Extraradical hyphae of the mycorrhizal fungus Glomus intraradices can hydrolyse organic phosphate

Organic phosphorus sources make up a large fraction of the total P in some soils. Vesicular–arbuscular mycorrhizal fungi provide a large surface area for the absorption of inorganic P. The question of whether or not they have direct access to organic P by producing extracellular phosphatases has hitherto been controversial because experiments had not been performed in the absence of other soil microorganisms. We used a split-dish in vitro carrot mycorrhiza system free from contaminating microorganisms. The extraradical hyphae of Glomus intraradices hydrolysed both 5-bromo-4-chloro-3-indolyl phosphate and phenolphthalein diphosphate. Moreover, they transferred significantly more P to roots when they had access to inositol hexaphosphoric acid (phytate) than when they did not. Thus we show unequivocally that extraradical hyphae of G. intraradices can hydrolyse organic P, and, further, that the resultant inorganic P can be taken up and transported to host roots.     

Thiols of Cu-treated maize plants inoculated with the arbuscular-mycorrhizal fungus Glomus intraradices

Mycorrhizal colonization of roots, fresh weight, content of cysteine, γ-glutamylcysteine (γEC). glutathione (GSH), thiol groups in Cu-binding peptides (CuBP), and the uptake of Cu were measured in roots and shoots of maize ( Zea mays L., cv. Honeycomb F-1) grown in quartz sand, with Cu at 0, 4.5, 9, 15 and 30 μg g⁻¹ added with or without inoculum of the arbuscular-mycorrhizal fungus (AMF) Glomus intraradices . In control plants (no Cu added) AMF significantly reduced shoot growth, but did not affect root growth. At an external Cu supply of 9 μg (g quartz sand)⁻¹ or higher, both mycorrhizal colonization and growth of roots and shoots of mycorrhizal and non-mycorrhizal plants were significantly reduced.
With up to 9 μg Cu g⁻¹, mycorrhizal colonization increased the content of cysteine, γEC and GSH in the roots. However, the amount of thiols in CuBPs was not increased by mycorrhizal colonization in Cu-treated plants and no differences in Cu uptake were detected between non-mycorrhizal and mycorrhizal plants. A CuBP-complex with a relative molecular mass of 7300 and a SH:Cu ratio of 1.77:1 was separated on a Sephadex G-50 column from both non-inoculated and inoculated roots of Cu-treated plants. HPLC chromatography of the CuBPs of both non-inoculated and inoculated roots resulted in a similar peak pattern, indicating that no additional CuBPs were formed by the fungus. In conclusion, our results do not support the idea that AMF protects maize from Cu-toxicity.     

Monoxenic in vitro production and colonization potential of AM fungus Glomus intraradices

The paper reports the establishment of mycorrhizal infection of a non-mycorrhizal Ri-T-DNA transformed carrot root when co-cultured with a surface sterilized sweet potato root segment colonized by arbuscular mycorrhizal (AM) fungus G. intraradices on minimal M medium. Extensive fungal hyphal emergence from each cut end of the mycorrhizal sweet potato root piece was observed in one week old cultures. These hyphae caused infection on contacting the transformed-carrot- root segment and produced many hyphae and spores both inside and outside the zone of the root after 6 week of growth. Axenically produced fungal propagules proliferated on the surface of fresh minimal M medium when sub-cultured without any root segment. On repeated sub-culturing, these propagules did not lose their ability to grow and produced many juvenile small spore-like vesicles during the non-symbiotic phase. Although these spores were morphologically and anatomically similar to their soil borne counter parts, they were much smaller. When placed in the vicinity of a fresh hairy root on the minimal medium or a Sudan grass seedling in sand culture, the axenically produced AM fungal propagules caused root infection, but the infection characteristics were significantly different to the original culture in terms of shape (spherical vs oval) and size (20 microm vs 45 microm) of the intraradical vesicles, and absence of 'H' branches. Sudan grass seedlings inoculated with the axenically cultured fungus showed significantly (P < 0.05) higher dry weights plant'. When compared to the plants inoculated with sand cultures, the growth parameters and the percentage infection were not significantly different. However, when both sources of inocula were used together, a synergistic effect on plant growth as well as root infection was observed.     

Breaking dormancy in spores of the arbuscular mycorrhizal fungus Glomus intraradices: a critical cold-storage period

To elucidate the effect of cold storage on spore dormancy in the arbuscular mycorrhizal (AM) fungus Glomus intraradices, spores were cold stratified at 4 degrees C, for either 0, 3, 7, 14, 90 or 120 days, prior to germination tests at 25 degrees C. The results showed that cold stratification longer than 14 days significantly increased spore germination. Moreover, the longer cold storage periods clearly reduced spore mortality from 90% to 50% and considerably altered the hyphal growth pattern. Long polarized hyphae were only observed after cold stratification periods longer than 14 days, involving consequences for root infectivity. The results clearly show that environmental factors, e.g., coldness, can affect the physiology of AM fungal spores.     

Recombination in Glomus intraradices, a supposed ancient asexual arbuscular mycorrhizal fungus

Background  

Arbuscular mycorrhizal fungi (AMF) are important symbionts of most plant species, promoting plant diversity and productivity. This symbiosis is thought to have contributed to the early colonisation of land by plants. Morphological stasis over 400 million years and the lack of an observed sexual stage in any member of the phylum Glomeromycota led to the controversial suggestion of AMF being ancients asexuals. Evidence for recombination in AMF is contradictory.     

Identification of heavy metal-induced genes encoding glutathione S-transferases in the arbuscular mycorrhizal fungus Glomus intraradices

Arbuscular mycorrhizal fungi are able to alleviate the stress for plants caused by heavy metal contamination of soil. To analyze the molecular response of arbuscular mycorrhizal fungi to these pollutants, a subtractive cDNA library was constructed using RNA from Glomus intraradices extraradical hyphae of a root organ culture treated with a mixture of Cd, Zn, and Cu. Screening by reverse Northern blot analysis indicated that, among 308 clones, 17% correspond to genes up-regulated by heavy metals. Sequence analysis of part of the clones resulted, amongst others, in the identification of six genes putatively coding for glutathione S-transferases belonging to two different classes of these enzymes. Expression analyses indicated that the genes are differentially expressed during fungal development and that their RNA accumulation dramatically increases in extraradical hyphae grown in a heavy metal-containing solution.     

Influence of the endomycorrhizal fungus Glomus mosseae on the development of peanut pod rot disease in Egypt

Glomus mosseae and the two pod rot pathogens Fusarium solani and Rhizoctonia solani and subsequent effects on growth and yield of peanut (Arachis hypogaea L.) plants were investigated in a greenhouse over a 5-month period. At plant maturity, inoculation with F. solani and/or R. solani significantly reduced shoot and root dry weights, pegs and pod number and seed weight of peanut plants. In contrast, the growth response and biomass of peanut plants inoculated with G. mosseae was significantly higher than that of non-mycorrhizal plants, both in the presence and absence of the pathogens. Plants inoculated with G. mosseae had a lower incidence of root rot, decayed pods, and death than non-mycorrhizal ones. The pathogens either alone or in combination reduced root colonization by the mycorrhizal fungus. Propagule numbers of each pathogen isolated from pod shell, seed, carpophore, lower stem and root were significantly lower in mycorrhizal plants than in the non-mycorrhizal plants. Thus, G. mosseae protected peanut plants from infection by pod rot fungal pathogens. Accepted: 10 February 2000     

Molecular characterization of chromosome termini of the arbuscular mycorrhizal fungus Glomus intraradices (Glomeromycota)

The minimum chromosome number of Glomus intraradices was assessed through cloning and sequencing of the highly divergent telomere-associated sequences (TAS) and by pulsed field gel electrophoresis (PFGE). The telomere of G. intraradices, as in other filamentous fungi, consists of TTAGGG repeats, this was confirmed using Bal31 nuclease time course reactions. Telomere length was estimated to be roughly 0.9 kb by Southern blots on genomic DNA and a telomere probe. We have identified six classes of cloned chromosomal termini based on the TAS. An unusually high genetic variation was observed within two of the six TAS classes. To further assess the total number of chromosome termini, we used telomere fingerprinting. Surprisingly, all hybridization patterns showed smears, which demonstrate that TAS are remarkably variable in the G. intraradices genome. These analyses predict the presence of at least three chromosomes in G. intraradices while PFGE showed a pattern of four bands ranging from 1.2 to 1.5 Mb. Taken together, our results indicate that there are at least four chromosomes in G. intraradices but there are probably more. The information on TAS and telomeres in the G. intradicies will be essential for making a physical map of the G. intraradices genome and could provide molecular markers for future studies of genetic variation among nuclei in these multigenomic fungi.     

Hydrogen peroxide accumulation in Medicago truncatula roots colonized by the arbuscular mycorrhiza-forming fungus Glomus intraradices

The diaminobenzidine (DAB) staining technique was used to examine the accumulation of H₂O₂ in parts of roots of Medicago truncatula Gaertn. colonized by the arbuscular mycorrhiza (AM)-forming fungus Glomus intraradices Schenk and Smith. At the cellular level, the combination of bright-field and fluorescence microscopy revealed that a brownish stain, indicative of H₂O₂ accumulation was present within cortical root cells in the space occupied by arbuscules. Accumulation of H₂O₂ was especially pronounced in cells containing arbuscules that were clumped and less branched. Moreover, H₂O₂ accumulated around hyphal tips attempting to penetrate a host cell. In contrast, no H₂O₂ accumulation was observed in hyphal tips growing along the middle lamella, or in appressoria or vesicles. On the basis of these findings we suggest that a locally restricted oxidative burst is involved in the temporal and spatial control of the intracellular colonization of M. truncatula cells by the AM-forming fungus G. intraradices. Received: 1 October 1998 / Accepted: 22 December 1998     

Fenpropimorph slows down the sterol pathway and the development of the arbuscular mycorrhizal fungus Glomus intraradices

The direct impact of fenpropimorph on the sterol biosynthesis pathway of Glomus intraradices when extraradical mycelia alone are in contact with the fungicide was investigated using monoxenic cultures. Bi-compartmental Petri plates allowed culture of mycorrhizal chicory roots in a compartment without fenpropimorph and exposure of extraradical hyphae to the presence of increasing concentrations of fenpropimorph (0, 0.02, 0.2, 2, 20 mg l⁻¹). In the fungal compartment, sporulation, hyphal growth, and fungal biomass were already reduced at the lowest fungicide concentration. A decrease in total sterols, in addition to an increase in the amount of squalene and no accumulation of abnormal sterols, suggests that the sterol pathway is severely slowed down or that squalene epoxidase was inhibited by fenpropimorph in G. intraradices. In the root compartment, neither extraradical and intraradical development of the arbuscular mycorrhizal (AM) fungus nor root growth was affected when they were not in direct contact with the fungicide; only hyphal length was significantly affected at 2 mg l⁻¹ of fenpropimorph. Our results clearly demonstrate a direct impact of fenpropimorph on the AM fungus by a perturbation of its sterol metabolism.     

Microsatellites for disentangling underground networks: strain-specific identification of Glomus intraradices, an arbuscular mycorrhizal fungus

The underground network of arbuscular mycorrhizal (AM) fungi is decisive for the above-ground diversity of many plant ecosystems, but tools to investigate the population structure of AM fungi are sorely lacking. Here, we present a bioinformatics approach to identify microsatellite markers in the AM fungus Glomus intraradices. Based on 1958 contigs of this fungus, assembled from public databases, we identified 842 microsatellites. One hundred of them were subjected to closer scrutiny by designing flanking primers and performing an extensive screen to identify polymorphic loci. We obtained 18 polymorphic microsatellite markers, and we found that seven out of eight individual single-spore cultures of G. intraradices could readily be identified by at least five allelic differences, as compared to all other strains. Two single-spore cultures, however, nominally originating from completely different locations, displayed identity at all 18 loci, suggesting with 99.999999% probability that they represent a single clone.     

GiFRD encodes a protein involved in anaerobic growth in the arbuscular mycorrhizal fungus Glomus intraradices

Fumarate reductase is a protein involved in the maintenance of redox balance during oxygen deficiency. This enzyme irreversibly catalyzes the reduction of fumarate to succinate and requires flavin cofactors as electron donors. Two examples are the soluble mitochondrial and the cytosolic fumarate reductases of Saccharomyces cerevisiae encoded by the OSM1 and FRDS1 genes, respectively. This work reports the identification and characterization of the gene encoding cytosolic fumarate reductase enzyme in the arbuscular mycorrhizal fungus, Glomus intraradices and the establishment of its physiological role. Using a yeast expression system, we demonstrate that G. intraradices GiFRD encodes a protein that has fumarate reductase activity which can functionally substitute for the S. cerevisiae fumarate reductases. Additionally, we showed that GiFRD transformants are not affected by presence of salt in medium, indicating that the presence of this gene has no effect on yeast behavior under osmotic stress. The fact that GiFRD expression and enzymatic activity was present only in asymbiotic stage confirmed existence of at least one anaerobic metabolic pathway in this phase of fungus life cycle. This suggests that the AMF behave as facultative anaerobes in the asymbiotic stage.