Genetic diversity and host plant preferences revealed by simple sequence repeat and mitochondrial markers in a population of the arbuscular mycorrhizal fungus Glomus intraradices

Arbuscular mycorrhizal fungi (AMF) are important symbionts of plants that improve plant nutrient acquisition and promote plant diversity. Although within-species genetic differences among AMF have been shown to differentially affect plant growth, very little is actually known about the degree of genetic diversity in AMF populations. This is largely because of difficulties in isolation and cultivation of the fungi in a clean system allowing reliable genotyping to be performed. A population of the arbuscular mycorrhizal fungus Glomus intraradices growing in an in vitro cultivation system was studied using newly developed simple sequence repeat (SSR), nuclear gene intron and mitochondrial ribosomal gene intron markers. The markers revealed a strong differentiation at the nuclear and mitochondrial level among isolates. Genotypes were nonrandomly distributed among four plots showing genetic subdivisions in the field. Meanwhile, identical genotypes were found in geographically distant locations. AMF genotypes showed significant preferences to different host plant species (Glycine max, Helianthus annuus and Allium porrum) used before the fungal in vitro culture establishment. Host plants in a field could provide a heterogeneous environment favouring certain genotypes. Such preferences may partly explain within-population patterns of genetic diversity.     

Phosphate uptake, transport and transfer by the arbuscular mycorrhizal fungus Glomus intraradices is stimulated by increased carbohydrate availability

* The influence of carbohydrate availability to mycorrhizal roots on uptake, metabolism and translocation of phosphate (P) by the fungus was examined in axenic cultures of transformed carrot (Daucus carota) roots in symbiosis with Glomus intraradices. * 14C-labelled carbohydrates, 33P-phosphate and energy dispersive X-ray microanalysis were used to follow the uptake and transfer of C and P in the arbuscular mycorrhizal (AM) symbiosis. * The uptake of P by the extraradical mycelium (ERM) and its translocation to the mycorrhizal roots was stimulated and the metabolic and spatial distribution of P within the fungus were altered in response to increased carbohydrate availability. Sucrose supply resulted in a decrease of polyphosphates and an increased incorporation into phospholipids and other growth-related P pools and also caused elevated cytoplasmic P levels in the intraradical mycelium (IRM) within the root and higher cytoplasmic P levels in the root cortex. * These findings indicate that the uptake of P by the fungus and its transfer to the host is also stimulated by the transfer of carbon from plant to fungus across the mycorrhizal interface.     

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.     

Intraradical dynamics of two coexisting isolates of the arbuscular mycorrhizal fungus Glomus intraradices sensu lato as estimated by real-time PCR of mitochondrial DNA

Real-time PCR in nuclear ribosomal DNA (nrDNA) is becoming a well-established tool for the quantification of arbuscular mycorrhizal (AM) fungi, but this genomic region does not allow the specific amplification of closely related genotypes. The large subunit of mitochondrial DNA (mtDNA) has a higher-resolution power, but mtDNA-based quantification has not been previously explored in AM fungi. We applied real-time PCR assays targeting the large subunit of mtDNA to monitor the DNA dynamics of two isolates of Glomus intraradices sensu lato coexisting in the roots of medic (Medicago sativa). The mtDNA-based quantification was compared to quantification in nrDNA. The ratio of copy numbers determined by the nrDNA- and mtDNA-based assays consistently differed between the two isolates. Within an isolate, copy numbers of the nuclear and the mitochondrial genes were closely correlated. The two quantification approaches revealed similar trends in the dynamics of both isolates, depending on whether they were inoculated alone or together. After 12 weeks of cultivation, competition between the two isolates was observed as a decrease in the mtDNA copy numbers of one of them. The coexistence of two closely related isolates, which cannot be discriminated by nrDNA-based assays, was thus identified as a factor influencing the dynamics of AM fungal DNA in roots. Taken together, the results of this study show that real-time PCR assays targeted to the large subunit of mtDNA may become useful tools for the study of coexisting AM fungi.     

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.     

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.     

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.     

Production and specificity of polyclonal antibodies against soluble proteins from the arbuscular mycorrhizal fungus Glomus intraradices

Rabbit polyclonal antibodies were produced against a soluble protein fraction from a vesicle and spore mixture of the arbuscular mycorrhizal fungus (AMF) Glomus intraradices. The protocol for isolation of vesicles and spores from plant roots was optimized to minimize debris contamination. Protein extract purification and preparation for immunization was adapted to increase protein content and immunogenicity. Active antisera were produced starting from the second boost immunization. Antibodies obtained were specific for surface antigens of AMF and revealed different patterns of soluble protein antigens in G. intraradices, G. constrictum and an unidentified Glomus species. Accepted: 6 December 2000     

The arbuscular mycorrhizal fungus Glomus intraradices is haploid and has a small genome size in the lower limit of eukaryotes

The genome size, complexity, and ploidy of the arbuscular mycorrhizal fungus (AMF) Glomus intraradices was determined using flow cytometry, reassociation kinetics, and genomic reconstruction. Nuclei of G. intraradices from in vitro culture, were analyzed by flow cytometry. The estimated average length of DNA per nucleus was 14.07+/-3.52 Mb. Reassociation kinetics on G. intraradices DNA indicated a haploid genome size of approximately 16.54 Mb, comprising 88.36% single copy DNA, 1.59% repetitive DNA, and 10.05% fold-back DNA. To determine ploidy, the DNA content per nucleus measured by flow cytometry was compared with the genome estimate of reassociation kinetics. G. intraradices was found to have a DNA index (DNA per nucleus per haploid genome size) of approximately 0.9, indicating that it is haploid. Genomic DNA of G. intraradices was also analyzed by genomic reconstruction using four genes (Malate synthase, RecA, Rad32, and Hsp88). Because we used flow cytometry and reassociation kinetics to reveal the genome size of G. intraradices and show that it is haploid, then a similar value for genome size should be found when using genomic reconstruction as long as the genes studied are single copy. The average genome size estimate was 15.74+/-1.69 Mb indicating that these four genes are single copy per haploid genome and per nucleus of G. intraradices. Our results show that the genome size of G. intraradices is much smaller than estimates of other AMF and that the unusually high within-spore genetic variation that is seen in this fungus cannot be due to high ploidy.     

The interface between the arbuscular mycorrhizal fungus Glomus intraradices and root cells of Panax quinquefolius: a Paris-type mycorrhizal association

Two major types of arbucular mycorrhizal associations, the Arum-type and the Paris-type, have been identified based on morphological features. Although the Paris-type is the most common, it is the Arum-type that has been most intensively studied in terms of structure/function because of its prevalence in agronomically important plant species. In this study, the interface between the host cell cytoplasm and intracellular hyphae (extensive hyphal coils and arbusculate coils), which typify the Paris-type mycorrhiza, was studied. Using immunofluorescence techniques combined with laser scanning confocal microscopy, dramatic changes in the cytoskeleton in colonized cells were observed. Changes in the positioning of both host cell microtubules and actin filaments occurred in colonized plant cells. Both microtubules and actin filaments were associated with the hyphal coils and the arbusculate coils. An interfacial matrix, of host origin, was demonstrated between hyphal coils and arbusculate coils using various affinity techniques. It formed an apoplastic compartment consisting of cellulose and pectins between the fungus and host cell cytoplasm. There was less labelling adjacent to the fine branches of arbusculate coils compared to the hyphal coils. These observations show some similarities to those seen with Arum-type mycorrhizas.     

Ultrastructural localization of heavy metals in the extraradical mycelium and spores of the arbuscular mycorrhizal fungus Glomus intraradices

Arbuscular mycorrhizal fungi, obligate symbionts of most plant species, are able to accumulate heavy metals, thereby, protecting plants from metal toxicity. In this study, the ultrastructural localization of Zn, Cu, and Cd in the extraradical mycelium and spores of the arbuscular mycorrhizal fungus Glomus intraradices grown in monoxenic cultures was investigated. Zinc, Cu, or Cd was applied to the extraradical mycelium to final concentrations of 7.5, 5.0, or 0.45 mmol/L, respectively. Samples were collected at time 0, 8 h, and 7 days after metal application and were prepared for rapid freezing and freeze substitution. Metal content in different subcellular locations (wall, cytoplasm, and vacuoles), both in hyphae and spores, was determined by energy-dispersive X-ray spectroscopy. In all treatments and fungal structures analysed, heavy metals accumulated mainly in the fungal cell wall and in the vacuoles, while minor changes in metal concentrations were detected in the cytoplasm. Incorporation of Zn into the fungus occurred during the first 8 h after metal addition with no subsequent accumulation. On the other hand, Cu steadily accumulated in the spore vacuoles over time, whereas Cd steadily accumulated in the hyphal vacuoles. These results suggest that binding of metals to the cell walls and compartmentalization in vacuoles may be essential mechanisms for metal detoxification.