Arbuscular Mycorrhizal Fungus Gigaspora Rosea Research Articles

Crescimento e atributos fisiológicos de mudas de mirtileiro inoculadas com fungos micorrízicosarbusculares

ABSTRACT: High-quality seedlings are one of the key factors in achieving high yield and precocity of blueberries. The inoculation of arbuscular mycorrhizal fungi (AMF) can enhance the development of seedlings in the nursery, ensuring more vigorous seedlings in a shorter time. This study evaluated the effect of inoculation of arbuscular mycorrhizal fungi on the development of ‘PowderBlue’blueberryseedlings. The treatments were arranged in a 4 x 2 factorial scheme, in which the first factor was the arbuscular mycorrhizal fungi Gigaspora rosea, Glomus clarum, G. rosea + G. clarum, and a control level without mycorrhizae, while the second factor consisted of usingindole-3-butyric acid(IBA) and a control level without IBA. Semi-hardwood cuttings were planted in pots containing sterilized soil and kept in a greenhouse for 660 days. The percentage of rooted cuttings, plant height, root system length, shoot dry mass, root dry mass, total dry mass, peroxidase (POD) and superoxide dismutase (SOD) enzyme activities, SPAD index, mycorrhizal efficiency and dependence, number of spores, and soil basal respiration were evaluated. Plants inoculated with G. clarum without IBA and inoculated with G. rosea with IBA showed higher dry matter and SOD and POD enzyme activities, but the use of IBA had a negative effect on the fungus. The inoculation of blueberry cuttings with G. clarummay help seedlingdevelopment, thus improving biometric and biochemical parameters. Furthermore, the plant regulator IBAwas essential in establishing the symbiosis between blueberry and the AMF G. rosea.

A Survey of the Gene Repertoire of Gigaspora rosea Unravels Conserved Features among Glomeromycota for Obligate Biotrophy.

Arbuscular mycorrhizal (AM) fungi are a diverse group of soil fungi (Glomeromycota) that form the most ancient mutualistic association termed AM symbiosis with a majority of land plants, improving their nutrition uptake and resistance to stresses. In contrast to their great ecological implications, the knowledge of the molecular biological mechanisms involved is still scant, partly due to the limited genomic resources available. Here, we describe the gene repertoire of a new AM fungus Gigaspora rosea (Diversisporales). Among the 86332 non-redundant virtual transcripts assembled, 15346 presented similarities with proteins in the Refseq database and 10175 were assigned with GO terms. KOG and Interpro domain annotations clearly showed an enrichment of genes involved in signal transduction in G. rosea. KEGG pathway analysis indicates that most primary metabolic processes are active in G. rosea. However, as for Rhizophagus irregularis, several metabolic genes were not found, including the fatty acid synthase (FAS) gene. This finding supports the hypothesis that AM fungi depend on the lipids produced by their hosts. Furthermore, the presence of a large number of transporters and 100s of secreted proteins, together with the reduced number of plant cell wall degrading enzymes could be interpreted as an evolutionary adaptation to its mutualistic obligate biotrophy. The detection of meiosis-related genes suggests that G. rosea might use a cryptic sexual process. Lastly, a phylogeny of basal fungi clearly shows Glomeromycota as a sister clade to Mucoromycotina, not only to the Mucorales or Mortierellales. The characterization of the gene repertoire from an AM fungal species belonging to the order of Diversisporales and its comparison with the gene sets of R. irregularis (Glomerales) and Gigaspora margarita (Diversisporales), reveal that AM fungi share several features linked to mutualistic obligate biotrophy. This work contributes to lay the foundation for forthcoming studies into the genomics of Diversisporales, and also illuminates the utility of comparing gene repertoires of species from Diversisporales and other clades of Glomeromycota to gain more insights into the genetics and evolution of this fungal group.

Erratum to: Involvement of autoregulation in the interaction between rhizobial nodulation and AM fungal colonization in soybean roots

Soybean plants autoregulate to suppress excessive nodulation. It has been revealed recently that the autoregulation of various legumes controls both nodulation and arbuscular mycorrhizal (AM) fungal colonization. We investigated the involvement of autoregulation in the interaction between rhizobial nodulation and AM fungal colonization. We used a wild-type soybean cv. Enrei and its hypernodulating mutant Kanto100, defective in the autoregulation. We included four different treatments: an uninoculated control, inoculation with rhizobium Bradyrhizobium japonicum alone, inoculation with AM fungus Gigaspora rosea alone, and dual inoculation with rhizobium and AM fungus. In both Enrei and Kanto100, AM fungal colonization enhanced the weight and N2 fixation of nodules, suggesting that autoregulation of host plant is not involved in the stimulatory effect of AM fungal colonization on rhizobial nodulation. In plants with the AM fungus alone, the AM fungal colonization of Enrei was comparable to that of Kanto100. In plants with dual inoculation, however, this was significantly (P < 0.05) lower than in Kanto100. To confirm the control of AM fungal colonization by the autoregulation of host plant, a reciprocal grafting experiment was performed between Enrei and Kanto100. In plants with the AM fungus alone, AM fungal colonization was comparable among Enrei (shoot)/Enrei (root), Enrei/Kanto100, Kanto100/Enrei, and Kanto100/Kanto100 grafts. In plants with dual inoculation, however, AM fungal colonization of Enrei/Enrei and Enrei/Kanto100 grafts was significantly (P < 0.05) lower than that of Kanto100/Enrei and Kanto100/Kanto100. These results indicate that rhizobial nodulation suppresses AM fungal colonization, and the autoregulation of host plant, initiated by nodulation, is involved in this phenomenon.

Strigolactones affect development in primitive plants. The missing link between plants and arbuscular mycorrhizal fungi?

Strigolactones affect development in primitive plants. The missing link between plants and arbuscular mycorrhizal fungi?

Both the arbuscular mycorrhizal fungus Gigaspora rosea and Frankia increase root system branching and reduce root hair frequency in Alnus glutinosa

Alnus glutinosa is an important pioneer species that forms effective symbioses with Frankia and ecto and arbuscular mycorrhizal fungi (AMF). There is evidence that Frankia and AMF interact and the focus of this study was to investigate how interactions affected root system and root hair development. A. glutinosa seedlings were grown in pots in soil pre-inoculated with the AMF Gigaspora rosea. Seedlings were inoculated with Frankia either immediately on transfer to AMF-inoculated pots (day 0) on day 15 or on day 30 following AMF inoculation so the effect of timing of inoculation on interactions could be determined. Seedlings were harvested in batches at intervals of 10, 15, 20, 25 and 30 days after the commencement of each treatment. Both G. rosea and Frankia increased root branching and effects were greater when both were present. By contrast, both G. rosea and Frankia decreased root hair numbers markedly. Effects on root hair development were not a consequence of phosphorous, as P levels were not changed significantly in seedlings colonised by G. rosea or nodulated by Frankia. Effects are not due to differences in root system size but conceivably could offset some of the carbon costs incurred by the symbioses.

GR24, a synthetic analog of strigolactones, stimulates the mitosis and growth of the arbuscular mycorrhizal fungus Gigaspora rosea by boosting its energy metabolism.

Arbuscular mycorrhizal (AM) fungi are obligate biotrophs that participate in a highly beneficial root symbiosis with 80% of land plants. Strigolactones are trace molecules in plant root exudates that are perceived by AM fungi at subnanomolar concentrations. Within just a few hours, they were shown to stimulate fungal mitochondria, spore germination, and branching of germinating hyphae. In this study we show that treatment of Gigaspora rosea with a strigolactone analog (GR24) causes a rapid increase in the NADH concentration, the NADH dehydrogenase activity, and the ATP content of the fungal cell. This fully and rapidly (within minutes) activated oxidative metabolism does not require new gene expression. Up-regulation of the genes involved in mitochondrial metabolism and hyphal growth, and stimulation of the fungal mitotic activity, take place several days after this initial boost to the cellular energy of the fungus. Such a rapid and powerful action of GR24 on G. rosea cells suggests that strigolactones are important plant signals involved in switching AM fungi toward full germination and a presymbiotic state.

The fine‐tuning of heavy metals in mycorrhizal fungi

The fine‐tuning of heavy metals in mycorrhizal fungi

Strigolactones stimulate arbuscular mycorrhizal fungi by activating mitochondria.

The association of arbuscular mycorrhizal (AM) fungi with plant roots is the oldest and ecologically most important symbiotic relationship between higher plants and microorganisms, yet the mechanism by which these fungi detect the presence of a plant host is poorly understood. Previous studies have shown that roots secrete a branching factor (BF) that strongly stimulates branching of hyphae during germination of the spores of AM fungi. In the BF of Lotus, a strigolactone was found to be the active molecule. Strigolactones are known as germination stimulants of the parasitic plants Striga and Orobanche. In this paper, we show that the BF of a monocotyledonous plant, Sorghum, also contains a strigolactone. Strigolactones strongly and rapidly stimulated cell proliferation of the AM fungus Gigaspora rosea at concentrations as low as 10 −13 M. This effect was not found with other sesquiterperne lactones known as germination stimulants of parasitic weeds. Within 1 h of treatment, the density of mitochondria in the fungal cells increased, and their shape and movement changed dramatically. Strigolactones stimulated spore germination of two other phylogenetically distant AM fungi, Glomus intraradices and Gl. claroideum. This was also associated with a rapid increase of mitochondrial density and respiration as shown with Gl. intraradices. We conclude that strigolactones are important rhizospheric plant signals involved in stimulating both the pre-symbiotic growth of AM fungi and the germination of parasitic plants.

Flavonoids exhibit fungal species and genus specific effects on the presymbiotic growth of Gigaspora and Glomus

The effect of the flavonoids chrysin, isorhamnetin, kaempferol, luteolin, morin and rutin on pre-symbiotic growth, such as spore germination, hyphal length, hyphal branching and the formation of auxiliary cells and secondary spores, of the arbuscular mycorrhizal fungi Gigaspora rosea, G. margarita, Glomus mosseae and G. intraradices was studied. According to the effect on each fungal growth parameter, the tested compounds could be classified to be genus and/or species specific or specific, for a certain developmental stage of pre-symbiotic growth. A clear arbuscular mycorrhizal genus specific, and even species specific, effect of some flavonoids was observed. However, this specificity of a flavonoid could not be generalized but differs depending on the pre-symbiotic stage of the AM fungus. Moreover, our show that for a better understanding of the role of compounds in the AM symbiosis, studies should not be conducted only with one AM fungus looking at one fungal growth parameter such as spore germination or hyphal length, but should be wider, including several growth parameters and several AM fungi.

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.

Root factors induce mitochondrial-related gene expression and fungal respiration during the developmental switch from asymbiosis to presymbiosis in the arbuscular mycorrhizal fungus Gigaspora rosea.

During spore germination, arbuscular mycorrhizal (AM) fungi show limited hyphal development in the absence of a host plant (asymbiotic). In the presence of root exudates, they switch to a new developmental stage (presymbiotic) characterized by extensive hyphal branching. Presymbiotic branching of the AM fungus Gigaspora rosea was induced in liquid medium by a semipurified exudate fraction from carrot (Daucus carota) root organ cultures. Changes in RNA accumulation patterns were monitored by differential display analysis. Differentially appearing cDNA fragments were cloned and further analyzed. Five cDNA fragments could be identified that show induced RNA accumulation 1 h after the addition of root exudate. Sequence similarities of two fragments to mammalian Nco4 and mitochondrial rRNA genes suggested that root exudates could influence fungal respiratory activity. To support this hypothesis, additional putative mitochondrial related-genes were shown to be induced by root exudates. These genes were identified after subtractive hybridization and putatively encode a pyruvate carboxylase and a mitochondrial ADP/ATP translocase. The gene GrosPyc1 for the pyruvate carboxylase was studied in more detail by cloning a cDNA and by quantifying its RNA accumulation. The hypothesis that respiratory activity of AM fungi is stimulated by root exudates was confirmed by physiological and cytological analyses in G. rosea and Glomus intraradices. Oxygen consumption and reducing activity of both fungi was induced after 3 and 2 h of exposition with the root factor, respectively, and the first respiration activation was detected in G. intraradices after approximately 90 min. In addition, changes in mitochondrial morphology, orientation, and overall biomass were detected in G. rosea after 4 h. In summary, the root-exuded factor rapidly induces the expression of certain fungal genes and, in turn, fungal respiratory activity before intense branching. This defines the developmental switch from asymbiosis to presymbiosis, first by gene activation (0.5-1 h), subsequently on the physiological level (1.5-3 h), and finally as a morphological response (after 5 h).

Effect of Garlic Mustard [Alliaria petiolata (Beib. Cavara &amp; Grande)] Extracts on Plants and Arbuscular Mycorrhizal (AM) Fungi

We examined the effects of garlic mustard (Alliaria petiolata) leachates on germination of arbuscular mycorrhizal (AM) fungal spores, colonization of plant roots by AM fungi and germination and root growth of monocot and dicot plants under laboratory conditions. In the field we examined the effect of garlic mustard on AM inoculum potential (MIP). Water leachates of garlic mustard prevented germination of spores of the AM fungus Gigaspora rosea, inhibited the formation of AM associations with tomato (Lycopesicum esculentum) and significantly reduced germination of tomato seeds. Garlic mustard leachates also reduced root length of tomato and sorghum (Sorghum bicolor) seedlings. Under field conditions we found a significant negative correlation between the density of garlic mustard and the mycorrhizal inoculum potential of the soil in which the plants grew. These results suggest that garlic mustard may reduce the competitive abilities of native plants by interfering with the formation of mycorrhizal associations and root growth.

EST-library construction using spore RNA of the arbuscular mycorrhizal fungus Gigaspora rosea

RNA was extracted from activated spores of the arbuscular mycorrhizal fungus Gigaspora rosea. Double-stranded cDNA was synthesised, digested and cloned into the vector lambda-ZAP express. Of the 1,500 clones obtained, 1.5% carried inserts of the rRNA gene cluster. After excision, inserts from 50 randomly selected clones were sequenced. Database searches revealed that 62% of the clones had similarities to already known sequences. These mainly code for proteins involved in translation and protein processing, replication and the cell cycle and cell signal transduction. One fragment probably belonged to a metallothionein-encoding gene which may be involved in heavy-metal binding. The method presented is an easy and rapid way to obtain short fragments of coding regions for expressed sequence tag libraries.

In vivo studies on the nuclear behavior of the arbuscular mycorrhizal fungus Gigaspora rosea grown under axenic conditions

The distribution and fate of nuclei of the arbuscular-my-corrhizal fungusGigaspora rosea during late stages of axenic cultures were studied in fixed cultures by transmitted light, conventional and confocal laser scanning microscopy, and in live cultures with two-photon fluorescence microscopy. Mature specimens not yet showing apical septation displayed oval-shaped nuclei localized in lateral positions of the hypha all along the germ-tube length. Beside these, round-shaped nuclei were found to migrate along the central germ-tube core. Some (rare) germ-tube areas, delimited by septa and containing irregularly shaped, much brighter fluorescent nuclei were also found. Specimens that had just initiated the septation process after germ-tube growth arrest displayed round or oval-shaped nuclei in several portions of the germ tubes. These hyphal areas often alternated with other septa-delimited cytoplasmic clusters which contained distorted, brightly fluorescent nuclei. Completely septated specimens mostly lacked nuclei along their germ tubes. However, highly fluorescent chromatin masses appeared within remnants of cytoplasmic material, often compressed between close septa. Our results provide a first clear picture of the in vivo distribution of nuclei along arbuscular mycorrhizal fungal germ tubes issued from resting spores, and suggest that selective areas of their coenocytic hyphae are under specific, single nuclear control. They indicate as well that random autolytic processes occur along senescingG. rosea germ tubes, probably as a consequence of the absence of a host root signal for mycorrhizal formation. Finally, the data presented here allow us to envisage the fate of nuclei released by the germinating spore after nonsymbiotic fungal growth arrest.

RNA Accumulation and Genes Expressed in Spores of the Arbuscular Mycorrhizal Fungus, Gigaspora rosea

RNA could be extracted from ungerminated spores of the arbuscular mycorrhizal fungus Gigaspora rosea with a maximum of yield 7 days after isolation from soil. cDNA was synthesized and used for library construction or as template for the amplification by polymerase chain reaction of fragments corresponding to several genes. Analysis of cDNA clones corresponding to fragments of ribosomal RNA genes indicated that the extracted RNA as starting material was of arbuscular mycorrhizal fungal origin. Polymerase chain reaction showed the presence of mRNA from genes encoding glyceraldehyde-3-phosphate dehydrogenase, β-tubulin and two types of ATPases; mRNA accumulation of the glyceraldehyde-3-phosphate dehydrogenase gene was analyzed further. This is the first report of identification of expressed arbuscular mycorrhizal fungal genes in spores.

RNA accumulation and genes expressed in spores of the arbuscular mycorrhizal fungus, Gigaspora rosea

RNA could be extracted from ungerminated spores of the arbuscular mycorrhizal fungus Gigaspora rosea with a maximum of yield 7 days after isolation from soil. cDNA was synthesized and used for library construction or as template for the amplification by polymerase chain reaction of fragments corresponding to several genes. Analysis of cDNA clones corresponding to fragments of ribosomal RNA genes indicated that the extracted RNA as starting material was of arbuscular mycorrhizal fungal origin. Polymerase chain reaction showed the presence of mRNA from genes encoding glyceraldehyde-3-phosphate dehydrogenase, β-tubulin and two types of ATPases; mRNA accumulation of the glyceraldehyde-3-phosphate dehydrogenase gene was analyzed further. This is the first report of identification of expressed arbuscular mycorrhizal fungal genes in spores.