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International audience; Modern agriculture is currently undergoing rapid changes in the face of the continuing growth of world population and many ensuing environmental challenges. Crop quality is becoming as important as crop yield and can be characterised by several parameters. For fruits and vegetables, quality descriptors can concern production cycle (e.g. conventional or organic farming), organoleptic qualities (e.g. sweet taste, sugar content, acidity) and nutritional qualities (e.g. mineral content, vitamins). For other crops, however, the presence of secondary metabolites such as anthocyanins or certain terpenes in the targeted tissues is of interest as well, especially for their human health properties. All plants are constantly interacting with microorganisms. These microorganisms include arbuscular mycorrhizal fungi as well as certain soil bacteria that provide ecosystem services related to plant growth, nutrition and quality parameters. This review is an update of current research on the single and combined (co-inoculation) use of arbuscular mycorrhizal fungi and plant growth-promoting rhizobacteria in crop production, with a focus on their positive impacts on crop quality traits (e.g. nutritional value, organoleptic properties). We also highlight the need to dissect mechanisms regulating plant-symbionts and symbiont-symbiont interactions, to develop farming practices and to study a broad range of interactions to optimize the symbiotic potential of root-associated microorganisms.

International audience; Crop inoculation with Glomus cubense isolate (INCAM-4, DAOM-241198) promotes yield in banana, cassava, forages, and others. Yield improvements range from 20 to 80% depending on crops, nutrient supply, and edaphoclimatic conditions. However, it is difficult to connect yield effects with G. cubense abundance in roots due to the lack of an adequate methodology to trace this taxon in the field. It is necessary to establish an accurate evaluation framework of its contribution to root colonization separated from native arbuscular mycorrhizal fungi (AMF). A taxon-discriminating primer set was designed based on the ITS nrDNA marker and two molecular approaches were optimized and validated (endpoint PCR and quantitative real-time PCR) to trace and quantify the G. cubense isolate in root and soil samples under greenhouse and environmental conditions. The detection limit and specificity assays were performed by both approaches. Different 18 AMF taxa were used for endpoint PCR specificity assay, showing that primers specifically amplified the INCAM-4 isolate yielding a 370 bp-PCR product. In the greenhouse, Urochloa brizantha plants inoculated with three isolates (Rhizophagus irregularis, R. clarus, and G. cubense) and environmental root and soil samples were successfully traced and quantified by qPCR. The AMF root colonization reached 41–70% and the spore number 4–128 per g of soil. This study demonstrates for the first time the feasibility to trace and quantify the G. cubense isolate using a taxon-discriminating ITS marker in roots and soils. The validated approaches reveal their potential to be used for the quality control of other mycorrhizal inoculants and their relative quantification in agroecosystems.

International audience; De manière générale, un couvert végétal se définit par une espèce ou une communauté d’espèces végétales recouvrant le sol de manière permanente ou temporaire. L’agriculteur a la possibilité de semer ces couverts, selon un choix raisonné, ou bien de laisser la végétation spontanée se développer. Il existe 3 types de couverts semés avec des objectifs différents pour l’exploitant : le couvert hivernal, installé pour faire face au phénomène d’érosion du sol important pendant cette période de repos végétatif ; les engrais verts, installés avec l’objectif d’amender naturellement la parcelle, en détruisant et en enfouissant les résidus du couvert qui libèrent ainsi de la matière organique plus ou moins rapidement, qui servira à alimenter la plante au cycle suivant, après des étapes de minéralisation ; les cultures intermédiaires pièges à nitrate (CIPAN), installées au tout début de l’automne pour capter dans le sol les nitrates en excès et des quantités d’eau importantes dans le but de limiter la lixiviation et la contamination des eaux souterraines et de surfaces par les nitrates.Les couverts et leurs intérêts en production végétaleLes espèces végétales les plus couramment utilisées dans ces couverts font essentiellement partie de trois familles présentant chacune différents avantages quant à leur utilisation. Nous retrouvons : les graminées (avoine, seigle, ray grass, brome, fétuque, orge…), avantageuses avec un rapport C/N élevé, produisent une biomasse (figure 1) importante bénéfique lors de la destruction ; les brassicacées (anciennement « crucifères ») (moutarde, navette fourragère, radis chinois, colza fourrager…) servent couramment de « pompe à azote » mais aussi de biofumigateur (figure 1), ce qui peut avoir un impact négatif sur certains champignons bénéfiques du sol (champignons mycorhiziens) si la proportion est trop importante. Elles présentent l’avantage de s’implanter rapidement, empêchant le développement important des adventices (appelées également fréquemment mauvaises herbes) ; les légumineuses (vesce, féverole, trèfle, luzerne…) sont de plus en plus utilisées, seules ou en mélange, pour leur capacité à réaliser une symbiose avec des bactéries du sol, leur permettant ainsi de fixer le diazote atmosphérique. Avec un rapport C/N alors très faible, le relargage de l’azote assimilé et des nutriments se fait très rapidement, et ceux-ci deviennent donc disponibles pour la culture.Les plantes de couvert sont fortement plébiscitées en polyculture pour couvrir les parcelles lorsqu’elles ne sont pas utilisées par une culture d’intérêt. L’utilisation de ces couverts d’interculture s’est développée notamment pour contrer des phénomènes de battance et d’érosion des sols, qualifiés d’anthropogènes car liés à l’exploitation, parfois intensive, des milieux agricoles et à la destruction de la couverture végétale naturelle.

Background: Wetlands in Neotropics harbor high fungal diversity, including arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE). This study describes the interaction of plant roots with AMF and DSE in a freshwater wetland belonging to a hotspot of biodiversity. Hypothesis: Differential root colonization between arbuscular mycorrhizal and dark septate endophyte fungi is influenced by plant species and abiotic conditions in a freshwater wetland. Studied species: Plant species colonized by arbuscular mycorrhizal and dark septate endophyte fungi. Methods: Properties of soils and the water column, floristic composition, root colonization by AMF and DSE, and molecular identification of AMF inside roots were studied. Results: Soils were Gleysol and flooded during the rainy season. Most of identified plant species were herbaceous, with Cyperus articulatus and Mimosa pigra as the dominant species. Seven of 8 analyzed plant species exhibited differential co-colonization between AMF and DSE. Repeated sampling for one year under flooding/dry conditions demonstrated that C. articulatus and M. pigra were mainly associated with DSE and AMF, respectively. A positive correlation between dissolved O2 in the water column and fungal colonization was observed in C. articulatus . Glomerales and Archaeosporales were molecularly identified inside roots containing arbuscules of M. pigra . Conclusions: Findings highlight differential coexistence between AMF and DSE in plant roots; fungal colonization was influenced by flooding/dry conditions in a neotropical wetland; the community of AMF inside arbusculated roots of M. pigra includes at least four clades.

prod ? SPE IPM UB INRA; Arbuscular mycorrhizal symbiosis occurs between obligate biotrophic fungi of the phylum Glomeromycota and most of land plants. The exchange of nutrients between host plants and arbuscular mycorrhizal fungi is presumed to be the main benefit for the two symbiotic partners. In this review article, we outline the current concepts of nutrient exchanges within this symbiosis (mechanisms and regulation). First, we focus on phosphorus and nitrogen transfer from the fungal partner to the host plant and on the reciprocal transfer of carbon compounds, with a highlight on a possible interplay between nitrogen and phosphorus nutrition during arbuscular mycorrhizal symbiosis. We further discuss potential mechanisms of regulation of these nutrient exchanges linked to membrane dynamics. The review finally addresses the common mycorrhizal networks formed by arbuscular mycorrhizal fungi, which inter-connect plants from similar and/or different species. Then the best way to integrate this knowledge and the ensuing potential benefits of arbuscular mycorrhiza in a sustainable agriculture is discussed. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Arbuscular mycorrhizal fungi (AMF) play a major role as biofertilizer for sustainable agriculture. Nevertheless, it is still poorly documented whether inoculated AMF can successfully establish in field soils as exotic AMF and improve plant growth and productivity. Further, the fate of an exogenous inoculum is still poorly understood. Here, we pre-inoculated two cultivars (Tasset and Gola) of the fruit tree Ziziphus mauritiana (jujube) with the exotic AM fungus Rhizophagus irregularis isolate IR27 before transplantation in the field. In two experiments, tracking and quantification of R. irregularis IR27 were assessed in a 13-month-old jujube and an 18-month-old jujube in two fields located in Senegal. Our results showed that the inoculant R. irregularis IR27 was quantitatively traced and discriminated from native R. irregularis isolates in roots by using a qPCR assay targeting a fragment of the RNA polymerase II gene (RPB1), and that the inoculum represented only fractions ranging from 11 to 15% of the Rhizophagus genus in the two plantations 13 and 18 months after transplantation, respectively. This study validates the use of the RPB1 gene as marker for a relative quantification of a mycorrhizal inoculant fungus isolate in the field.

The soil-borne nepovirus Grapevine fanleaf virus (GFLV), causal agent of grapevine fanleaf degeneration disease, spreads mainly via the nematode vector Xiphinema index. Since the arbuscular mycorrhizal (AM) fungus Rhizophagus intraradices can induce local and systemic protection against X. index (Hao et al., 2012), therefore, the objective of the present study was to investigate if AM may represent a means for limiting GFLV infection by reducing nematode vector attack. Three-week-old mycorrhizal or nonmycorrhizal grapevine rootstock SO4 (Vitis berlandieri × V. riparia) plants were inoculated with 10 or 100 viruliferous nematodes per plant under glasshouse conditions. Plants were harvested 35 and 90 days after inoculation with X. index (DAIN), prior to and after the appearance of fanleaf degeneration symptoms, respectively. Both levels of viruliferous X. index inhibited root growth of nonmycorrhizal plants at 90 DAIN, while the presence of arbuscular mycorrhizal colonization significantly decreased gall formation on roots at both time points and reduced nematode reproduction in soil at 90 DAIN. GFLV was not detected by one-step reverse transcription PCR in the roots of any plant at 35 DAIN after viruliferous X. index inoculation. At 90 DAIN with 10 viruliferous nematodes per plant, the virus was present in nonmycorrhizal roots but absent from mycorrhizal grapevine. The virus was detected in both mycorrhizal and nonmycorrhizal plants 90 DAIN. It is suggested that inhibition of X. index proliferation and penetration by AM may protect grapevine against GFLV at a low abundance of the viruliferous nematode in soils and so contribute to virus control under these conditions.

Prod 2017-372 SPE IPM INRA UB; National audience

The cessation of mowing in subalpine grasslands promotes the dominance of Festuca paniculata leading to the reduction in plant diversity. Moreover, it affects positively the abundance of Epichloe sp. inhabiting F. paniculata leaves and negatively the soil density of arbuscular mycorrhizal fungi (AMF). We explored how the cessation of mowing influences root AMF communities in F. paniculata and the neighboring plants, and Epichloe sp alkaloids. Thirteen AMF operational taxonomical units were found. The neighboring plants affected positively the abundances of Aalpin and GLOM_7 whereas the interaction plant/management type influenced significantly Claroide_1 , GLOM_1 and GLOM_7 . The N-formylnorloline, produced by Epichloe sp. increased in unmown grassland. Hence, the cessation of mowing, coinciding with the high abundance of endophyte alkaloid, affected root-associated AMF with differential responses at the abundance level. The N-formylnorloline could be one compound underpinning the dynamic of plant diversity with a resulting structuration of AMF in subalpine grasslands.

SPE IPM INRA SUPDAT; International audience; Many of the non-pathogenic endophytic bacteria that reside in plant roots promote plant growth as well as protection against pathogens attack. However, little is known about their mode of action in wheat. We have previously demonstrated the potential of Paenibacillus sp. strain B2 (PB2) to stimulate plant defense mechanisms via its paenimyxin lipo-polypeptide elicitor. Recently, we isolated the Curtobacterium plantarum strain EDS (EDS) from seeds of almost all wheat cultivars. In the present work, the ability of PB2 and EDS to promote wheat growth and protection against Septoria leaf blotch (SLB) was investigated. Results showed that PB2 is a general root external colonizer and cultivar-dependent endophyte. In the endophytic state and only in co-inoculation, it significantly increased the internal root colonization by EDS, resulting in an increase of root and aerial part fresh weights. qPCR analysis showed that, in the endophytic and nonendophytic states, PB2 conferred >= 59% protection against SLB by inducing systemic resistance which is characterized by the over expression of the pr1, lox, Aos, peroxidase, oxo and gst genes. Paenimyxin conferred 76% local protection characterized by the overexpression of the glu, lox, aos, pal, chs, oxo, and gst genes, and 82% systemic protection by chs. It was concluded that PB2 is potentially very interesting in the biocontrol of SLB and, in a mixture with EDS, in the wheat growth promoting. Genes involved in the flavonoid, salicylic acid, jasmonic acid, reactive oxygen species and basal defense pathways seem to play an important role in the resistance against SLB.

Paenibacillus sp. strain B2, isolated from the mycorrhizosphere of Sorghum bicolor , was previously shown to possess antagonistic activity against fungi and bacteria due to the secretion of the peptide paenimyxin. The effects of strain B2 and paenimyxin were assessed against grapevine pathogens as they potentially represent a non-polluting complement or an alternative biocontrol agent to existing disease management strategies. In this study, the effects of Paenibacillus sp. strain B2 and paenimyxin were analyzed in vitro or in planta on the aerial or root pathogens of grapevine Botrytis cinerea , Plasmopara viticola and Xiphinema index . The bacterium significantly reduced B. cinerea development in vitro , and substantially decreased X. index numbers in soil and gall formation in the root system of grapevine plants. In vitro , paenimyxin inhibited the mycelial growth of B. cinerea , the mobility of the zoospores of P. viticola and X . index activity. Supplying paenimyxin at the time of inoculation of leaf tissue with P. viticola decreased infection sites, compared to application prior to fungal inoculation. Using the P. viticola /grapevine pathosystem, we showed that paenimyxin acts as a biopesticide against the oomycete but does not trigger plant defense. Conversely, Paenibacillus sp. strain B2 can induce the expression of the defense genes CHI (chitinase), PR-3 (proteinase inhibitor) and LOX (lipoxygenase). Altogether, the results obtained highlight the interest of Paenibacillus sp. strain B2 and paenimyxin for the biocontrol of different grapevine diseases.

SPE UB; International audience; Plants interact with microbes whose ultimate aim is to exploit plant carbohydrates for their reproduction. Plant-microbe interactions (PMIs) are classified according to the nature of their trophic exchanges : while mutualistic microbes trade nutrients with plants, pathogens unilaterally divert carbohydrates. The early responses following microbe recognition and the subsequent control of plant sugar distribution are still poorly understood. To further decipher PMI functionality, we used tobacco cells treated with microbial molecules mimicking pathogenic or mutualistic PMIs, namely cryptogein, a defense elicitor, and chitotetrasaccharide (CO4), which is secreted by mycorrhizal fungi. CO4 was perceived by tobacco cells and triggered widespread transient signaling components such as a sharp cytosolic Ca2+ elevation, NtrbohD-dependent H2O2 production, and MAP kinase activation. These CO4-induced events differed from those induced by cryptogein, i.e., sustained events leading to cell death. Furthermore, cryptogein treatment inhibited glucose and sucrose uptake but not fructose uptake, and promoted the expression of NtSUT and NtSWEET sugar transporters, whereas CO4 had no effect on sugar uptake and only a slight effect on NtSWEET2B expression. Our results suggest that microbial molecules induce different signaling responses that reflect microbial lifestyle and the subsequent outcome of the interaction.

International audience; Arbuscular mycorrhizal (AM) symbiosis that associates roots of most land plants with soil-borne fungi (Glomeromycota), is characterized by reciprocal nutritional benefits. Fungal colonization of plant roots induces massive changes in cortical cells where the fungus differentiates an arbuscule, which drives proliferation of the plasma membrane. Despite the recognized importance of membrane proteins in sustaining AM symbiosis, the root microsomal proteome elicited upon mycorrhiza still remains to be explored. In this study, we first examined the qualitative composition of the root membrane proteome of Medicago truncatula after microsome enrichment and subsequent in depth analysis by GeLC-MS/MS. The results obtained highlighted the identification of 1226 root membrane protein candidates whose cellular and functional classifications predispose plastids and protein synthesis as prevalent organelle and function, respectively. Changes at the protein abundance level between the membrane proteomes of mycorrhizal and nonmycorrhizal roots were further monitored by spectral counting, which retrieved a total of 96 proteins that displayed a differential accumulation upon AM symbiosis. Besides the canonical markers of the periarbuscular membrane, new candidates supporting the importance of membrane trafficking events during mycorrhiza establishment/functioning were identified, including flotillin-like proteins. The data have been deposited to the ProteomeXchange with identifier PXD000875. Biological significance During arbuscular mycorrhizal symbiosis, one of the most widespread mutualistic associations in nature, the endomembrane system of plant roots is believed to undergo qualitative and quantitative changes in order to sustain both the accommodation process of the AM fungus within cortical cells and the exchange of nutrients between symbionts. Large-scale GeLC-MS/MS proteomic analysis of the membrane fractions from mycorrhizal and nonmycorrhizal roots of M. truncatula coupled to spectral counting retrieved around one hundred proteins that displayed changes in abundance upon mycorrhizal establishment. The symbiosis-related membrane proteins that were identified mostly function in signaling/membrane trafficking and nutrient uptake regulation. Besides extending the coverage of the root membrane proteome of M. truncatula, new candidates involved in the symbiotic program emerged from the current study, which pointed out a dynamic reorganization of microsomal proteins during the accommodation of AM fungi within cortical cells

International audience; Sulfur plays an essential role in plants' growth and development and in their response to various abiotic and biotic stresses despite its leachability and its very low abundance in the only form that plant roots can uptake (sulfate). It is part of amino acids, glutathione (GSH), thiols of proteins and peptides, membrane sulfolipids, cell walls and secondary products, so reduced availability can drastically alter plant growth and development. The nutritional benefits of symbiotic interactions can help the plant in case of S deficiency. In particular the arbuscular mycorrhizal (AM) interaction improves N, P and S plant nutrition, but the mechanisms behind these exchanges are not fully known yet. Although the transcriptional changes in the leguminous model plant Medicago truncatula have been already assessed in several biotic and/or abiotic conditions, S deficiency has not been considered so far. The aim of this work is to get a first overview on S-deficiency responses in the leaf and root tissues of plants interacting with the AM fungus Rhizophagus irregularis. Several hundred genes displayed significantly different transcript accumulation levels. Annotation and GO ID association were used to identify biological processes and molecular functions affected by sulfur starvation. Beside the beneficial effects of AM interaction, plants were greatly affected by the nutritional status, showing various differences in their transcriptomic footprints. Several pathways in which S plays an important role appeared to be differentially affected according to mycorrhizal status, with a generally reduced responsiveness to S deficiency in mycorrhized plants.

For almost 2 centuries it has been disputed whether Tuber aestivum and Tuber uncinatum constitute two different species of truffles. Molecular markers have been applied previously to contribute to resolving this question, coming to different conclusions. In this study, we address this question by analyzing the genetic structure of truffles assigned to either of the two putative species from a geographically broad sampling across Europe. We used an approach involving multigene phylogenies and coalescent analyses of nine regions from five genes. All tests conducted supported the conspecificity of Tuber aestivum and Tuber uncinatum.

Fluctuations in intracellular calcium levels generate signalling events and regulate different cellular processes. Whilst the implication of Ca2+ in plant responses during arbuscular mycorrhiza (AM) interactions is well documented, nothing is known about the regulation or role of this secondary messenger in the fungal symbiont. The spatio-temporal expression pattern of putatively Ca2+-related genes of Glomus intraradices BEG141 encoding five proteins involved in membrane transport and one nuclear protein kinase, was investigated during the AM symbiosis. Expression profiles related to successful colonization of host roots were observed in interactions of G. intraradices with roots of wild-type Medicago truncatula (line J5) compared to the mycorrhiza-defective mutant dmi3/Mtsym13. Symbiotic fungal activity was monitored using stearoyl-CoA desaturase and phosphate transporter genes. Laser microdissection based-mapping of fungal gene expression in mycorrhizal root tissues indicated that the Ca2+-related genes were differentially upregulated in arbuscules and/or in intercellular hyphae. The spatio-temporal variations in gene expression suggest that the encoded proteins may have different functions in fungal development or function during symbiosis development. Full-length cDNA obtained for two genes with interesting expression profiles confirmed a close similarity with an endoplasmic reticulum P-type ATPase and a Vcx1-like vacuolar Ca2+ ion transporter functionally characterized in other fungi and involved in the regulation of cell calcium pools. Possible mechanisms are discussed in which Ca2+-related proteins G. intraradices BEG141 may play a role in mobilization and perception of the intracellular messenger by the AM fungus during symbiotic interactions with host roots. (C) 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

International audience; The ectoparasitic dagger nematode (Xiphinema index), vector of Grapevine fanleaf virus (GFLV), provokes gall formation and can cause severe damage to the root system of grapevines. Mycorrhiza formation by Glomus (syn. Rhizophagus) intraradices BEG141 reduced both gall formation on roots of the grapevine rootstock SO4 (Vitis berlandierixV. riparia) and nematode number in the surrounding soil. Suppressive effects increased with time and were greater when the nematode was post-inoculated rather than co-inoculated with the arbuscular mycorrhizal (AM) fungus. Using a split-root system, decreased X. index development was shown in mycorrhizal and non-mycorrhizal parts of mycorrhizal root systems, indicating that both local and systemic induced bioprotection mechanisms were active against the ectoparasitic nematode. Expression analyses of ESTs (expressed sequence tags) generated in an SSH (subtractive suppressive hybridization) library, representing plant genes up-regulated during mycorrhiza-induced control of X. index, and of described grapevine defence genes showed activation of chitinase 1b, pathogenesis-related 10, glutathione S-transferase, stilbene synthase 1, 5-enolpyruvyl shikimate-3-phosphate synthase, and a heat shock proein 70-interacting protein in association with the observed local and/or systemic induced bioprotection against the nematode. Overall, the data suggest priming of grapevine defence responses by the AM fungus and transmission of a plant-mediated signal to non-mycorrhizal tissues. Grapevine gene responses during AM-induced local and systemic bioprotection against X. index point to biological processes that are related either to direct effects on the nematode or to protection against nematode-imposed stress to maintain root tissue integrity

Chapitre 1; International audience; All fungi forming a mutualistic symbiosis with plant roots called arbuscular mycorrhiza were formerly grouped together in one order, the Glomales, placed in the Zygomycota (Morton 1993). Based on molecular analyses suggestingthat arbuscular mycorrhizal fungi should be separated from other fungal taxa, they were transferred a decade ago to the Glomeromy-cota , a new phylum created specifically for them (Schu¨ ssler et al. 2001). Whilst members of this monophyletic group originated from the same common ancestor as the Ascomycota and Basidiomycota, they have no obvious affinity to other major extant phylogenetic groups in the kingdom Fungi (James et al. 2006) and they probably diverged from the other fungal lineages several hundred million years before plants colonized terrestrial habitats 400–500 million years ago (mya) (Heckman et al. 20 01). Glomeromycotan fungi are complex but extremely successful organisms. They establish a compatible interaction with plants by either avoiding or suppressing plant defence reactions whilst redirecting host metabolic flow to their benefit without being detrimental to their host. The mechanisms by which this bio-trophy is achieved are largely unknown but the Glomeromycota have accompanied land plants through evolution and survived across periods of important environmental change to become ecologically and agriculturally impor-tant symbionts which improve the overall fit-ness of very different plant taxa in terrestrial ecosystems world-wide (Smith and Read 2008 ). Substantial evidence has accumulated about how a rational use of the microsymbiont proper-ties should significantly contribute to decreasing fertilizer and pesticide use in agriculture (Gianinazzi et al. 20 10). Although the Glomeromycota show consid-erable diversity between or within morphologi-cally recognizable species (Rosendahl 2008), they share some singular biological traits which limit experimental approaches that can be exploited to characterize their complexity. One particularity is their reproduction through large asexual spores , each of which is a single cell harbouring several hundreds or thousands of nuclei. The main mechanism for filling a spore with so many nuclei appears to be a massive influx of nuclei from subtending myce-lium into the developing spore (Jany and Pawlowska 2010). No sexual stage is known for these fungi but, although they are assumed to only reproduce asexually, recent transcrip-tome analyses indicate that they do possess genetic information essential for sexual repro-duction and meiosis (Tisserant et al. 2012 ). Absence of a sexual cycle raises questions about how Glomeromycota deal with deleteri-ous mutations usually eliminated through meiosis and how they have adapted to new hosts or habitats during evolution. High poly-ploidy, with multiple gene copies in the same genome, has been speculated as a possible mechanism to buffer against mutational events (Pawlowska and Taylor 2004 ), whilst nuclear exchange through hyphal anastomosis between different individuals of a same species (Casana and Bonfante 1988; Giovannetti et al. 2001 ) provides the possibility for genetic flux and recombination events (Croll et al. 2009 ; Angelard and Sanders 2011). However, basic information concerning ploidy, karyosis, num-ber of chromosomes or whether meiosis does occur in the Glomeromycota is still lacking, and evidence for genetic exchange, recombination or segregation is very limited. Another particularity is that all glomeromy-cotan fungi are obligate symbionts which have so far proved to be incalcitrant to pure culture in the absence of a host root on which they depend as a carbon source. This introduces inherent limitations in the application of standard tech-niques like genetic transformation or mutant generation/characterization, and it greatly hin-ders advances in the knowledge about gene function in these organisms. As previously pointed out (Gianinazzi-Pearson et al. 2001), there is converging evidence that the Glomero-mycota are an unusual group of fungi, and information about their genome structure, com-plexity and function is essential to understand-ing the processes regulating their symbiotic attributes, their reproductive biology and their apparent stability during coevolution in symbi-osis with many different plant taxa. Despite the fact that the biology of glomeromycotan fungi makes them extremely difficult to manipulate experimentally, the advent of powerful molecu-lar techniques has considerably furthered research during the past decade. The present chapter updates on the state of the art on Glo-meromycota genomics within the past decade (Gianinazzi-Pearson et al. 20 01), as well as on progress made through targeted and high-throughput sequencing programmes into an understanding of their basic biology.

Although plant biotisation with arbuscular mycorrhizal fungi (AMF) is a promising strategy for improving plant health, a better knowledge regarding the molecular mechanisms involved is required. In this context, we sought to analyse the root proteome of grapevine rootstock Selection Oppenheim 4 (SO4) upon colonisation with two AMF. As expected, AMF colonisation stimulates plant biomass. At the proteome level, changes in protein amounts due to AMF colonisation resulted in 39 differentially accumulated two-dimensional electrophoresis spots in AMF roots relative to control. Out of them, 25 were co-identified in SO4 roots upon colonisation by Glomus irregulare and Glomus mosseae supporting the existence of conserved plant responses to AM symbiosis in a woody perennial species. Among the 18 proteins whose amount was reduced in AMF-colonised roots were proteins involved in glycolysis, protein synthesis and fate, defence and cell rescue, ethylene biosynthesis and purine and pyrimidine salvage degradation. The six co-identified proteins whose amount was increased had functions in energy production, signalling, protein synthesis and fate including proteases. Altogether these data confirmed that a part of the accommodation program of AMF previously characterized in annual plants is maintained within roots of the SO4 rootstock cuttings. Nonetheless, particular responses also occurred involving proteins of carbon metabolism, development and root architecture, defence and cell rescue, anthocyanin biosynthesis and P remobilization, previously reported as induced upon P-starvation. This suggests the occurrence of P reprioritization upon AMF colonization in a woody perennial plant species with agronomical interest.

International audience; Root colonization by arbuscular mycorrhizal (AM) fungi of Artemisia umbelliformis, investigated in natural and cultivated sites in the Southern Alps of France, showed typical structures (arbuscules, vesicles, hyphae) as well as spores and mycelia in its rhizosphere. Several native AM fungi belonging to different Glomeromycota genera were identified as colonizers of A. umbelliformis roots, including Glomus tenue, Glomus intraradices, G. claroideum/etunicatum and a new Acaulospora species. The use of the highly mycorrhizal species Trifolium pratense as a companion plant impacted positively on mycorrhizal colonization of A. umbelliformis under greenhouse conditions. The symbiotic performance of an alpine microbial community including native AM fungi used as inoculum on A. umbelliformis was evaluated in greenhouse conditions by comparison with mycorrhizal responses of two other alpine Artemisia species, Artemisia glacialis and Artemisia genipi Weber. Contrary to A. genipi Weber, both A. umbelliformis and A. glacialis showed a significant increase of P concentration in shoots. Volatile components were analyzed by GC–MS in shoots of A. umbelliformis 6 months after inoculation. The alpine microbial inoculum increased significantly the percentage of E-β-ocimene and reduced those of E-2-decenal and (E,E)-2-4-decadienal indicating an influence of alpine microbial inoculum on essential oil production. This work provides practical indications for the use of native AM fungi for A. umbelliformis field culture.

With the aim of obtaining new strategies to control plant diseases, we investigated the ability of antagonistic lipopolypeptides (paenimyxin) from Paenibacillus sp. strain B2 to elicit hydrogen peroxide (H 2 O 2 ) production and several defense-related genes in the model legume Medicago truncatula . For this purpose, M. truncatula cell suspensions were used and a pathosystem between M. truncatula and Fusarium acuminatum was established. In M. truncatula cell cultures, the induction of H 2 O 2 reached a maximum 20 min after elicitation with paenimyxin, whereas concentrations higher than 20 μM inhibited H 2 O 2 induction and this was correlated with a lethal effect. In plant roots incubated with different concentrations of paenimyxin for 24 h before inoculation with F. acuminatum , paenimyxin at a low concentration (ca. 1 μM) had a protective effect and suppressed 95% of the necrotic symptoms, whereas a concentration higher than 10 μM had an inhibitory effect on plant growth. Gene responses were quantified in M. truncatula by semiquantitative reverse transcription-PCR (RT-PCR). Genes involved in the biosynthesis of phytoalexins (phenylalanine ammonia-lyase, chalcone synthase, chalcone reductase), antifungal activity (pathogenesis-related proteins, chitinase), or cell wall (invertase) were highly upregulated in roots or cells after paenimyxin treatment. The mechanisms potentially involved in plant protection are discussed.

International audience; The beneficial effects of arbuscular mycorrhizal (AM) fungi on plant performance and soil health are essential for the sustainable management of agricultural ecosystems. Nevertheless, since the ‘first green revolution’, less attention has been given to beneficial soil microorganisms in general and to AM fungi in particular. Human society benefits from a multitude of resources and processes from natural and managed ecosystems, to which AM make a crucial contribution. These resources and processes, which are called ecosystem services, include products like food and processes like nutrient transfer. Many people have been under the illusion that these ecosystem services are free, invulnerable and infinitely available; taken for granted as public benefits, they lack a formal market and are traditionally absent from society’s balance sheet. In 1997, a team of researchers from the USA, Argentina and the Netherlands put an average price tag of US $33 trillion a year on these fundamental ecosystem services. The present review highlights the key role that the AM symbiosis can play as an ecosystem service provider to guarantee plant productivity and quality in emerging systems of sustainable agriculture. The appropriate management of ecosystem services rendered by AM will impact on natural resource conservation and utilisation with an obvious net gain for human society.

International audience; The rhizodeposition of plants dramatically influence the surrounding soil and its microflora. Root exudates have pronounced selective and promoting effects on specific microbial populations which are able to respond with chemotaxis and fast growth responses, such that only a rather small subset of the whole soil microbial diversity is finally colonizing roots successfully. The exudates carbon compounds provide readily available nutrient and energy sources for heterotrophic organisms but also contribute e.g. complexing agents, such as carboxylates, phenols or siderophores for the mobilization and acquisition of rather insoluble minerals. Root exudation can also quite dramatically alter the pH- and redox-milieu in the rhizosphere. In addition, not only specific stimulatory compounds, but also antimicrobials have considerable discriminatory effect on the rhizosphere microflora. In the “biased rhizosphere” concept, specific root associated microbial populations are favored based on modification of the root exudation profile. Rhizosphere microbes may exert specific plant growth promoting or biocontrol effects, which could be of great advantage for the plant host. Since most of the plant roots have symbiotic fungi, either arbuscular or ectomycorrhizal fungi, the impact of plants towards the rhizosphere extends also to the mycorrhizosphere. The selective effect of the roots towards the selection of microbes also extends towards the root associated and symbiotic fungi. While microbes are known to colonize plant roots endophytically, also mycorrhiza are now known to harbor closely associated bacterial populations even within their hyphae. The general part of the manuscript is followed by the more detailed presentation of specific examples for the selection and interaction of roots and microbes, such as in the rhizosphere of strawberry, potato and oilseed rape, where the soil-borne plant pathogen Verticillium dahliae can cause high yield losses; the potential of biocontrol by specific constituents of the rhizosphere microbial community is demonstrated. Furthermore, plant cultivar specificity of microbial communities is described in different potato lines including the case of transgenic lines. Finally, also the specific selective effect of different Medicago species on the selection of several arbuscular mycorrhizal taxa is presented.

International audience; * • Mechanisms of root penetration by arbuscular mycorrhizal (AM) fungi are unknown and investigations are hampered by the lack of transformation systems for these unculturable obligate biotrophs. Early steps of host infection by hemibiotrophic fungal phytopathogens, sharing common features with those of AM fungal colonization, depend on the transcription factor STE12. * • Using degenerated primers and rapid amplification of cDNA ends, we isolated the full-length cDNA of an STE12-like gene, GintSTE, from Glomus intraradices and profiled GintSTE expression by real-time and in situ RT-PCR. GintSTE activity and function were investigated by heterologous complementation of a yeast ste12Δ mutant and a Colletotrichum lindemuthianum clste12Δ mutant. * • Sequence data indicate that GintSTE is similar to STE12 from hemibiotrophic plant pathogens, especially Colletotrichum spp. Introduction of GintSTE into a noninvasive mutant of C. lindemuthianum restored fungal infectivity of plant tissues. GintSTE expression was specifically localized in extraradicular fungal structures and was up-regulated when G. intraradices penetrated roots of wild-type Medicago truncatula as compared with an incompatible mutant. * • Results suggest a possible role for GintSTE in early steps of root penetration by AM fungi, and that pathogenic and symbiotic fungi may share common regulatory mechanisms for invasion of plant tissues.

The effect of paenimyxin, a new biopesticide produced by Paenibacillus sp. strain B2, on the density of soil bacterial communities was assessed by colony counting and by 16S rDNA and nirK quantitative polymerase chain reaction (PCR). Paenimyxin had a negative effect on the bacterial colony-forming unit (CFU) number, which was significantly reduced 2 and 4 days after treatment. The effect of paenimyxin on cultivatable bacteria was negligible 7 days after treatment. Approximately 107 16S rDNA sequences per gram of soil (dry weight) were detected by quantitative PCR in all samples. Paenimyxin did not affect the quantification of 16S rDNA or of the denitrifying bacterial community. In addition, RISA fingerprinting showed that the genetic structure of the bacterial communities was significantly modified 2 days after paenimyxin application at 50 µM and 4 days after treatment at lower concentrations (0.5 and 5 µM). The impact of paenimyxin treatment on the genetic structure of soil bacterial communities was transient, as no effect could be observed after 7, 14 and 28 days when compared with the untreated control. Copyright © 2007 Society of Chemical Industry

Analysis of arbuscular mycorrhizal (AM) fungal diversity through morphological characters of spores and intraradicular hyphae has suggested previously that preferential associations occur between plants and AM fungi. A field experiment was established to investigate whether AM fungal diversity is affected by different host plants in upland grasslands. Indigenous vegetation from plots in an unimproved pasture was replaced with monocultures of either Agrostis capillaris or Lolium perenne. Modification of the diversity of AM fungi in these plots was evaluated by analysis of partial sequences in the large subunit (LSU) ribosomal RNA (rDNA) genes. General primers for AM fungi were designed for the PCR amplification of partial sequences using DNA extracted from root tissues of A. capillaris and L. perenne. PCR products were used to construct LSU rDNA libraries. Sequencing of randomly selected clones indicated that plant roots were colonised by AM fungi belonging to the genera Glomus, Acaulospora and Scutellospora. There was a difference in the diversity of AM fungi colonising roots of A. capillaris and L. perenne that was confirmed by PCR using primers specific for each sequence group. These molecular data suggest the existence of a selection pressure of plants on AM fungal communities.

To construct macro- and microarray tools suitable for expression profiling in root endosymbioses of the model legume Medicago truncatula, we PCR-amplified a total of 6048 cDNA probes representing genes expressed in uninfected roots, mycorrhizal roots and young root nodules [Nucleic Acids Res. 30 (2002) 5579]. Including additional probes for either tissue-specific or constitutively expressed control genes, 5651 successfully amplified gene-specific probes were used to grid macro- and to spot microarrays designated Mt6k-RIT (M. truncatula 6k root interaction transcriptome). Subsequent to a technical validation of microarray printing, we performed two pilot expression profiling experiments using Cy-labeled targets from Sinorhizobium meliloti-induced root nodules and Glomus intraradices-colonized arbuscular mycorrhizal roots. These targets detected marker genes for nodule and arbuscular mycorrhiza development, amongst them different nodule-specific leghemoglobin and nodulin genes as well as a mycorrhiza-specific phosphate transporter gene. In addition, we identified several dozens of genes that have so far not been reported to be differentially expressed in nodules or arbuscular mycorrhiza thus demonstrating that Mt6k-RIT arrays serve as useful tools for an identification of genes relevant for legume root endosymbioses. A comprehensive profiling of such candidate genes will be very helpful to the development of breeding strategies and for the improvement of cultivation management targeted at increasing legume use in sustainable agricultural systems. (C) 2003 Elsevier B.V. All rights reserved.

International audience; We report on a large-scale expressed sequence tag(EST) sequencing and analysis program aimed atcharacterizing the sets of genes expressed in rootsof the model legumeMedicago truncatuladuringinteractions with either of two microsymbionts,the nitrogen-Æxing bacteriumSinorhizobium melilotior the arbuscular mycorrhizal fungusGlomusintraradices.We have designed speciÆc tools forinsilicoanalysis of EST data, in relation to chimericcDNA detection, EST clustering, encoded proteinprediction, and detection of differential expression.Our 21 473 5¢- and 3¢-ESTs could be grouped into6359 EST clusters, corresponding to distinct virtualgenes, along with 52 498 otherM.truncatulaESTsavailable in the dbEST (NCBI) database that wererecruited in the process. These clusters were manu-ally annotated, using a speciÆcally developed anno-tation interface. Analysis of EST cluster distributionin variousM.truncatulacDNA libraries, supportedby a reÆnedRtest to evaluate statistical signiÆ-cance and by `electronic northern' representation,enabled us to identify a large number of novelgenes predicted to be up- or down-regulated duringeither symbiotic root interaction. Thesein silicoanalyses provide a Ærst global view of the geneticprograms for root symbioses inM.truncatula.Asearchable database has been built and can beaccessed through a public interfaceWe report on a large-scale expressed sequence tag(EST) sequencing and analysis program aimed atcharacterizing the sets of genes expressed in rootsof the model legumeMedicago truncatuladuringinteractions with either of two microsymbionts,the nitrogen-Æxing bacteriumSinorhizobium melilotior the arbuscular mycorrhizal fungusGlomusintraradices.We have designed speciÆc tools forinsilicoanalysis of EST data, in relation to chimericcDNA detection, EST clustering, encoded proteinprediction, and detection of differential expression.Our 21 473 5¢- and 3¢-ESTs could be grouped into6359 EST clusters, corresponding to distinct virtualgenes, along with 52 498 otherM.truncatulaESTsavailable in the dbEST (NCBI) database that wererecruited in the process. These clusters were manu-ally annotated, using a speciÆcally developed anno-tation interface. Analysis of EST cluster distributionin variousM.truncatulacDNA libraries, supportedby a reÆnedRtest to evaluate statistical signiÆ-cance and by `electronic northern' representation,enabled us to identify a large number of novelgenes predicted to be up- or down-regulated duringeither symbiotic root interaction. Thesein silicoanalyses provide a Ærst global view of the geneticprograms for root symbioses inM.truncatula.Asearchable database has been built and can beaccessed through a public interface

Fluorescence in situ hybridization (FISH) was applied to interphasic nuclei isolated from spores of four species of AM fungi : Scutellospora castanea, Glomus mosseae, Glomus intraradices and Gigaspora rosea. Ribosomal DNA loci were visualized using digoxigenin-labeled 25 S rDNA probes obtained by nested PCR. Several hybridization sites were detected per nucleus and an internuclear variability was observed in the number of loci. This is the first report of successful application of FISH to analyse the genomes of glomalean fungi.

International audience; A full-length cDNA encoding Neurospora crassa calmodulin was isolated from a lambdaZAP II cDNA expression library. The open reading frame encodes a protein of 148 amino acid residues with a calculated M(r) of 16,865 Da. Using site-directed mutagenesis, the complete cDNA was ligated into a trc promoter-regulated bacterial expression vector to allow expression of N. crassa calmodulin in E. coli. The expressed protein was found to be identical to the native protein on the basis of some of its biochemical properties. Finally, Southern analysis of restriction digests of genomic DNA indicates that calmodulin is encoded by a single-copy gene.

Calmodulin is a small Ca 2+ -binding protein proposed to act as the intracellular Ca 2+ receptor that translates Ca 2+ signals into cellular responses. We have constructed mutant yeast calmodulins in which the Ca 2+ -binding loops have been altered by site-directed mutagenesis. Each of the mutant proteins has a dramatically reduced affinity for Ca 2+ ; one does not bind detectable levels of 45 Ca 2+ either during gel filtration or when bound to a solid support. Furthermore, none of the mutant proteins change conformation even in the presence of high Ca 2+ concentrations. Surprisingly, yeast strains relying on any of the mutant calmodulins not only survive but grow well. In contrast, yeast strains deleted for the calmodulin gene are not viable. Thus, calmodulin is required for growth, but it can perform its essential function without the apparent ability to bind Ca 2+ .

A calcium, calmodulin-dependent protein kinase activity has been partially purified by calmodulin-Sepharose affinity chromatography from the soluble fraction of Neurospora crassa . The phosphorylated peptide has an apparent molecular mass on SDS-polyacrylamide gel of 47 kDa. The apparent half maximal phosphorylation is obtained after 1.5 min at 30° C in the presence of calcium and calmodulin. The apparent half maximal activation of the phosphorylation is obtained at 1 μM calcium, and 0.1 or 0.2 μM calmodulin from bovine brain or Neurospora , respectively. The 32 P incorporation is enhanced about 10-fold by calmodulin.

Fluctuations in intracellular (Ca2+) calcium levels generate signaling events and regulate different cellular processes. Whilst the implication of Ca2+ in plant cell responses during arbuscular mycorrhiza (AM) interactions is well documented, nothing is known about the regulation or role of this secondary meesenger in the fungal symbiont. The molecular basis of fungal calcium homeostasis in the AM symbiosis was analyzed by investigating the expression of Ca2+-related fungal genes. In a first study, G. mosseae genes putatively encoding a MAP3k-like protein kinase (Gm2) and a P-type ATPase (Gm152) were investigated. Both Ca2+-related genes were up-regulated by A. sinicum root exudates, suggesting a role in early interactions prior to symbiosis establishment. The full-length cDNA sequence of Gm152 obtained from germinating spores of G. mosseae confirmed its identity. The role of Ca2+ in fungal processes leading to establishment of an AM symbiosis was investigated in more detail in G. intraradices-M. truncatula interactions. Enhanced expression of genes encoding six membrane transport proteins and one nuclear protein kinase, selected from the G. intraradices transcriptome database, was related to colonization of wild-type M. truncatula (line J5) roots and not observed with the mycorrhiza-resistant mutant dmi3/Mtsym13. Laser microdissection mapping of transcripts indicated that the Ca2+-related G. intraradices genes were differentially up-regulated in arbuscules and/or in intercellular hyphae. The tempo-spatial variations in fungal gene expression suggest different roles in the development or functioning of the AM symbiosis. Full-length cDNA of three G. intraradices genes putatively encoding a PMR-like endoplasmic reticulum P-type ATPase, a VCX1-like vacuolar Ca2+ ion transporter and a nuclear CCaMK were obtained for functional analyses in yeast mutants to gain insight into their role in the mycorrhizal symbiosis. Possible mechanisms are discussed in which Ca2+-related proteins of G. intraradices may play a role in the mobilization and perception of the intracellular messenger by the AM fungus during symbiotic interactions with host roots; Les fluctuations du taux de calcium (Ca2+) intracellulaire sont impliquées dans les événements de signalisation et de régulation de différents processus cellulaires. Alors que le role du Ca2+ dans la réponse des plantes lors des interactions mycorhiziennes à arbuscules (MA) interactions est bien documentée, il n’existe aucune information concernant la régulation ou le rôle de ce messager secondaire chez le symbiote fongique. La base moléculaire de l'homéostasie calcique fongique dans la symbiose MA a été analysée en étudiant l'expression de gènes fongiques liés au Ca2+. Dans un premier temps, des gènes de G. mosseae codant putativement pour une protéine kinase-like MAP3k (Gm2) et une P-type ATPase (Gm152) ont été étudiés. L’expression des deux gènes est stimulée par les exudats racinaires d’A. sinicum, suggérant un rôle dans les interactions précoces avant l'établissement de la symbiose. L’obtention de la séquence d'ADNc pleine longueur de Gm152 a confirmé son identité. Une étude plus approfondie du rôle de Ca2+ dans les processus fongiques impliqués dans la symbiose MA a été réalisée chez G. intraradices. L'expression de sept gènes fongiques encodant six protéines de transport membranaire calcique et une protéine kinase nucléaire, sélectionnés du séquençage transcriptomique du G. intraradices, était stimulée lors de la colonisation des racines de M. truncatula type sauvage (lignée J5) mais pas chez le mutant non-mycorhizienne dmi3/Mtsym13. La cartographie par microdissection laser des transcrits des gènes fongiques a indiqué une activation différentielle dans les arbuscules et/ou dans hyphes intercellulaires. Les variations tempo-spatiales de l'expression des gènes fongiques suggèrent des roles différents dans le développement ou le fonctionnement de la symbiose MA. L’ADNc pleine longueur a été obtenue de trois gènes de G. intraradices encodant un PMR1-like réticulum endoplasmique ATPase, un VCX1-like transporteur ionique vacuolaire et un CCaMK nucléaire pour des analyses fonctionnelles chez la levure afin de mieux comprendre leur rôle dans la symbiose MA. Les mécanismes par lesquels les protéines liées au Ca2+ pourraient jouer un rôle chez G. intraradices dans la mobilisation et la perception du messager secondaire au cours des interactions MA sont discutés