Unraveling the diversity of hyphal explorative traits among Rhizophagus irregularis genotypes.

Differences in functioning among various genotypes of arbuscular mycorrhizal (AM) fungi can determine their fitness under specific environmental conditions, although knowledge of the underlying mechanisms still is very fragmented. Here we compared seven homokaryotic isolates (genotypes) of Rhizophagus irregularis, aiming to characterize the range of intraspecific variability with respect to hyphal exploration of organic nitrogen (N) resources, and N supply to plants. To this end we established two experiments (one in vitro and one in open pots) and used 15N-chitin as the isotopically labeled organic N source. In Experiment 1 (in vitro), mycelium of all AM fungal genotypes transferred a higher amount of 15N to the plants than the passive transfer of 15N measured in the non-mycorrhizal (NM) controls. Noticeably, certain genotypes (e.g., LPA9) showed higher extraradical mycelium biomass production but not necessarily greater 15N acquisition than the others. Experiment 2 (in pots) highlighted that some of the AM fungal genotypes (e.g., MA2, STSI) exhibited higher rates of targeted hyphal exploration of chitin-enriched zones, indicative of distinct N exploration patterns from the other genotypes. Importantly, there was a high congruence of hyphal exploration patterns between the two experiments (isolate STSI always showing highest efficiency of hyphal exploration and isolate L23/1 being consistently the lowest), despite very different (micro) environmental conditions in the two experiments. This study suggests possible strategies that AM fungal genotypes employ for efficient N acquisition, and how to measure them. Implications of such traits for local mycorrhizal community assembly still need to be understood.

Mycorrhizal response of Solanum tuberosum to homokaryotic versus dikaryotic arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal fungi (AMF) are obligate plant symbionts of most land plants. In these organisms, thousands of nuclei that are either genetically similar (homokaryotic) or derived from two distinct parents (dikaryotic) co-exist in a large syncytium. Here, we investigated the impact of these two nuclear organizations on the mycorrhizal response of potatoes (Solanum tuberosum) by inoculating four potato cultivars with eight Rhizophagus irregularis strains individually (four homokaryotic and four dikaryotic). By evaluating plant and fungal fitness-related traits four months post inoculation, we found that AMF genetic organization significantly affects the mycorrhizal response of host plants. Specifically, homokaryotic strains lead to higher total, shoot, and tuber biomass and a higher number of tubers, compared to dikaryotic strains. However, fungal fitness-related traits showed no clear differences between homokaryotic and dikaryotic strains. Nucleotype content analysis of single spores confirmed that the nucleotype ratio of AMF heterokaryon spores can shift depending on host identity. Together, these findings continue to highlight significant ecological differences derived from the two distinct genetic organizations in AMF.

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.

Early branching arbuscular mycorrhizal fungus Paraglomus occultum carries a small and repeat-poor genome compared to relatives in the Glomeromycotina.

The arbuscular mycorrhizal fungi (AMFs) are obligate root symbionts in the subphylum Glomeromycotina that can benefit land plants by increasing their soil nutrient uptake in exchange for photosynthetically fixed carbon sources. To date, annotated genome data from representatives of the AMF orders Glomerales, Diversisporales and Archaeosporales have shown that these organisms have large and highly repeated genomes, and no genes to produce sugars and fatty acids. This led to the hypothesis that the most recent common ancestor (MRCA) of Glomeromycotina was fully dependent on plants for nutrition. Here, we aimed to further test this hypothesis by obtaining annotated genome data from a member of the early diverging order Paraglomerales (Paraglomus occultum). Genome analyses showed this species carries a 39.6 Mb genome and considerably fewer genes and repeats compared to most AMF relatives with annotated genomes. Consistent with phylogenies based on ribosomal genes, our phylogenetic analyses suggest P. occultum as the earliest diverged branch within Glomeromycotina. Overall, our analyses support the view that the MRCA of Glomeromycotina carried hallmarks of obligate plant biotrophy. The small genome size and content of P. occultum could either reflect adaptive reductive processes affecting some early AMF lineages, or indicate that the high gene and repeat family diversity thought to drive AMF adaptability to host and environmental change was not an ancestral feature of these prominent plant symbionts.

The arbuscular mycorrhizal fungus Rhizophagus irregularis harmonizes nuclear dynamics in the presence of distinct abiotic factors.

Arbuscular mycorrhizal fungi (AMF) are widespread obligate root symbionts that assist plants in obtaining nutrients and protection against environmental stresses. In the model species Rhizophagus irregularis, heterokaryotic strains (AMF dikaryons) carry thousands of nuclei originating from two parental strains whose frequency varies depending on strains and host identity. Here, using digital droplet PCR, we demonstrate that surrounding abiotic factors (temperature, phosphorus, and pH) also change the nuclear dynamics of such strains in root organ cultures. Furthermore, when spatially separated portions of the AMF mycelium grow under different abiotic conditions, all the produced spores carry highly similar nuclear ratios. Overall, these findings demonstrate that abiotic stressors impact the nuclear organization of a widespread group of multinucleate plant symbionts, and reveal remarkable mechanisms of nuclear ratio harmonization across the mycelium in these prominent symbionts.

Homo- and Dikaryons of the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis Differ in Life History Strategy.

Arbuscular mycorrhizal fungi (AMF) are obligate plant symbionts that have the potential to improve crop yield. These multinucleate organisms are either "homokaryotic" or "dikaryotic". In AMF dikaryons, thousands of nuclei originating from two parental strains coexist in the same cytoplasm. In other fungi, homokaryotic and dikaryotic strains show distinct life history traits (LHTs), such as variation in growth rates and fitness. However, how such traits compare between dikaryons and homokaryons of AMF is unknown. To address this, we measured 20 LHT of four dikaryons and five homokaryons of the model fungus Rhizophagus irregularis across root organ cultures of three host plants (carrot, chicory, and tobacco). Our analyses show that dikaryons have clearly distinct life history strategies (LHSs) compared to homokaryons. In particular, spores of homokaryons germinate faster and to a higher proportion than dikaryons, whereas dikaryons grow significantly faster and create a more complex hyphal network irrespective of host plant species. Our study links AMF nuclear status with key LHT with possible implications for mycorrhizal symbiotic functioning.

Co-inoculation with arbuscular mycorrhizal fungi differing in carbon sink strength induces a synergistic effect in plant growth.

Arbuscular mycorrhizal (AM) fungi play a key role in determining ecosystem functionality. Understanding how diversity in the fungal community affects plant productivity is therefore an important question in ecology. Current research has focused on understanding the role of functional complementarity in the fungal community when the host plant faces multiple stress factors. Fewer studies, however, have investigated how variation in traits affecting nutrient exchange can impact the plant growth dynamics, even in the absence of environmental stressors. Combining experimental data and a mathematical model based on ordinary differential equations, we investigate the role played by carbon sink strength on plant productivity. We simulate and measure plant growth over time when the plant is associated with two fungal isolates with different carbon sink strength, and when the plant is in pairwise association with each of the isolates alone. Overall, our theoretical as well as our experimental results show that co-inoculation with fungi with different carbon sink strength can induce positive non-additive effects (or synergistic effects) in plant productivity. Fungi with high carbon sink strength are able to quickly establish a fungal community and increase the nutrient supply to the plant, with a consequent positive impact on plant growth rate. On the other side, fungi with low carbon sink strength inflict lower carbon costs to the host plant, and support maximal plant productivity once plant biomass is large. As AM fungi are widely used as organic fertilizers worldwide, our findings have important implications for restoration ecology and agricultural management.

Commercial arbuscular mycorrhizal fungal inoculant failed to establish in a vineyard despite priority advantage.

BACKGROUND: Arbuscular mycorrhizal (AM) fungi associate with most plants and can increase nutrient uptake. As a result, commercial inoculants called "biofertilizers" containing AM fungi have been developed and marketed to increase plant performance. However, successful establishment of these inoculants remains a challenge, and may be negatively impacted by competition with fungi already present (priority effects). Perennial agriculture may be more amenable if inoculants can be successfully established on crops prior to field planting. METHODS: Here, we inoculate grapevine (Vitis vinifera) with a commercial inoculant in three treatments designed to manipulate the strength and direction of priority effects and quantified the abundance of the fungal strain before and after introduction using droplet digital PCR (ddPCR). RESULTS: We found that the introduced strain did not establish in any treatment, even with priority advantage, and inoculated vines did not differ in performance from non-inoculated vines. Fungal abundance was not greater than in pre-inoculation soil samples during any of the five years sampled and may have been impaired by high available phosphorus levels in the soil. This study highlights the need to understand and evaluate how the management of the agricultural system will affect establishment before introduction of "biofertilizers", which is often unpredictable.

Host identity influences nuclear dynamics in arbuscular mycorrhizal fungi.

The arbuscular mycorrhizal fungi (AMF) are involved in one of the most ecologically important symbioses on the planet, occurring within the roots of most land plants.1 Knowledge of even basic elements of AM fungal biology is still poor, with the discovery that AMF may in fact have a sexual life cycle being only very recently reported.2-5 AMF produce asexual spores that contain up to several thousand individual haploid nuclei6 of either largely uniform genotypes (AMF homokaryons) or nuclei originating from two parental genotypes2-5 (AMF dikaryons or heterokaryons). In contrast to the sexual dikaryons in the phyla Ascomycota and Basidiomycota,7,8 in which pairs of nuclei coexist in single hyphal compartments, AMF dikaryons carry several thousand nuclei in a coenocytic mycelium. Here, we set out to better understand the dynamics of this unique multinucleate condition by combining molecular analyses with advanced microscopy and modeling. Herein, we report that select AMF dikaryotic strains carry the distinct nucleotypes in equal proportions to one another, whereas others show an unequal distribution of parental nucleotypes. In both cases, the relative proportions within a given strain are inherently stable. Simulation models suggest that AMF dikaryons may be maintained through nuclear cooperation dynamics. Remarkably, we report that these nuclear ratios shift dramatically in response to plant host identity, revealing a previously unknown layer of genetic complexity and dynamism within the intimate interactions that occur between the partners of a prominent terrestrial symbiosis.

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.

Parasexual and Sexual Reproduction in Arbuscular Mycorrhizal Fungi: Room for Both.

Arbuscular mycorrhizal fungi (AMF) harbor thousands of nuclei in a large syncytium at all times. Although mating processes have not been observed in AMF, their cells and genomes show many signatures of sexual reproduction. Here, we describe how some of these signatures could also arise from parasexual processes in these widespread plant symbionts. As such, parasexual and sexual evolution could both be at play in generating nuclear diversity in AMF.

Codependency between plant and arbuscular mycorrhizal fungal communities: what is the evidence?

That arbuscular mycorrhizal (AM) fungi covary with plant communities is clear, and many papers report nonrandom associations between symbiotic partners. However, these studies do not test the causal relationship, or 'codependency', whereby the composition of one guild affects the composition of the other. Here we outline underlying requirements for codependency, compare important drivers for both plant and AM fungal communities, and assess how host preference - a pre-requisite for codependency - changes across spatiotemporal scales and taxonomic resolution for both plants and AM fungi. We find few examples in the literature designed to test for codependency and those that do have been conducted within plots or mesocosms. Also, while plants and AM fungi respond similarly to coarse environmental filters, most variation remains unexplained, with host identity explaining less than 30% of the variation in AM fungal communities. These results combined question the likelihood of predictable co-occurrence, and therefore evolution of codependency, between plant and AM fungal taxa across locations. We argue that codependency is most likely to occur in homogeneous environments where specific plant - AM fungal pairings have functional consequences for the symbiosis. We end by outlining critical aspects to consider moving forward.

In vitro Propagation of Arbuscular Mycorrhizal Fungi May Drive Fungal Evolution.

Transformed root cultures (TRC) are used to mass produce arbuscular mycorrhizal (AM) fungal propagules in vitro. These propagules are then used in research, agriculture, and ecological restoration. There are many examples from other microbial systems that long-term in vitro propagation leads to domesticated strains that differ genetically and functionally. Here, we discuss potential consequences of in TRC propagation on AM fungal traits, and how this may affect their functionality. We examine weather domestication of AM fungi has already happened and finally, we explore whether it is possible to overcome TRC-induced domestication.

Contrasting common measures of arbuscular mycorrhizal fungal root colonization.

Estimating the abundance of arbuscular mycorrhizal fungi relies entirely on indirect methods, meaning all measures are associated with some variability. The most common methods use microscopic estimates of the relative proportion of root length colonized by fungal structures. These methods typically examine root subsamples. While such methods are inexpensive and relatively simple, significant variation within single root system means there is opportunity for sampling bias. We evaluated the two most common methods of percent root length colonization for AM fungi both as a subsample and for the entire root system of flax plants. We compared these measures to a novel technique that returns projected fungal surface area (fungal coverage), by using microphotography and imaging analysis. Both microscopic methods overestimated the colonization intensity compared to image analysis. Among the microscopic methods, the method which incorporated colonization intensity (Trouvelot) was significantly more similar to imaging method results, than the method that is based on the presence/absence of the fungus (McGonigle).

The role of in vitro cultivation on symbiotic trait and function variation in a single species of arbuscular mycorrhizal fungus.

In vitro propagation of AM fungi using transformed root cultures (TRC) is commonly used to obtain pure AM fungal propagules for use in research and industry. Early observations indicate that such an artificial environment can alter traits and function of AM fungi over time. We hypothesized that increased in vitro cultivation may promote ruderal strategies in fungi by enhancing propagule production and reducing mutualistic quality. To examine the effect of in vitro cultivation on the trait and function of AM fungi, we inoculated plants with 11 Rhizoglomus irregulare isolates which fell along a cultivation gradient spanning 80 generations. We harvested plants at 10, 20 and 30 d post inoculation to observe differences in fungal and plant traits post infection. In vitro cultivation led to increased spore production but reduced plant shoot phosphorus. Our results indicate that in vitro propagation may indirectly select for traits that affect symbiotic quality.

Mycorrhizal response in crop versus wild plants.

We proposed a theoretical framework predicting mutualistic outcomes for the arbuscular mycorrhizal (AM) symbiosis based on host provenance (crop versus wild). To test the framework, we grew two isolates of Rhizoglomus irregulare (commercial versus an isolate locally isolated), with five crop plants and five wild plants endemic to the region that co-occur with the locally sourced fungus. While inoculation with either isolate had no effect on plant biomass, it decreased leaf P content, particularly for wild plants. All plants associating with the commercial fungus had lower leaf P. Overall, our data shows that wild plants may be more sensitive to differences in mutualistic quality among fungal isolates.

Accumulation of heavy metals by wild edible mushrooms with respect to soil substrates in the Athens metropolitan area (Greece).

Six wild edible mushroom species, Agaricus bisporus, A. bitorquis, A. gennadii, Coprinus comatus, Psathyrella candolleana and Volvopluteus gloiocephalus, were collected from the Greater Athens area (Greece), together with their soil substrates (two depth-layers) for studying bioaccumulation of heavy metals in a densely populated urban environment. Total and bioavailable Cr, Cu, Fe, Mn, Ni, Pb and Zn concentrations in soils were assessed along with their respective concentrations in mushrooms, and were evaluated in conjunction with soil properties, including K, P and Na content, CaCO3 equivalent percentage, mechanical composition, pH and organic matter. In particular, Cu, Pb, Zn and Ni displayed a high variability in their total and bioavailable concentrations measured in the upper soil layer. Relatively high Pb and Ni contents were measured in mushrooms, while Cu, Zn, Fe and Mn concentrations varied considerably. No significant correlations were detected between total concentrations of heavy metals in soils and mushrooms, whereas bioavailable fractions for several metals were significantly correlated with their respective content in A. bisporus, C. comatus, P. candolleana and V. gloiocephalus. K, Na and P concentrations in soils were associated to the content of several metals in fruit-bodies. The effect of soil texture on metals accumulation by mushrooms was species-dependent since high correlations were found for V. gloiocephalus and C. comatus only. Interactions between metals content in fruit-bodies seem to be species-specific except for Ni vs. Fe and Mn vs. Fe, which are positively correlated in all cases. Overaccumulation of metals in fruit-bodies was established only in respect to the bioavailable fractions of Cu, Mn, Zn and Ni in soil. The levels of toxicity for Pb were exceeded in mushrooms of five species, whereas the rest of the heavy metals (with the exception of Cu) were detected at lower contents than their recommended dietary allowances or tolerable upper intake levels.

The role of in vitro cultivation on asymbiotic trait variation in a single species of arbuscular mycorrhizal fungus.

Cultivating arbuscular mycorrhizal (AM) fungi in vitro is an efficient way to produce material for industry and research. However, such artificial growing conditions may impose selective pressure on fungi grown in vitro over many generations. We hypothesized that isolates subjected to long term propagation in vitro may develop increasingly ruderal traits. We proposed a predictive framework for the effect of in vitro cultivation on asymbiotic AM fungal traits. Using photomicrography and image processing, we analyzed morphology and growth traits for 14 isolates representing an in vitro cultivation gradient from 0 to >80 generations in vitro. We investigated the range of trait variation among asymbiotic growth of arbuscular mycorrhizal (AM) fungus isolates (Rhizoglomus irregulare). Spore dormancy was strongly associated with in vitro cultivation. We observed extremely high levels of inter-isolate variation for most fungal traits, but this was not related to time in vitro. Our results indicate that intra-specific diversity may have a strong ecological role in AM fungal communities.

Site specificity in establishment of a commercial arbuscular mycorrhizal fungal inoculant.

While establishment and persistence of arbuscular mycorrhizal (AM) fungal inoculants in agricultural fields are critical to their success, little is known about how farming practices can affect their establishment in field. We developed a probe assay specific to a commercial AM fungal inoculant (Rhizoglomus irregulare DAOM197198) and tested its establishment among different grain cropping practices in the field. Establishment of the fungus was not related to cropping, or inoculation practices. Instead, establishment was site specific over the two growing seasons. Our results show that it is not yet possible to predict inoculation success in the field and use of biofertilizers requires further research under field conditions to identify key factors involved in establishment and persistence.