Strigolactones induce hyphal branching in AM fungi at very low concentrations, suggesting a highly sensitive perception system for strigolactones present in AM fungi. However, little is known about the structural requirements of strigolactones for hyphal branching in AM fungi. Here, we tested a series of natural and synthetically modified strigolactones as well as non-strigolactone-type germination stimulants for hyphal branching-inducing activity in germinating spores of the AM fungus Gigaspora margarita. All tested compounds with a tricyclic lactone coupled to a methylbutenolide via an enol ether bond showed activity, but differed in the active concentration and in the branching pattern of hyphae. Truncation of the A- and AB-rings in the tricyclic ABC lactone of strigolactones resulted in a drastic reduction in hyphal branching activity. Although the connection of the C-ring in the tricyclic lactone to the methylbutenolide D-ring was shown to be essential for hyphal branching, the bridge structure in the C–D part was found not necessarily to be enol ether, being replaceable with either alkoxy or imino ethers. These structural requirements in AM fungi are very similar but not identical to those observed in root parasitic weeds, especially with respect to the enol ether bridge in the C–D part.

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.

Produção de ácido indol acético por microorganismos solubilizadores de fosfato e sua interação com fungos micorrízicos arbusculares

Indolacetic acid (IAA) production was evaluated under in vitro conditions by five P-solubilizing fungi (PSF), identified as Aspergillus sp. and four P-solubilizing bacteria (PSB) of the Enterobacteriaceae family. The bacteria and fungus isolates were incubated in a liquid medium at 28ºC for two and four days, respectively. Moreover, two PSB isolates of the Enterobacteriaceae family (PSB 8 and PSB 56) were evaluated in relation to their ability to stimulate or inhibit spore germination and hyphal growth of two arbuscular mycorrhizal fungi (AMF) species under in vitro conditions. Two assays were carried out in Petri dishes with agar (0.8%), the first using the AMF Gigaspora margarita and the second with the AMF Scutellospora sp. The treatments were: inoculation of PSB 8; PSB 56 and uninoculated control. P-solubilizing isolates produced different amounts of IAA and most PSF isolates produced higher IAA than PSB. From the 18th day of incubation of Scutellospora sp., a significant increase in hyphal growth in the treatment inoculated with PSB 8 was observed. In contrast, PSB 56 inhibited the hyphal growth of Gigaspora margarita from the 24th day of incubation.

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.

Transcriptome analysis of arbuscular mycorrhizal roots during development of the prepenetration apparatus.

Information on changes in the plant transcriptome during early interaction with arbuscular mycorrhizal (AM) fungi is still limited since infections are usually not synchronized and plant markers for early stages of colonization are not yet available. A prepenetration apparatus (PPA), organized in epidermal cells during appressorium development, has been reported to be responsible for assembling a trans-cellular tunnel to accommodate the invading fungus. Here, we used PPAs as markers for cell responsiveness to fungal contact to investigate gene expression at this early stage of infection with minimal transcript dilution. PPAs were identified by confocal microscopy in transformed roots of Medicago truncatula expressing green fluorescent protein-HDEL, colonized by the AM fungus Gigaspora margarita. A PPA-targeted suppressive-subtractive cDNA library was built, the cDNAs were cloned and sequenced, and, consequently, 107 putative interaction-specific genes were identified. The expression of a subset of 15 genes, selected by reverse northern dot blot screening, and five additional genes, potentially involved in PPA formation, was analyzed by real-time reverse transcription-polymerase chain reaction and compared with an infection stage, 48 h after the onset of the PPA. Comparison of the expression profile of G. margarita-inoculated wild type and the mycorrhiza-defective dmi3-1 mutant of M. truncatula revealed that an expansin-like gene, expressed in wild-type epidermis during PPA development, can be regarded as an early host marker for successful mycorrhization. A putative Avr9/Cf-9 rapidly elicited gene, found to be up-regulated in the mutant, suggests novel regulatory roles for the DMI3 protein in the early mycorrhization process.

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.

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.

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.

Stimulatory effect of peat on spore germination and hyphal growth of arbuscular mycorrhizal fungus Gigaspora margarita

The effects of peat extract solutions on spore germination and hyphal growth of the arbuscular mycorrhizal fungus Gigaspora margarita Becker & Hall were investigated. Spores were incubated in extract solutions of peat from China and peat moss from Canada, which were prepared at a weight ratio of 1:10, 1:20, 1:30, 1:50 and 1:100 for each substrate according to the weight ratio of water. Compared with peat and peat moss solutions, solutions of KH2PO4 and (NH4)2SO4 at various concentrations were used as media to investigate the effect of phosphorus (P) and nitrogen (N). Spores in the peat solutions showed the highest values for germination percentage, hyphal length and number of auxiliary cells, and these values were significantly higher than the values recorded for the other treatments. In contrast, in the peat moss extract solutions, which had a lower content of N and higher content of P than the peat solutions, the germination and hyphal growth of spores decreased. Spore growth was promoted with increased concentrations of the N solutions. However, P solutions exerted an inhibitory effect on spore germination and hyphal growth. Peat solutions showed a high N content and low P content, which might be related to the promotion of spore growth.

Competition and substrate colonization strategies of three polyxenically grown arbuscular mycorrhizal fungi

Intra- and extraradical colonization competition and hyphal interactions among arbuscular mycorrhizal fungi (AMF) Glomus intraradices, Glomus proliferum and Gigaspora margarita were investigated in two in vitro experimental systems. AMF were polyxenically cultured with a Ri T-DNA transformed carrot root organ culture (ROC) in either big Petri plates containing three culture compartments and a common hyphal compartment (i.e. an independent host root for each AMF) or two by two in the culture compartment of regular bicompartmented Petri dishes (i.e. a common host root and a common hyphal compartment). Maps of the extraradical mycelial development of the three AMF were obtained. Two distinct substrate colonization strategies (Glomus-type and Gigaspora-type) were identified, reflecting intrinsic differences among AMF genera/families. Our data reveal a general lack of antagonism between the isolates when extraradical hyphae explore and exploit the substrate outside the root influence zone; however certain growth restrictions were imposed by Gi. margarita extraradical mycelium when developing near the host root and by G. proliferum intraradical hyphae. This work highlights once more the appropriateness of AM in vitro culture systems to perform in vivo studies on the biology of this symbiosis and opens new avenues to the formulation of in vitro AMF inoculants.

Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi

Arbuscular mycorrhizal (AM) fungi form mutualistic, symbiotic associations with the roots of more than 80% of land plants. The fungi are incapable of completing their life cycle in the absence of a host root. Their spores can germinate and grow in the absence of a host, but their hyphal growth is very limited. Little is known about the molecular mechanisms that govern signalling and recognition between AM fungi and their host plants. In one of the first stages of host recognition, the hyphae of AM fungi show extensive branching in the vicinity of host roots before formation of the appressorium, the structure used to penetrate the plant root. Host roots are known to release signalling molecules that trigger hyphal branching, but these branching factors have not been isolated. Here we have isolated a branching factor from the root exudates of Lotus japonicus and used spectroscopic analysis and chemical synthesis to identify it as a strigolactone, 5-deoxy-strigol. Strigolactones are a group of sesquiterpene lactones, previously isolated as seed-germination stimulants for the parasitic weeds Striga and Orobanche. The natural strigolactones 5-deoxy-strigol, sorgolactone and strigol, and a synthetic analogue, GR24, induced extensive hyphal branching in germinating spores of the AM fungus Gigaspora margarita at very low concentrations.

The mycorrhizal fungus Gigaspora margarita possesses a CuZn superoxide dismutase that is up-regulated during symbiosis with legume hosts.

A full-length cDNA showing high similarity to previously described CuZn superoxide dismutases (SODs) was identified in an expressed sequence tag collection from germinated spores of the arbuscular mycorrhizal fungus Gigaspora margarita (BEG 34). The corresponding gene sequence, named GmarCuZnSOD, is composed of four exons. As revealed by heterologous complementation assays in a yeast mutant, GmarCuZnSOD encodes a functional polypeptide able to confer increased tolerance to oxidative stress. The GmarCuZnSOD RNA was differentially expressed during the fungal life cycle; highest transcript levels were found in fungal structures inside the roots as observed on two host plants, Lotus japonicus and Medicago truncatula. These structures also reacted positively to 3,3'-diaminobenzidine, used to localize H2O2 accumulation. This H2O2 is likely to be produced by CuZnSOD activity since treatment with a chelator of copper ions, generally used to inhibit CuZnSODs, strongly reduced the 3,3'-diaminobenzidine deposits. A slight induction of GmarCuZnSOD gene expression was also observed in germinated spores exposed to L. japonicus root exudates, although the response showed variation in independent samples. These results provide evidence of the occurrence, in an arbuscular mycorrhizal fungus, of a functional SOD gene that is modulated during the life cycle and may offer protection as a reactive oxygen species-inactivating system against localized host defense responses raised in arbuscule-containing cells.

Flavonoids exclusively present in mycorrhizal roots of white clover exhibit a different effect on arbuscular mycorrhizal fungi than flavonoids exclusively present in non-mycorrhizal roots of white clover

The flavonoids 5,6,7,8,9-hydroxy chalcone, 3,7-hydroxy-4′methoxy flavone, 5,6,7,8-hydroxy-4′-methoxy flavone and 3,5,6,7,4′-hydroxy flavone can be detected only in non-mycorrhizal roots of white clover, but not in mycorrhizal roots, whereas the flavonoids acacetin, quercetin and rhamnetin are only present in mycorrhizal roots. We tested the effect of several concentrations of these compounds on spore germination, hyphal growth, hyphal branching, formation of clusters of auxiliary cells and of secondary spores of the arbuscular mycorrhizal fungi Gigaspora rosea, Gigaspora margarita, Glomus mosseae and Glomus intraradices. Our results indicate that depending on the flavonoid, the tested compounds are involved at different stages in the regulation of mycorrhization. This hypothesis is strengthened by their differing effect on several AM fungal growth parameters. Furthermore, our study provides more data on the AM fungus genus/species specificity of flavonoids.

Effects of pH on mycorrhizal colonisation and nutrient uptake in cowpea under conditions that minimise confounding effects of elevated available aluminium

Effects of the arbuscular mycorrhizal (AM) fungi Gigaspora margarita and Glomus etunicatum on the growth of cowpea (Vigna unguiculata L. Walp.) were assessed at low pH by growing plants, with and without AM inoculation, individually in pots containing a mixture of sand and soil adjusted to pH 4.7, 4.9 or 5.2 at the start of the experiment, and with soluble aluminium (Al) concentrations at a sub-toxic level for the plant. Cowpea grew poorly in the absence of AM colonisation, particularly at pH 4.7. Growth was enhanced both by increasing the pH and by inoculating with the AM fungi, with plant responses greater with inoculation. The relative growth improvement by mycorrhizas (mycorrhizal growth response) was highest at pH 4.7, and decreased as the pH increased, although effects were not always significant. Gi. margarita was much more effective than G. etunicatum. There were differential effects of the two fungi on uptake of mineral elements. Plants inoculated with Gi. margarita took up a range of elements, including P and Zn as well as Al, to a much greater extent than those inoculated with G. etunicatum, regardless of medium pH. The effectiveness of Gi. margarita in increasing plant growth was closely correlated with colonised root length.

Substratos e fungo micorrízico arbuscular em mudas micropropagadas de bananeira na fase de aclimatação

Visando a avaliar diferentes substratos na formação de mudas de bananeira e seu efeito na resposta da planta à inoculação do fungo micorrízico Gigaspora margarita, foi conduzido um experimento em estufa de aclimatação da Biofábrica CAMPO - CPA/Embrapa Mandioca e Fruticultura, Cruz das Almas, Bahia. Foram testados 13 substratos, com e sem inoculação do fungo, em plântulas na fase de aclimatação. A inoculação foi realizada no momento do transplante e, após 55 dias de cultivo, obtiveram-se dados de crescimento, nutrição mineral e colonização micorrízica. O fungo micorrízico arbuscular (FMA) Gigaspora margarita colonizou intensamente e mostrou-se benéfico para o desenvolvimento das mudas de bananeira, sendo o seu efeito modulado pelo substrato de crescimento; o substrato turfa + vermiculita + 5% de esterco destacou-se entre os melhores para a formação de mudas normais e sadias, mas só quando associado à inoculação do FMA; o uso de substrato comercial Rendmax Citrus promoveu o melhor desenvolvimento das mudas, mas inibiu a colonização e o efeito da micorriza; a complementação mineral do Rendmax Citrus não se mostrou necessária para o cultivo de mudas de bananeira; o uso de vermicomposto mostrou-se promissor para a produção de mudas de bananeira, permitindo o efeito da inoculação com FMA.

Utilização de Gigaspora margarita em plantas micropropagadas de bananeira em diferentes estádios de enraizamento

Visando a avaliar o efeito de fungos micorrízicos arbusculares (FMA) na diminuição do tempo de formação de mudas de bananeira micropropagadas, foi conduzido um experimento em estufa de aclimatação da Biofábrica CAMPO - CPA/Embrapa Mandioca e Fruticultura, Cruz das Almas, Bahia. Foram testadas plântulas em três diferentes estádios de desenvolvimento do sistema radicular, inoculadas ou não com o fungo Gigaspora margarita e cultivadas em dois diferentes substratos à base de turfa, vermiculita e esterco. A inoculação foi realizada no momento do transplantio para a estufa e as plantas, cultivadas por 55 dias, quando foram coletadas para obtenção dos dados de avaliação. O fungo Gigaspora margarita colonizou intensamente e mostrou- se benéfico para o desenvolvimento das mudas de bananeira, sendo seu efeito modulado pelo substrato de crescimento; o substrato turfa + vermiculita + 5% de esterco, quando associado à inoculação do FMA, promoveu a formação de mudas normais e sadias; o início da fase de aclimatação de mudas micropropagadas de bananeira pode ser antecipado pelo uso da inoculação com fungos micorrízicos arbusculares, em substrato adequado.

Identification and spore proliferation of an arbuscular mycorrhizal fungus Gigaspora margarita isolated from Shiiba village, Miyazaki, Japan

Identification and spore proliferation of an arbuscular mycorrhizal fungus Gigaspora margarita isolated from Shiiba village, Miyazaki, Japan