Diversity and species composition of arbuscular mycorrhizal fungi across maize fields in the southern part of Belgium.

Arbuscular mycorrhizal fungi (AMF) are key actors among soil microbial inhabitants, forming beneficial associations with most horticultural plants and crops (e.g., maize). For maize, the world most cultivated cereal, data on AMF species diversity in fields is sparse and even totally nonexistent in the southern part of Belgium where maize represents 8% of the cultivated area. In the present study, 14 maize fields in South Belgium under conventional, conversion, or organic management were analyzed for AMF diversity and species composition using 454 pyrosequencing. A large part (54%) of the 49 AMF species observed were unknown or have not been described in the literature. AMF diversity highly varied among fields, with the number of species ranging between 1 and 37 according to the field. A statistically significant effect of management was measured on AMF diversity, with the highest Hill index values (diversity and richness) under the organic management system compared with conventional management or conversion. Our results suggest a positive effects of organic management on AMF diversity in maize. They also highlight the rather high diversity or richness of AMF and the large portion of sequences not yet ascribed to species, thereby emphasizing a need to intensify AMF identification in cropping systems.

Common Mycorrhizal Network Induced JA/ET Genes Expression in Healthy Potato Plants Connected to Potato Plants Infected by Phytophthora infestans.

Most plants are connected belowground via common mycorrhizal networks (CMNs). In their presence, the transmission of warning signals from diseased to uninfected plants has been reported. However, current studies have all been conducted in pots making it difficult to discriminate direct from indirect contribution of hyphae to the transmission of the signals. Here, we conducted an in vitro study with potato plantlets connected by a CMN of the arbuscular mycorrhizal fungus Rhizophagus irregularis. The plantlets were grown in physically separated compartments and their connection ensured only by the CMN. The donor potato plantlets were infected by Phytophthora infestans and defense genes analyzed 24, 48 and 120 h post-infection (hpi) in the uninfected receiver potato plantlets. Twenty-four hpi by the pathogen, PAL, PR-1b, ERF3, and LOX genes were significantly upregulated, whereas no significant transcript variation was noticed 48 and 120 hpi. The exact nature of the warning signals remains unknown but was not associated to microorganisms other than the AMF or to diffusion mechanisms through the growth medium or induced by volatile compounds. The defense response appeared to be transitory and associated with the jasmonic acid or ethylene pathway. These findings demonstrate the direct involvement of hyphae in the transmission of warning signals from diseased to uninfected potato plantlets and their indubitable role in providing a route for activating defense responses in uninfected plants.

Community composition of arbuscular mycorrhizal fungi associated with native plants growing in a petroleum-polluted soil of the Amazon region of Ecuador.

Arbuscular mycorrhizal fungi (AMF) are worldwide distributed plant symbionts. However, their occurrence in hydrocarbon-polluted environments is less investigated, although specific communities may be present with possible interest for remediation strategies. Here, we investigated the AMF community composition associated with the roots of diverse plant species naturally recolonizing a weathered crude oil pond in the Amazon region of Ecuador. Next generation 454 GS-Junior sequencing of an 800 bp LSU rRNA gene PCR amplicon was used. PCR amplicons were affiliated to a maximum-likelihood phylogenetic tree computed from 1.5 kb AMF reference sequences. A high throughput phylogenetic annotation approach, using an evolutionary placement algorithm (EPA) allowed the characterization of sequences to the species level. Fifteen species were detected. Acaulospora species were identified as dominant colonizers, with 73% of relative read abundance, Archaeospora (19.6%) and several genera from the Glomeraceae (Rhizophagus, Glomus macrocarpum-like, Sclerocystis, Dominikia and Kamienskia) were also detected. Although, a diverse community belonging to Glomeraceae was revealed, they represented <10% of the relative abundance in the Pond. Seventy five % of the species could not be identified, suggesting possible new species associated with roots of plants under highly hydrocarbon-polluted conditions.

Transcriptional Changes in Mycorrhizal and Nonmycorrhizal Soybean Plants upon Infection with the Fungal Pathogen Macrophomina phaseolina.

Macrophomina phaseolina is a soil-borne fungal pathogen with a wide host range that causes charcoal rot in soybean [Glycine max (L.) Merr.]. Control of the disease is a challenge, due to the absence of genetic resistance and effective chemical control. Alternative or complementary measures are needed, such as the use of biological control agents, in an integrated approach. Several studies have demonstrated the role of arbuscular mycorrhizal fungi (AMF) in enhancing plant resistance or tolerance to biotic stresses, decreasing the symptoms and pressure caused by various pests and diseases, including M. phaseolina in soybean. However, the specific contribution of AMF in the regulation of the plant response to M. phaseolina remains unclear. Therefore, the objective of the present study was to investigate, under strict in-vitro culture conditions, the global transcriptional changes in roots of premycorrhized soybean plantlets challenged by M. phaseolina (+AMF+Mp) as compared with nonmycorrhizal soybean plantlets (-AMF+Mp). MapMan software was used to distinguish transcriptional changes, with special emphasis on those related to plant defense responses. Soybean genes identified as strongly upregulated during infection by the pathogen included pathogenesis-related proteins, disease-resistance proteins, transcription factors, and secondary metabolism-related genes, as well as those encoding for signaling hormones. Remarkably, the +AMF+Mp treatment displayed a lower number of upregulated genes as compared with the -AMF+Mp treatment. AMF seemed to counteract or balance costs upon M. phaseolina infection, which could be associated to a negative impact on biomass and seed production. These detailed insights in soybean-AMF interaction help us to understand the complex underlying mechanisms involved in AMF-mediated biocontrol and support the importance of preserving and stimulating the existing plant-AMF associates, via adequate agricultural practices, to optimize their agro-ecological potential.

Arbuscular Mycorrhizal Fungal Community Composition in Carludovica palmata, Costus scaber and Euterpe precatoria from Weathered Oil Ponds in the Ecuadorian Amazon.

Arbuscular mycorrhizal fungi (AMF) are ubiquitous to most natural and anthropized ecosystems, and are often found in polluted environments. However, their occurrence and community composition in highly weathered petroleum-polluted soils has been infrequently reported. In the present study, two ponds of weathered crude oil and their surrounding soil from the Charapa field in the Amazon region of Ecuador were selected and root colonization by AMF of their native plants investigated. The AMF community was further analyzed in three selected plant species (i.e., Carludovica palmata, Costus scaber and Euterpe precatoria) present in the two ponds and the surrounding soil. A fragment covering partial SSU, the whole ITS and partial LSU rDNA region was amplified (i.e., 1.5 kb), cloned and sequenced from the roots of each host species. AMF root colonization exceeded 56% in all plant species examined and no significant difference was observed between sites or plants. For AMF community analysis, a total of 138 AMF sequences were obtained and sorted into 32 OTUs based on clustering (threshold ≥97%) by OPTSIL. The found OTUs belonged to the genera Rhizophagus (22%), Glomus (31%), Acaulospora (25%) and Archaeospora (22%). Glomus and Archaeospora were always present regardless of the plant species or the site. Acaulospora was found in the three plant species and in the two ponds while Rhizophagus was revealed only in the surrounding soil in one plant species (Euterpe precatoria). Our study contributed to the molecular community composition of AMF and revealed an unexpected high presence of four AMF genera which have established a symbiosis with roots of native plants from the Amazon forest under high polluted soil conditions.

Dynamics of Short-Term Phosphorus Uptake by Intact Mycorrhizal and Non-mycorrhizal Maize Plants Grown in a Circulatory Semi-Hydroponic Cultivation System.

A non-destructive cultivation system was developed to study the dynamics of phosphorus (Pi) uptake by mycorrhizal and non-mycorrhizal maize plantlets. The system consisted of a plant container connected via silicon tubes to a glass bottle containing a nutrient solution supplemented with Pi. The nutrient solution is pumped with a peristaltic pump to the upper part of the container via the silicon tubes and the solution percolate through the plantlet container back into the glass bottle. Pi is sampled from the glass bottle at regular intervals and concentration evaluated. Maize plantlets were colonized by the AMF Rhizophagus irregularis MUCL 41833 and Pi uptake quantified at fixed intervals (9, 21, and 42 h) from the depletion of the Pi in the nutrient solution flowing through the plantlets containers. Plants and fungus grew well in the perlite substrate. The concentration of Pi in the bottles followed an almost linear decrease over time, demonstrating a depletion of Pi in the circulating solution and a concomitant uptake/immobilization by the plantlet-AMF associates in the containers. The Pi uptake rate was significantly increased in the AMF-colonized plantlets (at 9 and 21 h) as compared to non-colonized plantlets, although no correlation was noticed with plant growth or P accumulation in shoots. The circulatory semi-hydroponic cultivation system developed was adequate for measuring Pi depletion in a nutrient solution and by corollary Pi uptake/immobilization by the plant-AMF associates. The measurements were non-destructive so that the time course of Pi uptake could be monitored without disturbing the growth of the plant and its fungal associate. The system further opens the door to study the dynamics of other micro and macro-nutrients as well as their uptake under stressed growth conditions such as salinity, pollution by hydrocarbon contaminants or potential toxic elements.

Rhizophagus irregularis MUCL 41833 can colonize and improve P uptake of Plantago lanceolata after exposure to ionizing gamma radiation in root organ culture.

Long-lived radionuclides such as (90)Sr and (137)Cs can be naturally or accidentally deposited in the upper soil layers where they emit ß/γ radiation. Previous studies have shown that arbuscular mycorrhizal fungi (AMF) can accumulate and transfer radionuclides from soil to plant, but there have been no studies on the direct impact of ionizing radiation on AMF. In this study, root organ cultures of the AMF Rhizophagus irregularis MUCL 41833 were exposed to 15.37, 30.35, and 113.03 Gy gamma radiation from a (137)Cs source. Exposed spores were subsequently inoculated to Plantago lanceolata seedlings in pots, and root colonization and P uptake evaluated. P. lanceolata seedlings inoculated with non-irradiated AMF spores or with spores irradiated with up to 30.35 Gy gamma radiation had similar levels of root colonization. Spores irradiated with 113.03 Gy gamma radiation failed to colonize P. lanceolata roots. P content of plants inoculated with non-irradiated spores or of plants inoculated with spores irradiated with up to 30.35 Gy gamma radiation was higher than in non-mycorrhizal plants or plants inoculated with spores irradiated with 113.03 Gy gamma radiation. These results demonstrate that spores of R. irregularis MUCL 41833 are tolerant to chronic ionizing radiation at high doses.

The induction of Ethylene response factor 3 (ERF3) in potato as a result of co-inoculation with Pseudomonas sp. R41805 and Rhizophagus irregularis MUCL 41833 - a possible role in plant defense.

Colonization of plant rhizosphere/roots by beneficial microorganisms (e.g. plant growth promoting rhizobacteria - PGPR, arbuscular mycorrhizal fungi - AMF) confers broad-spectrum resistance to virulent pathogens and is known as induced systemic resistance (ISR) and mycorrhizal-induced resistance (MIR). ISR or MIR, an indirect mechanism for biocontrol, involves complex signaling networks that are regulated by several plant hormones, the most important of which are salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). In the present study, we investigated if inoculation of potato plantlets with an AMF (Rhizophagus irregularis MUCL 41833) and a PGPR (Pseudomonas sp R41805) either alone or in combination, could elicit host defense response genes in the presence or absence of Rhizoctonia Solani EC-1, a major potato pathogen. RT-qPCR revealed the significant expression of ethylene response factor 3 (EFR3) in mycorrhized potato plantlets inoculated with Pseudomonas sp R41805 and also in mycorrhized potato plantlets inoculated with Pseudomonas sp R41805 and challenged with R. solani. The significance of ethylene response factors (ERFs) in pathogen defense has been well documented in the literature. The results of the present study suggest that the dual inoculation of potato with PGPR and AMF may play a part in the activation of plant systemic defense systems via ERF3.

Cryopreservation of arbuscular mycorrhizal fungi from root organ and plant cultures.

Long-term maintenance of arbuscular mycorrhizal fungi (AMF) by in vitro or in vivo subcultivation is often expensive and time-consuming and could present the risk of contaminations and possibly morphological, physiological, and genetic variations over time. Recently, in vitro produced AMF isolates belonging to the genus Rhizophagus were successfully cryopreserved at -130 °C following encapsulation-drying. Here, this method was tested on 12 single species cultures belonging to six different genera (i.e., Rhizophagus, Glomus, Claroideoglomus, Septoglomus, Paraglomus, and Gigaspora) produced in vitro or in vivo. Their viability was estimated, after 1 month of cryopreservation at -130 °C, by the percentage of potentially infective beads (i.e., the percentage of beads that contained at least one germinated propagule) for the in vitro produced species and the percentage of infective beads (i.e., the percentage of beads that contained at least one propagule able to colonize a new host plant in pot culture) for the in vivo produced species. With the exception of Gigaspora sp. MUCL 52331 and Septoglomus constrictus PER 7.2, no significant differences were observed in the viability of the single species cultures before and after cryopreservation. These results, thus, demonstrated the suitability of the cryopreservation method by encapsulation-drying for AMF species belonging to different genera and produced in vitro or in vivo. This method opens the door to the long-term preservation at ultra-low temperature of a large number of AMF species and for the preservation of species that are still recalcitrant to in vitro cultivation.

Maintenance and preservation of ectomycorrhizal and arbuscular mycorrhizal fungi.

Short- to long-term preservation of mycorrhizal fungi is essential for their in-depth study and, in the case of culture collections, for safeguarding their biodiversity. Many different maintenance/preservation methods have been developed in the last decades, from soil- and substrate-based maintenance to preservation methods that reduce (e.g., storage under water) or arrest (e.g., cryopreservation) growth and metabolism; all have advantages and disadvantages. In this review, the principal methods developed so far for ectomycorrhizal and arbuscular mycorrhizal fungi are reported and described given their distinct biology/ecology/evolutionary history. Factors that are the most important for their storage are presented and a protocol proposed which is applicable, although not generalizable, for the long-term preservation at ultra-low temperature of a large panel of these organisms. For ECM fungi, isolates should be grown on membranes or directly in cryovials until the late stationary growth phase. The recommended cryopreservation conditions are: a cryoprotectant of 10% glycerol, applied 1-2 h prior to cryopreservation, a slow cooling rate (1 °C min(-1)) until storage below -130 °C, and fast thawing by direct plunging in a water bath at 35-37 °C. For AMF, propagules (i.e., spores/colonized root pieces) isolated from cultures in the late or stationary phase of growth should be used and incorporated in a carrier (i.e., soil or alginate beads), preferably dried, before cryopreservation. For in vitro-cultured isolates, 0.5 M trehalose should be used as cryoprotectant, while isolates produced in vivo can be preserved in dried soil without cryoprotectant. A fast cryopreservation cooling rate should be used (direct immersion in liquid nitrogen or freezing at temperatures below -130 °C), as well as fast thawing by direct immersion in a water bath at 35 °C.

Cryopreservation of in vitro-produced Rhizophagus species has minor effects on their morphology, physiology, and genetic stability.

Cryogenic storage is considered to be the most convenient method to maintain phenotypic and genetic stability of organisms. A cryopreservation technique based on encapsulation-drying of in vitro-produced arbuscular mycorrhizal fungi has been developed at the Glomeromycota In Vitro Collection. In this study, we investigated fungal morphology (i.e., number and size of spores, number of branched absorbing structures (BAS), hyphal length, and number of anastomosis per hyphal length), activity of acid phosphatase and alkaline phosphatase in extraradical hyphae, and variation in amplified fragment length polymorphism (AFLP) profiles of in vitro-produced isolates of five Rhizophagus species maintained by cryopreservation for 6 months at -130 °C and compared to the same isolates preserved at 27 °C. Isolates were stable after 6 months cryopreservation. Comparing isolates, the number of BAS increased significantly in one isolate, and hyphal length decreased significantly in another isolate. No other morphological variable was impacted by the mode of preservation. Phosphatase activities in extraradical hyphae and AFLP profiles were not influenced by cryopreservation. These findings indicate that cryopreservation at -130 °C of encapsulated-dried and in vitro-produced Rhizophagus isolates (i.e., Rhizophagus irregularis, Rhizophagus fasciculatus, Rhizophagus diaphanous, and two undefined isolates) is a suitable alternative for their long-term preservation.

Preservation at ultra-low temperature of in vitro cultured arbuscular mycorrhizal fungi via encapsulation-drying.

At present, over 300 species of arbuscular mycorrhizal fungi (AMF) have been identified, most of which being stored in international collections. Their maintenance is mostly achieved in greenhouse via continuous culture on trap plants or in vitro in association with excised root organs. Both methods are work-intensive and for the former present the risk of unwanted contaminations. The in vitro root organ culture of AMF has become an alternative preventing contamination. Nevertheless, the risk for somaclonal variation during the sub-cultivation process cannot be excluded. A method for the long-term conservation that guarantees the stability of the biological material is thus highly demanded to preserve the microorganisms and their genetic stability. Here, 12 AMF isolates cultured in vitro in association with excised carrot roots were encapsulated in alginate beads and subsequently cryopreserved. Several protocols were tested taking into consideration culture age, alginate bead pre-drying, and rate of decrease in temperature. The viability of the AMF isolates was estimated by the percentage of potentially infective beads (%PIB) that measure the % of beads that contain at least one germinated propagule. Thermal behaviour of alginate beads was analysed by a differential thermal calorimeter before and after drying to estimate the frozen and unfrozen water during the cryopreservation process. It was shown that the spore damage was directly related to ice formation during cryopreservation. The encapsulation and culture age were also determinant parameters for the successful cryopreservation. Irrespective of the AMF isolate, the optimal procedure for cryopreservation comprised five steps: (1) the encapsulation of propagules (i.e. spores and mycorrhizal root pieces) isolated from 5m old cultures, (2) the incubation overnight in trehalose (0.5M), (3) the drying during 48h at 27°C, (4) the cryopreservation in the freezer at -130°C following a two-step decrease in temperature: a fast decrease (∼12°Cmin(-1)) from room temperature (+20°C) to -110°C followed by a slow decrease in temperature (∼1°Cmin(-1)) from -110°C to -130°C, and (5) the direct thawing in a water bath (+35°C). The % PIB was above 70 % for all the isolates and even above 95% for 11 out of the 12 isolates after several months of storage at ultra-low temperature. All the isolates kept their capacity to associate to an excised carrot root in vitro and to reproduce the fungal life cycle with the production of several hundreds to thousands of spores after 2m. This method opens the door for the long-term maintenance at ultra-low temperature of AMF isolates within international repositories.

Transcriptional regulation of defence genes and involvement of the WRKY transcription factor in arbuscular mycorrhizal potato root colonization.

The establishment of arbuscular mycorrhizal associations causes major changes in plant roots and affects significantly the host in term of plant nutrition and resistance against biotic and abiotic stresses. As a consequence, major changes in root transcriptome, especially in plant genes related to biotic stresses, are expected. Potato microarray analysis, followed by real-time quantitative PCR, was performed to detect the wide transcriptome changes induced during the pre-, early and late stages of potato root colonization by Glomus sp. MUCL 41833. The microarray analysis revealed 526 up-regulated and 132 down-regulated genes during the pre-stage, 272 up-regulated and 109 down-regulated genes during the early stage and 734 up-regulated and 122 down-regulated genes during the late stage of root colonization. The most important class of regulated genes was associated to plant stress and in particular to the WRKY transcription factors genes during the pre-stage of root colonization. The expression profiling clearly demonstrated a wide transcriptional change during the pre-, early and late stages of root colonization. It further suggested that the WRKY transcription factor genes are involved in the mechanisms controlling the arbuscular mycorrhizal establishment by the regulation of plant defence genes.

The sterol biosynthesis inhibitor molecule fenhexamid impacts the vegetative compatibility of Glomus clarum.

The vegetative compatibility of the arbuscular mycorrhizal fungus (AMF) Glomus clarum MUCL 46238 was evaluated after continuous exposure to fenhexamid, a sterol biosynthesis inhibitor (SBI). Three lineages of this AMF were cultured in vitro for five generations in association with Ri T-DNA transformed carrot roots in the presence of 0, 5 or 10 mg l(-1) of fenhexamid. Whatever the AMF generation, fenhexamid at 5 and 10 mg l(-1) had no significant impact on the number of spores produced. However, vegetative compatibility tests (VCT) conducted with spores from the three lineages, in the presence of 10 mg l(-1) of fenhexamid, impacted the anastomosis process. At this concentration, the morphology of the germ tubes was modified. In addition, nitrotetrazolium-trypan blue staining revealed that 10 mg l(-l) of fenhexamid significantly reduced the probability of fusion between the germ tubes regardless of the culture conditions (i.e. absence or presence of fenhexamid) preceding the VCT. Our results demonstrated that spore production was not affected by fenhexamid, while anastomosis between germ tubes was decreased. This suggested that high concentrations, accumulation or repeated application of this SBI fungicide may impact the community structure of AMF in soil.

Methods for large-scale production of AM fungi: past, present, and future.

Many different cultivation techniques and inoculum products of the plant-beneficial arbuscular mycorrhizal (AM) fungi have been developed in the last decades. Soil- and substrate-based production techniques as well as substrate-free culture techniques (hydroponics and aeroponics) and in vitro cultivation methods have all been attempted for the large-scale production of AM fungi. In this review, we describe the principal in vivo and in vitro production methods that have been developed so far. We present the parameters that are critical for optimal production, discuss the advantages and disadvantages of the methods, and highlight their most probable sectors of application.

Do arbuscular mycorrhizal fungi with contrasting life-history strategies differ in their responses to repeated defoliation?

Arbuscular mycorrhizal (AM) fungi obligatorily depend on carbon (C) resources provided via the plant and therefore fluctuations in C availability may strongly and differently affect AM fungi with different life-history strategies (LHS). In the present study, we examined the effect of repeated defoliation of in vitro grown barrel medic (Medicago truncatula) on the spore and auxiliary cell (AC) production dynamics of a presumed r-strategist (Glomus intraradices) and a presumed K-strategist (Dentiscutata reticulata). Glomus intraradices modulated the production of spores directly to C availability, showing direct investment in reproduction as expected for r-strategists. In contrast, AC production of D. reticulata was not affected after a single defoliation and thus showed higher resistance to fluctuating C levels, as expected for K-strategists. Our results demonstrate that plant defoliation affects the production of extraradical C storage structures of G. intraradices and D. reticulata differently. Our results contribute towards revealing differences in LHS among AM fungal species, a step further towards understanding their community dynamics in natural ecosystems and agroenvironments.

Impact of multispores in vitro subcultivation of Glomus sp. MUCL 43194 (DAOM 197198) on vegetative compatibility and genetic diversity detected by AFLP.

Vegetative compatibility and amplified fragment length polymorphism (AFLP) genotyping of in vitro multispores clonal lineages, issued from the same ancestor culture of the arbuscular mycorrhizal fungal strain MUCL 43194 and subcultured several generations in different locations, was assessed. Vegetative compatibility was studied by confronting the germ tubes of two spores from the same or different clonal lineages and stained with nitrotetrazolium blue-Trypan blue and diamidinophenylindole to detect hyphal fusions and nuclei, respectively. Further AFLP analysis of single spores was performed to assess the genetic profile and Dice similarity between clonal lineages. Germ tubes of spores distant by as many as 69 generations were capable of fusing. The anastomosis frequencies averaged 69% between spores from the same clonal lineage, 57% between spores from different clonal lineages, and 0% between spores belonging to different strains. The AFLP patterns showed similarities averaging 92% within clonal lineages and 86% between clonal lineages. Each spore presented unique genotype and some of them shared more markers with spores from different lineages than within the same lineage. We showed that MUCL 43194 maintained self-recognition for long periods of subcultures in vitro and that spores involved in compatibility tests had different genotypes. Our findings suggest that MUCL 43194 maintains genetic diversity by means of anastomoses.

Fast track in vitro mycorrhization of potato plantlets allow studies on gene expression dynamics.

Root colonization by arbuscular mycorrhizal (AM) fungi is a dynamic process involving major changes in plant gene expression. Here, the expression of a phosphate transporter gene (PT3) and several defense genes, already known to be involved in the various stages of AM establishment, were monitored in the mycelium donor plant (MDP) in vitro culture system associating potato plantlets with an AM fungus. This system allows fast and homogenous mycorrhization of seedlings at their early stage of development by growing the plantlets in active mycelial networks, but has never been validated for gene expression analysis. Here, QRT-PCR analyses were conducted in parallel to pre- (1 day), early (2 and 3 days), and late (6, 9, and 15 days) stages of root colonization. We observed the induction of a plant gene marker of AM root colonization (PT3) at the late stage and the induction of MAPK and PAL genes at the early and late stages of root colonization. We also demonstrated the induction of PR1 and PR2 genes at pre- and late stages and of GST1 and Lox genes at a late stage of root colonization. These results validated the MDP in vitro culture system as an optimal tool to study gene expression analysis during the AM fungi establishment. This system further opened the door to investigate gene networks associated with the plants-AM fungi symbiosis.

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.