Rhizophagus irregularis, the model fungus in arbuscular mycorrhiza research, forms dimorphic spores.

Rhizophagus irregularis is the model species for arbuscular mycorrhizal fungi (AMF) research and the most widely propagated species for commercial plant biostimulants. Using asymbiotic and symbiotic cultivation systems initiated from single spores, advanced microscopy, Sanger sequencing of the glomalin gene, and PacBio sequencing of the partial 45S rRNA gene, we show that four strains of R. irregularis produce spores of two distinct morphotypes, one corresponding to the morphotype described in the R. irregularis protologue and the other having the phenotype of R. fasciculatus. The two spore morphs are easily distinguished by spore colour, thickness of the subtending hypha, thickness of the second wall layer, lamination of the innermost layer, and the dextrinoid reaction of the two outer spore wall layers to Melzer's reagent. The glomalin gene of the two spore morphs is identical and that of the PacBio sequences of the partial SSU-ITS-LSU region (2780 bp) obtained from single spores of the R. cf fasciculatus morphotype has a median pairwise similarity of 99.8% (SD = 0.005%) to the rDNA ribotypes of R. irregularis DAOM 197198. Based on these results, we conclude that the model AMF species R. irregularis is dimorphic, which has caused taxonomic confusion in culture collections and possibly in AMF research.

Inoculum Sources Modulate Mycorrhizal Inoculation Effect on Tamarix articulata Development and Its Associated Rhizosphere Microbiota.

(1) Background: Soil degradation is an increasingly important problem in many parts of the world, particularly in arid and semiarid areas. Arbuscular mycorrhizal fungi (AMF) isolated from arid soils are recognized to be better adapted to these edaphoclimatic conditions than exogenous ones. Nevertheless, little is known about the importance of AMF inoculum sources on Tamarix articulata development in natural saline soils. Therefore, the current study aims at investigating the efficiency of two AMF-mixed inoculums on T. articulata growth, with consideration of its rhizosphere microbiota. (2) Methods: indigenous inoculum made of strains originating from saline soils and a commercial one were used to inoculate T. articulata in four saline soils with different salinity levels under microcosm conditions with evaluation of rhizosphere microbial biomasses. (3) Results: Our findings showed that indigenous inoculum outperforms the commercial one by 80% for the mycorrhizal rate and 40% for plant biomasses, which are correlated with increasing shoot phosphorus content. Soil microbial biomasses increased significantly with indigenous mycorrhizal inoculum in the most saline soil with 46% for AMF, 25% for saprotrophic fungi and 15% for bacterial biomasses. (4) Conclusion: Present results open the way towards the preferential use of mycorrhizal inoculum, based on native AMF, to perform revegetation and to restore the saline soil microbiota.

Nuclear Dynamics in the Arbuscular Mycorrhizal Fungi.

Arbuscular mycorrhizal fungi (AMF) are plant root symbionts that continuously carry thousands of nuclei in their spores and hyphae. This unique cellular biology raises fundamental questions regarding their nuclear dynamics. This review aims to address these by synthesizing current knowledge of nuclear content and behavior in these ubiquitous soil fungi. Overall, we find that that nuclear counts, as well as the nuclei shape and organization, vary drastically both within and among species in this group. By comparing these features with those of other fungi, we highlight unique aspects of the AMF nuclear biology that require further attention. The potential implications of the observed nuclear variability for the biology and evolution of these widespread plant symbionts are discussed.

Ultra-low input transcriptomics reveal the spore functional content and phylogenetic affiliations of poorly studied arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal fungi (AMF) are a group of soil microorganisms that establish symbioses with the vast majority of land plants. To date, generation of AMF coding information has been limited to model genera that grow well axenically; Rhizoglomus and Gigaspora. Meanwhile, data on the functional gene repertoire of most AMF families is non-existent. Here, we provide primary large-scale transcriptome data from eight poorly studied AMF species (Acaulospora morrowiae, Diversispora versiforme, Scutellospora calospora, Racocetra castanea, Paraglomus brasilianum, Ambispora leptoticha, Claroideoglomus claroideum and Funneliformis mosseae) using ultra-low input ribonucleic acid (RNA)-seq approaches. Our analyses reveals that quiescent spores of many AMF species harbour a diverse functional diversity and solidify known evolutionary relationships within the group. Our findings demonstrate that RNA-seq data obtained from low-input RNA are reliable in comparison to conventional RNA-seq experiments. Thus, our methodology can potentially be used to deepen our understanding of fungal microbial function and phylogeny using minute amounts of RNA material.

Impact of soil salinity on arbuscular mycorrhizal fungi biodiversity and microflora biomass associated with Tamarix articulata Vahll rhizosphere in arid and semi-arid Algerian areas.

Soil salinization is an increasingly important problem in many parts of the world, particularly under arid and semi-arid areas. Unfortunately, the knowledge about restoration of salt affected ecosystems using mycorrhizae is limited. The current study aims to investigate the impact of salinity on the microbial richness of the halophytic plant Tamarix articulata rhizosphere. Soil samples were collected from natural sites with increasing salinity (1.82-4.95 ds.m(-1)). Six arbuscular mycorrhizal fungi (AMF) species were isolated from the different saline soils and identified as Septoglomus constrictum, Funneliformis mosseae, Funneliformis geosporum, Funneliformis coronatum, Rhizophagus fasciculatus, and Gigaspora gigantea. The number of AMF spores increased with soil salinity. Total root colonization rate decreased from 65 to 16% but remained possible with soil salinity. Microbial biomass in T. articulata rhizosphere was affected by salinity. The phospholipid fatty acids (PLFA) C16:1ω5 as well as i15:0, a15:0, i16:0, i17:0, a17:0, cy17:0, C18:1ω7 and cy19:0 increased in high saline soils suggesting that AMF and bacterial biomasses increased with salinity. In contrast, ergosterol amount was negatively correlated with soil salinity indicating that ectomycorrhizal and saprotrophic fungal biomasses were reduced with salinity. Our findings highlight the adaptation of arbuscular and bacterial communities to natural soil salinity and thus the potential use of mycorrhizal T. articulata trees as an approach to restore moderately saline disturbed arid lands.

Arbuscular mycorrhizal fungal inoculation protects Miscanthus × giganteus against trace element toxicity in a highly metal-contaminated site.

Arbuscular mycorrhizal fungus (AMF)-assisted phytoremediation could constitute an ecological and economic method in polluted soil rehabilitation programs. The aim of this work was to characterize the trace element (TE) phytoremediation potential of mycorrhizal Miscanthus × giganteus. To understand the mechanisms involved in arbuscular mycorrhizal symbiosis tolerance to TE toxicity, the fatty acid compositions and several stress oxidative biomarkers were compared in the roots and leaves of Miscanthus × giganteus cultivated under field conditions in either TE-contaminated or control soils. TEs were accumulated in greater amounts in roots, but the leaves were the organ most affected by TE contamination and were characterized by a strong decrease in fatty acid contents. TE-induced oxidative stress in leaves was confirmed by an increase in the lipid peroxidation biomarker malondialdehyde (MDA). TE contamination decreased the GSSG/GSH ratio in the leaves of exposed plants, while peroxidase (PO) and superoxide dismutase (SOD) activities were increased in leaves and in whole plants, respectively. AMF inoculation also increased root colonization in the presence of TE contamination. The mycorrhizal colonization determined a decrease in SOD activity in the whole plant and PO activities in leaves and induced a significant increase in the fatty acid content in leaves and a decrease in MDA formation in whole plants. These results suggested that mycorrhization is able to confer protection against oxidative stress induced by soil pollution. Our findings suggest that mycorrhizal inoculation could be used as a bioaugmentation technique, facilitating Miscanthus cultivation on highly TE-contaminated soil.

Extreme diversification of the mating type-high-mobility group (MATA-HMG) gene family in a plant-associated arbuscular mycorrhizal fungus.

Arbuscular mycorrhizal fungi (AMF) are important plant symbionts that have long been considered evolutionary anomalies because of their apparent long-term lack of sexuality, but recent explorations of available DNA sequence have challenged this notion by revealing the presence of homologues of fungal mating type-high-mobility group (MATA-HMG) and core meiotic genes in these organisms. To obtain more insights into the sexual potential of AMF, homologues of MATA-HMGs were sought in the transcriptome of three AMF isolates, and their functional and evolutionary trajectories were studied in genetically divergent strains of Rhizophagus irregularis using conventional and quantitative PCR procedures. Our analyses revealed the presence of at least 76 homologues of MATA-HMGs in R. irregularis isolates. None of these was found to be surrounded by genes generally found near other known fungal mating type loci, but here we report the presence of a 9-kb-long region in the AMF R. irregularis harbouring a total of four tandem-repeated MATA-HMGs; a feature that highlights a potentially elevated intragenomic diversity in this AMF species. The present study provides intriguing insights into the genome evolution of R. irregularis, and represents a stepping stone for understanding the potential of these fungi to undergo cryptic sex.

Microwave-assisted technology for the clearing and staining of arbuscular mycorrhizal fungi in roots.

The use of microwave irradiation as a source of energy to clear and stain intra-radical arbuscular mycorrhizal fungi propagules has been tested on a variety of indigenous and cultivated herbaceous plants. The aim of the study was to evaluate the efficiency of microwave irradiation on root softening, fungi tissue staining, and preservation of DNA integrity for subsequent molecular analyses. The proposed methodology has been adapted from the standard procedures used to detect and quantify mycorrhizal root colonization levels. Using a domestic microwave oven, tissue clearing and staining required together between 30 s and 1.5 min of microwave treatment to be completed, depending the diameter size of the roots. The well-performing chemical stains tested were acid fuchsin, trypan blue, and aniline blue. The acid fuchsin clearing and staining processes, as performed, were also demonstrated to preserve DNA integrity for further molecular analyses. Irradiation by microwaves has been used with success in our laboratory within the frame of several studies. It offers considerable time saving over traditional method, reducing processing times from several hours to a few minutes while decreasing considerably the amount of chemicals and energy required to perform analyses.

Spore development and nuclear inheritance in arbuscular mycorrhizal fungi.

BACKGROUND: A conventional tenet of classical genetics is that progeny inherit half their genome from each parent in sexual reproduction instead of the complete genome transferred to each daughter during asexual reproduction. The transmission of hereditary characteristics from parents to their offspring is therefore predictable, although several exceptions are known. Heredity in microorganisms, however, can be very complex, and even unknown as is the case for coenocytic organisms such as Arbuscular Mycorrhizal Fungi (AMF). This group of fungi are plant-root symbionts, ubiquitous in most ecosystems, which reproduce asexually via multinucleate spores for which sexuality has not yet been observed. RESULTS: We examined the number of nuclei per spore of four AMF taxa using high Z-resolution live confocal microscopy and found that the number of nuclei was correlated with spore diameter. We show that AMF have the ability, through the establishment of new symbioses, to pass hundreds of nuclei to subsequent generations of multinucleated spores. More importantly, we observed surprising heterogeneity in the number of nuclei among sister spores and show that massive nuclear migration and mitosis are the mechanisms by which AMF spores are formed. We followed spore development of Glomus irregulare from hyphal swelling to spore maturity and found that the spores reached mature size within 30 to 60 days, and that the number of nuclei per spores increased over time. CONCLUSIONS: We conclude that the spores used for dispersal of AMF contain nuclei with two origins, those that migrate into the spore and those that arise by mitosis in the spore. Therefore, these spores do not represent a stage in the life cycle with a single nucleus, raising the possibility that AMF, unlike all other known eukaryotic organisms, lack the genetic bottleneck of a single-nucleus stage.

Phosphate transporter genes as reliable gene markers for the identification and discrimination of arbuscular mycorrhizal fungi in the genus glomus.

An inorganic phosphate transporter gene sequence (852-bp section) allowed discrimination between 10 Glomus fungal species represented by 25 strains. It was particularly valuable in differentiating between morphologically similar species with nucleotide and amino acid sequence differences higher than 3%. This gene is proposed as a reliable barcode for the Glomeromycetes.

Copper release from chemical root-control baskets in hardwood tree production.

The City of Montreal, Canada, evaluated the environmental impact and usefulness of in-ground copper (Cu)-treated baskets in controlling root growth of hardwood trees in nursery culture. Using baskets planted with 5-yr-old Norway maple (Acer platanoides L.) trees, the amount and temporal pattern of Cu release from the basket surface into soil was determined for two copper formulations: Cu metal powder and Cu(OH)2. Release of both Cu formulations from the basket surface decreased exponentially over time, with Cu concentration at the basket surface dropping to 2% of the initial Cu applied by the end of the second field season. Total Cu content increased significantly in the soil around the baskets (from 7 to 28 mg Cu kg(-1) soil) and in the baskets (from 7 to 50-70 mg Cu kg(-1) soil) over the two years of the study. Three levels of phosphorus application (33, 66, and 100% of the regular nursery rate of 465 kg ha(-1) yr(-1)) did not affect release of Cu from the basket surface. The release of Cu metal at 28 and 105 d in the field was significantly increased by inoculation with the symbiotic arbuscular mycorrhizal fungus (AMF) Glomus intraradices Schenck & Smith; however, AMF inoculation had no affect on Cu(OH)2 release. Trees grown in Cu-treated baskets and inoculated with G. intraradices had similar colonization to non-inoculated trees, suggesting that inoculation was not very effective and that AMF inoculum was already present in the root ball of the trees at planting. After two years, copper basket-grown trees had significantly less root colonization than isolated control trees growing in the open field. This strongly suggests that conditions inside the baskets were not favorable to AMF.

Development of Acaulospora rehmii spore and hyphal swellings under root-organ culture.

A strain of Acaulospora rehmii was, for the first time, successfully grown in vitro on Ri T-DNA transformed carrot roots allowing the in situ observation of Acaulospora spore development and of extraradical thin-walled hyphal swellings. The sporogenous hypha developed intercalarly along thin-walled coenocytic hyphae. The distal part of the sporogenous hypha swelled slightly as a sporiferous saccule primordium followed by the differentiation of a lateral spore primordium along the neck of the sporogenous hypha. Both structures matured simultaneously, and the sporiferous saccule began to collapse after spore maturation and complete differentiation of the spore wall. Several of the in situ observations on in vitro differentiated A. rehmii spores are concordant with previous ontogenic studies done on other Acaulospora species obtained from in vivo cultures. New and original observations on the early developmental stages of sporiferous saccules and spores and on the occurrence of small diameter intercalary hyphal swellings provide additional elements in the study of Acaulospora sporulation process and life cycle.