Arbuscular Mycorrhizal Fungal Assemblages Significantly Shifted upon Bacterial Inoculation in Non-Contaminated and Petroleum-Contaminated Environments.

Arbuscular mycorrhizal fungi (AMF) have been shown to reduce plant stress and improve their health and growth, making them important components of the plant-root associated microbiome, especially in stressful conditions such as petroleum hydrocarbons (PHs) contaminated environments. Purposely manipulating the root-associated AMF assemblages in order to improve plant health and modulate their interaction with the rhizosphere microbes could lead to increased agricultural crop yields and phytoremediation performance by the host plant and its root-associated microbiota. In this study, we tested whether repeated inoculations with a Proteobacteria consortium influenced plant productivity and the AMF assemblages associated with the root and rhizosphere of four plant species growing either in non-contaminated natural soil or in sediments contaminated with petroleum hydrocarbons. A mesocosm experiment was performed in a randomized complete block design in four blocks with two factors: (1) substrate contamination (contaminated or not contaminated), and (2) inoculation (or not) with a bacterial consortium composed of ten isolates of Proteobacteria. Plants were grown in a greenhouse over four months, after which the effect of treatments on plant biomass and petroleum hydrocarbon concentrations in the substrate were determined. MiSeq amplicon sequencing, targeting the 18S rRNA gene, was used to assess AMF community structures in the roots and rhizosphere of plants growing in both contaminated and non-contaminated substrates. We also investigated the contribution of plant identity and biotope (plant roots and rhizospheric soil) in shaping the associated AMF assemblages. Our results showed that while inoculation caused a significant shift in AMF communities, the substrate contamination had a much stronger influence on their structure, followed by the biotope and plant identity to a lesser extent. Moreover, inoculation significantly increased plant biomass production and was associated with a decreased petroleum hydrocarbons dissipation in the contaminated soil. The outcome of this study provides knowledge on the factors influencing the diversity and community structure of AMF associated with indigenous plants following repeated inoculation of a bacterial consortium. It highlights the dominance of soil chemical properties, such as petroleum hydrocarbon presence, over biotic factors and inputs, such as plant species and microbial inoculations, in determining the plant-associated arbuscular mycorrhizal fungi communities.

Plant Identity Shaped Rhizospheric Microbial Communities More Strongly Than Bacterial Bioaugmentation in Petroleum Hydrocarbon-Polluted Sediments.

Manipulating the plant-root microbiota has the potential to reduce plant stress and promote their growth and production in harsh conditions. Community composition and activity of plant-roots microbiota can be either beneficial or deleterious to plant health. Shifting this equilibrium could then strongly affect plant productivity in anthropized areas. In this study, we tested whether repeated bioaugmentation with Proteobacteria influenced plant productivity and the microbial communities associated with the rhizosphere of four plant species growing in sediments contaminated with petroleum hydrocarbons (PHCs). A mesocosm experiment was performed in randomized block design with two factors: (1) presence or absence of four plants species collected from a sedimentation basin of a former petrochemical plant, and (2) bioaugmentation or not with a bacterial consortium composed of ten isolates of Proteobacteria. Plants were grown in a greenhouse over 4 months. MiSeq amplicon sequencing, targeting the bacterial 16S rRNA gene and the fungal ITS, was used to assess microbial community structures of sediments from planted or unplanted microcosms. Our results showed that while bioaugmentation caused a significant shift in microbial communities, presence of plant and their species identity had a stronger influence on the structure of the microbiome in PHCs contaminated sediments. The outcome of this study provides knowledge on the diversity and behavior of rhizosphere microbes associated with indigenous plants following repeated bioaugmentation, underlining the importance of plant selection in order to facilitate their efficient management, in order to accelerate processes of land reclamation.

Comparative genomics of Rhizophagus irregularis, R. cerebriforme, R. diaphanus and Gigaspora rosea highlights specific genetic features in Glomeromycotina.

Glomeromycotina is a lineage of early diverging fungi that establish arbuscular mycorrhizal (AM) symbiosis with land plants. Despite their major ecological role, the genetic basis of their obligate mutualism remains largely unknown, hindering our understanding of their evolution and biology. We compared the genomes of Glomerales (Rhizophagus irregularis, Rhizophagus diaphanus, Rhizophagus cerebriforme) and Diversisporales (Gigaspora rosea) species, together with those of saprotrophic Mucoromycota, to identify gene families and processes associated with these lineages and to understand the molecular underpinning of their symbiotic lifestyle. Genomic features in Glomeromycotina appear to be very similar with a very high content in transposons and protein-coding genes, extensive duplications of protein kinase genes, and loss of genes coding for lignocellulose degradation, thiamin biosynthesis and cytosolic fatty acid synthase. Most symbiosis-related genes in R. irregularis and G. rosea are specific to Glomeromycotina. We also confirmed that the present species have a homokaryotic genome organisation. The high interspecific diversity of Glomeromycotina gene repertoires, affecting all known protein domains, as well as symbiosis-related orphan genes, may explain the known adaptation of Glomeromycotina to a wide range of environmental settings. Our findings contribute to an increasingly detailed portrait of genomic features defining the biology of AM fungi.

Independent mitochondrial and nuclear exchanges arising in Rhizophagus irregularis crossed-isolates support the presence of a mitochondrial segregation mechanism.

BACKGROUND: Arbuscular mycorrhizal fungi (AMF) are members of the phylum Glomeromycota, an early divergent fungal lineage that forms symbiotic associations with the large majority of land plants. These organisms are asexual obligate biotrophs, meaning that they cannot complete their life cycle in the absence of a suitable host. These fungi can exchange genetic information through hyphal fusions (i.e. anastomosis) with genetically compatible isolates belonging to the same species. The occurrence of transient mitochondrial length-heteroplasmy through anastomosis between geographically distant Rhizophagus irregularis isolates was previously demonstrated in single spores resulting from crossing experiments. However, (1) the persistence of this phenomenon in monosporal culture lines from crossed parental isolates, (2) its correlation with nuclear exchanges and (3) the potential mechanisms responsible for mitochondrial inheritance are still unknown. Using the AMF model organism R. irregularis, we tested whether the presence of a heteroplasmic state in progeny spores was linked to the occurrence of nuclear exchanges and whether the previously observed heteroplasmic state persisted in monosporal in vitro crossed-culture lines. We also investigated the presence of a putative mitochondrial segregation apparatus in Glomeromycota by identifying proteins similar to those found in other fungal groups. RESULTS: We observed the occurrence of biparental inheritance both for mitochondrial and nuclear markers tested in single spores obtained from crossed-isolates. However, only one parental mitochondrial DNA and nuclear genotype were recovered in each monosporal crossed-cultures, with an overrepresentation of certain mitochondrial haplotypes. These results strongly support the presence of a nuclear-independent mitochondrial segregation mechanism in R. irregularis. Furthermore, a nearly complete set of genes was identified with putative orthology to those found in other fungi and known to be associated with the mitochondrial segregation in Saccharomyces cerevisiae and filamentous fungi. CONCLUSIONS: Our findings suggest that mitochondrial segregation might take place either during spore formation or colony development and that it might be independent of the nuclear segregation machinery. We present the basic building blocks for a better understanding of the mitochondrial inheritance process and segregation in these important symbiotic fungi. The comprehension of these processes is of great importance since it has been shown that different segregated lines of the same isolate can have variable effects on the host plant.

Culture-Dependent and -Independent Methods Capture Different Microbial Community Fractions in Hydrocarbon-Contaminated Soils.

Bioremediation is a cost-effective and sustainable approach for treating polluted soils, but our ability to improve on current bioremediation strategies depends on our ability to isolate microorganisms from these soils. Although culturing is widely used in bioremediation research and applications, it is unknown whether the composition of cultured isolates closely mirrors the indigenous microbial community from contaminated soils. To assess this, we paired culture-independent (454-pyrosequencing of total soil DNA) with culture-dependent (isolation using seven different growth media) techniques to analyse the bacterial and fungal communities from hydrocarbon-contaminated soils. Although bacterial and fungal rarefaction curves were saturated for both methods, only 2.4% and 8.2% of the bacterial and fungal OTUs, respectively, were shared between datasets. Isolated taxa increased the total recovered species richness by only 2% for bacteria and 5% for fungi. Interestingly, none of the bacteria that we isolated were representative of the major bacterial OTUs recovered by 454-pyrosequencing. Isolation of fungi was moderately more effective at capturing the dominant OTUs observed by culture-independent analysis, as 3 of 31 cultured fungal strains ranked among the 20 most abundant fungal OTUs in the 454-pyrosequencing dataset. This study is one of the most comprehensive comparisons of microbial communities from hydrocarbon-contaminated soils using both isolation and high-throughput sequencing methods.

Arbuscular mycorrhizal fungal diversity associated with Eleocharis obtusa and Panicum capillare growing in an extreme petroleum hydrocarbon-polluted sedimentation basin.

Arbuscular mycorrhizal fungi (AMF) have been extensively studied in natural and agricultural ecosystems, but little is known about their diversity and community structure in highly petroleum-polluted soils. In this study, we described an unexpected diversity of AMF in a sedimentation basin of a former petrochemical plant, in which petroleum hydrocarbon (PH) wastes were dumped for many decades. We used high-throughput PCR, cloning and sequencing of 18S rDNA to assess the molecular diversity of AMF associated with Eleocharis obtusa and Panicum capillare spontaneously inhabiting extremely PH-contaminated sediments. The analyses of rhizosphere and root samples over two years showed a remarkable AMF richness comparable with that found in temperate natural ecosystems. Twenty-one taxa, encompassing the major families within Glomeromycota, were detected. The most abundant OTUs belong to genera Claroideoglomus, Diversispora, Rhizophagus and Paraglomus. Both plants had very similar overall community structures and OTU abundances; however, AMF community structure differed when comparing the overall OTU distribution across the two years of sampling. This could be likely explained by variations in precipitations between 2011 and 2012. Our study provides the first view of AMF molecular diversity in soils extremely polluted by PH, and demonstrated the ability of AMF to colonize and establish in harsh environments.

Intraisolate mitochondrial genetic polymorphism and gene variants coexpression in arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal fungi (AMF) are multinucleated and coenocytic organisms, in which the extent of the intraisolate nuclear genetic variation has been a source of debate. Conversely, their mitochondrial genomes (mtDNAs) have appeared to be homogeneous within isolates in all next generation sequencing (NGS)-based studies. Although several lines of evidence have challenged mtDNA homogeneity in AMF, extensive survey to investigate intraisolate allelic diversity has not previously been undertaken. In this study, we used a conventional polymerase chain reaction -based approach on selected mitochondrial regions with a high-fidelity DNA polymerase, followed by cloning and Sanger sequencing. Two isolates of Rhizophagus irregularis were used, one cultivated in vitro for several generations (DAOM-197198) and the other recently isolated from the field (DAOM-242422). At different loci in both isolates, we found intraisolate allelic variation within the mtDNA and in a single copy nuclear marker, which highlighted the presence of several nonsynonymous mutations in protein coding genes. We confirmed that some of this variation persisted in the transcriptome, giving rise to at least four distinct nad4 transcripts in DAOM-197198. We also detected the presence of numerous mitochondrial DNA copies within nuclear genomes (numts), providing insights to understand this important evolutionary process in AMF. Our study reveals that genetic variation in Glomeromycota is higher than what had been previously assumed and also suggests that it could have been grossly underestimated in most NGS-based AMF studies, both in mitochondrial and nuclear genomes, due to the presence of low-level mutations.

Mitochondrial genome rearrangements in glomus species triggered by homologous recombination between distinct mtDNA haplotypes.

Comparative mitochondrial genomics of arbuscular mycorrhizal fungi (AMF) provide new avenues to overcome long-lasting obstacles that have hampered studies aimed at understanding the community structure, diversity, and evolution of these multinucleated and genetically polymorphic organisms.AMF mitochondrial (mt) genomes are homogeneous within isolates, and their intergenic regions harbor numerous mobile elements that have rapidly diverged, including homing endonuclease genes, small inverted repeats, and plasmid-related DNA polymerase genes (dpo), making them suitable targets for the development of reliable strain-specific markers. However, these elements may also lead to genome rearrangements through homologous recombination, although this has never previously been reported in this group of obligate symbiotic fungi. To investigate whether such rearrangements are present and caused by mobile elements in AMF, the mitochondrial genomes from two Glomeraceae members (i.e., Glomus cerebriforme and Glomus sp.) with substantial mtDNA synteny divergence,were sequenced and compared with available glomeromycotan mitochondrial genomes. We used an extensive nucleotide/protein similarity network-based approach to investigated podiversity in AMF as well as in other organisms for which sequences are publicly available. We provide strong evidence of dpo-induced inter-haplotype recombination, leading to a reshuffled mitochondrial genome in Glomus sp. These findings raise questions as to whether AMF single spore cultivations artificially underestimate mtDNA genetic diversity.We assessed potential dpo dispersal mechanisms in AMF and inferred a robust phylogenetic relationship with plant mitochondrial plasmids. Along with other indirect evidence, our analyses indicate that members of the Glomeromycota phylum are potential donors of mitochondrial plasmids to plants.

Detection of a transient mitochondrial DNA heteroplasmy in the progeny of crossed genetically divergent isolates of arbuscular mycorrhizal fungi.

Nonself fusion and nuclear genetic exchange have been documented in arbuscular mycorrhizal fungi (AMF), particularly in Rhizophagus irregularis. However, mitochondrial transmission accompanying nonself fusion of genetically divergent isolates remains unknown. Here, we tested the hypothesis that mitochondrial DNA (mtDNA) heteroplasmy occurs in the progeny of spores, obtained by crossing genetically divergent mtDNAs in R. irregularis isolates. Three isolates of geographically distant locations were used to investigate nonself fusions and mtDNA transmission to the progeny. We sequenced two additional mtDNAs of two R. irregularis isolates and developed isolate-specific size-variable markers in intergenic regions of these isolates and those of DAOM-197198. We achieved three crossing combinations in pre-symbiotic and symbiotic phases. Progeny spores per crossing combination were genotyped using isolate-specific markers. We found evidence that nonself recognition occurs between isolates originating from different continents both in pre-symbiotic and symbiotic phases. Genotyping patterns of individual spores from the progeny clearly showed the presence of markers of the two parental mtDNA haplotypes. Our results demonstrate that mtDNA heteroplasmy occurs in the progeny of the crossed isolates. However, this heteroplasmy appears to be a transient stage because all the live progeny spores that were able to germinate showed only one mtDNA haplotype.

Trichoderma harzianum might impact phosphorus transport by arbuscular mycorrhizal fungi.

Trichoderma sp. is a biocontrol agent active against plant pathogens via mechanisms such as mycoparasitism. Recently, it was demonstrated that Trichoderma harzianum was able to parasitize the mycelium of an arbuscular mycorrhizal (AM) fungus, thus affecting its viability. Here, we question whether this mycoparasitism may reduce the capacity of Glomus sp. to transport phosphorus ((33)P) to its host plant in an in vitro culture system. (33)P was measured in the plant and in the fungal mycelium in the presence/absence of T. harzianum. The viability and metabolic activity of the extraradical mycelium was measured via succinate dehydrogenase and alkaline phosphatase staining. Our study demonstrated an increased uptake of (33)P by the AM fungus in the presence of T. harzianum, possibly related to a stress reaction caused by mycoparasitism. In addition, the disruption of AM extraradical hyphae in the presence of T. harzianum affected the (33)P translocation within the AM fungal mycelium and consequently the transfer of (33)P to the host plant. The effects of T. harzianum on Glomus sp. may thus impact the growth and function of AM fungi and also indirectly plant performance by influencing the source-sink relationship between the two partners of the symbiosis.

Mycoparasitism of arbuscular mycorrhizal fungi: a pathway for the entry of saprotrophic fungi into roots.

Within the rhizosphere, arbuscular mycorrhizal (AM) fungi interact with a cohort of microorganisms, among which is the biological control agent, Trichoderma spp. This fungus parasitizes a wide range of phytopathogenic fungi, a phenomenon also reported in the extraradical mycelium (ERM) of AM fungi. Here, we question whether the mycoparasitism of the ERM could be extended to the intraradical mycelium (IRM), thus representing a pathway for the entry of Trichoderma harzianum within the root. Microcosm experiments allowing interactions between Glomus sp. MUCL 41833 placed in a clade that contains the recently described species Glomus irregulare and T. harzianum were set up under in vitro autotrophic culture conditions using potato as a host. A microscope camera-imaging system, coupled with succinate dehydrogenase staining, was used to assess the mycoparasitism in the ERM and IRM. Trichoderma harzianum colonized the ERM of the AM fungus and spread into the IRM, before exiting into the root cells. Intrahyphal growth of T. harzianum caused protoplasm degradation, decreasing the ERM and IRM viability. ERM of the AM fungus represented a pathway for the entry of T. harzianum into the roots of potato. It further sets off the debate on the susceptibility of the AM fungi of being infected by microorganisms from the rhizosphere.

Extraradical mycelium network of arbuscular mycorrhizal fungi allows fast colonization of seedlings under in vitro conditions.

Actively growing extraradical hyphae extending from mycorrhizal plants are an important source of inoculum in soils which has seldom been considered in vitro to inoculate young plantlets. Seedlings of Medicago truncatula were grown in vitro in the extraradical mycelium network extending from mycorrhizal plants. After 3, 6, 9, 12, and 15 days of contact with the mycelium, half of the seedlings were harvested and analyzed for root colonization. The other half was carefully transplanted in vitro on a suitable growth medium and mycelium growth and spore production were evaluated for 4 weeks. Seedlings were readily colonized after 3 days of contact with the mycelium. Starting from 6 days of contact, the newly colonized seedlings were able to reproduce the fungal life cycle, with the production of thousands of spores within 4 weeks. The fast mycorrhization process developed here opens the door to a broad range of in vitro studies for which either homogenous highly colonized seedlings or mass-produced in vitro inoculum is necessary.

Hyphal healing mechanism in the arbuscular mycorrhizal fungi Scutellospora reticulata and Glomus clarum differs in response to severe physical stress.

The hyphal healing mechanism (HHM) has been shown to differ between Gigasporaceae and Glomeraceae. However, this process has not been considered under (severe) physical stress conditions. Scutellospora reticulata and Glomus clarum strains were cultured monoxenically. The impact of long distance separating cut extremities of hyphae and of multiple injuries within hyphae on the HHM was monitored. For long distances (>5000 microm) separating cut extremities, hyphae healing was observed in half the cases in S. reticulata and was absent in G. clarum. For multiple-injured hyphae, the HHM was always oriented towards the complete recovery of hyphae integrity in S. reticulata, while in G. clarum, the growing hyphal tips (GHTs) could indifferently reconnect cut sections, by-pass cut sections or develop into the environment. Hyphae behaviour under severe physical stress clearly differentiated S. reticulata from G. clarum, suggesting that both fungi have developed different strategies for colony growth to survive under adverse conditions.

Arbuscular mycorrhizal fungi reveal distinct patterns of anastomosis formation and hyphal healing mechanisms between different phylogenic groups.

The significance of anastomosis formation and the hyphal healing mechanism (HHM) for functionality and integrity of the arbuscular mycorrhizal (AM) fungal mycelial network remains poorly documented. Four Glomeraceae and three Gigasporaceae were cultured monoxenically. Anastomosis formation was assessed using the grid line method, while HHM was time-lapse monitored. In intact mycelial networks, the number of anastomosis per hyphal length was higher for Glomeraceae than for Gigasporaceae strains. Glomeraceae strains studied always formed anastomosis between different hyphae, whereas anastomosis in the Gigasporaceae more often concerned hyphal bridges within the same hyphae. In both families the HHM corresponded to a four-step process; first septum formation; second initiation of growing hyphal tips (GHTs); third GHT elongation, orientation and contact; and fourth GHT fusion and cytoplasmic/protoplasmic flux re-establishment. These four steps differentiated Glomeraceae from Gigasporaceae. The type and number of anastomosis per hyphal length, and the HHM differed considerably between Glomeraceae and Gigasporaceae families representing a supplementary character that distinguishes these two families and may be of significance in ecological studies of AM fungi.