Impact of intercropping cultures on truffle production and soil microbial communities in Mediterranean oak orchards

Truffle orchards are agroecosystems that play an economic, cultural and structural role in Mediterranean landscapes. In Southern France, some truffle growers associate secondary production in truffle orchards, such as the intercropping of aromatic and medicinal plants (AMP). Recent works described AMPs as acting positively on soil microbial activity. We hypothesized that this beneficial impact of AMPs may also improve truffle production through direct and indirect mechanisms involving biotic and abiotic soil parameters. The survey, conducted among truffle growers, indicates that AMP intercropping was mainly limited to lavender (Lavandula hybrida) production, and occasionally to rosemary (Rosmarinus officinalis) and thyme (Thymus vulgaris). According to one-third of the truffle growers, the production of truffles was improved and for 40% this intercropping allowed a contribution of organic matter and a protection against the drought. The study aimed at better testing this reported effect and exploring the mechanisms underlying this pattern by characterizing soil physicochemical and microbial community changes induced by AMP culture. A multidisciplinary approach associating microbiology and molecular ecology has been conducted in truffle orchards with a historical practice of AMP intercropping culture. We report a positive of AMP culture on Tuber populations, with no major change on soil microbial community diversity. Nevertheless, we identify microbial taxa indicators associated with truffle production and/or plant types. The investigation of a wider range of truffle-based agroecosystems may provide important guidelines for the development of intercropping culture to improve the productivity and sustainability of truffle orchards

Transcriptome analysis of Azospirillum lipoferum during its interaction with rice

The associative symbiosis between Plant Growth Promoting Rhizobacteria of the genus Azospirillum and cereals have mainly been studied from an agronomic and economic point of view, and several studies showed that plant morphological and metabolic changes depend on both bacterial and plant genotypes. However, if the specificity in the Rhizobium-legume symbiosis has been well characterized, the question of whether specificity occurs in the Azospirillum-plant associative symbiosis remains controversial. In this context, the overall gene expression of A. lipoferum 4B during its interaction with roots of two rice varieties (cv. Cigalon, cv. Nipponbare) was analyzed in order to characterize (i) genes differentially regulated in response to plant regardless of the variety and (ii) genes displaying a varietydependent regulation. Results of the transcriptomic analysis show that presence of the host plant triggers stress response systems, a large number of putative transcriptional regulators, signal transduction pathways, and many proteins of unknown function. This indicates a reprogramming of bacterial gene expression, due to adaptation to host plant. Genes specifically expressed during the interaction with one of the two varieties could be identified, suggesting the existence of specificity in the associative symbiosis between Azospirillum and cereals. (Texte intégral

Mycorrhizal networks and nitrogen fluxes between Pterocarpus officinalis and Taro in swamp forests of Guadeloupe

Swamp forests of Pterocarpus officinalis (jacq.) form remarkable monodominant forest stands growing on temporarily or permanently flooded soils in mangrove hinterland areas, along rivers and in wet depressions in the mountains of the Caribbean and Guiana regions. In Guadeloupe, smallholder farmers traditionally cultivate flooded Taro (Colocasia esculenta (L.) Schott) monocultures under the canopy of P. officinalis stands in the swamp forests. Taro corms and unrolled leaves are commonly consumed in Guadeloupe. The understorey culture of Taro is conducted without pesticides and fertilizers, which could be partly due to the net input of nitrogen into the soil by P. officinalis through its ability to fix atmospheric nitrogen. Furthermore, the mycorrhizal networks could favor the transfer of fixed nitrogen from P. officinalis to the intercropped Taro. Taro cultivation is conducted during the dry season to facilitate their planting between mature Pterocarpus trees and their harvest when the marshy soils are dewatered. The sampling of roots and leaves on Taro and two cohorts of P. officinalis (mature trees and seedlings) were made during the dry season in two swamp forest sites located at Gosier (approx. 0.1 ha in area) and Belle-Plaine (approx. 0.5 ha in area) in the island of Grande-Terre, Guadeloupe. The arbuscular mycorrhizal (AM) fungal community was compared between Taro and two cohorts of Pterocarpus, by using pyrosequencing of partial 18S rDNA gene. We also compared natural abundance of 13C and 15N contents in leaves of the two cohorts of Pterocarpus, Taro and surrounding non-nitrogen-fixing plant species, in order to estimate what proportions of N and C were transferred to Taro. Of the 210,676 sequences, 37,631 sequences were assigned to a total of 215 OTUs belonging to the orders of Glomerales, Paraglomerales, Archeosporales and Diversisporales. A low AM fungal community membership was observed between P. officinalis and C. esculenta. However, certain AM fungal community taxa overlapped between both plants, notably predominant Funneliformis OTUs, suggesting a potential common AM network. The isotopic analyses did not show any direct link between the mycorrhizal status of both associated species and the transfer of N and C between P. officinalis and Taro. The proportion of N derived from atmospheric fixation in P. officinalis varied according to the study site, from about 80 to 95% in adult trees and from 50 to 80% in young seedlings while we estimated that 35% of fixed N was transferred from Pterocarpus seedlings to Taro in mixed stands of both species. The implementation of experiments under controlled conditions are needed to demonstrate the possible role of mycorrhizal networks in the transfer of nutrients between P. officinalis and Taro

Assessment of functional diversity and structure of phytate-hydrolysing bacterial community in Lolium perenne rhizosphere

Background and aims Plant growth is frequently limited by the availability of inorganic phosphorus (P) in the soil. In most soils, a considerable amount of the soil P is bound to organic molecules. Of these, phytate is the most abundant identifiable organic P form, but is not readily available to plants. In contrast, microorganisms have been shown to degrade phytate with high efficiency. The current study aims to characterize the members of the phytate-hydrolysing bacterial community in rhizosphere, and the molecular and enzymatic ability of these bacteria to degrade phytate. Methods and results The phytate-hydrolysing bacterial community was characterized from the rhizosphere of plants cultivated in the presence or absence of phytate supplementation. Major changes in the bacterial community structure were observed with both culturedependent and -independent methods, which highlighted the predominance of Proteobacteria and Actinobacteria. Phytase activity was detected for a range of rhizobacterial isolates as well as the presence of, β-propeller phytases (BPP) for both isolates and directly in a soil sample. Conclusion Awide taxonomic range of functional phytate utilizers have been discovered, in soil bacterial taxa that were previously not well known for their ability to utilise phytate as P or C sources. This study provides new insights into microbial carbon and phosphorus cycling in soil. (Résumé d'auteur

Host specificity of the plant growth-promoting cooperation between Azospirillum and rice

1. Introduction. Host specificity is a fundamental concept in understanding evolutionary processes leading to intimate interactions between bacteria and plants. In the case of Plant Growth-Promoting Rhizobacteria (PGPR) specificity appears to be controlled either by a strain-specific bacterial adaptation to non-specific traits of the host plant or by non-specific bacterial adaptation to genotype-specific properties of the host plant (1). Thus, we hypothesize that these adaptations result in the regulation of a large number of genes, independently of their direct involvement in phytostimulation. These regulations may depend on the bacterial strain/plant genotype combination. 2. Objectives. This work aims at identifying genes involved in reciprocal adaptation of partners and those involved in host specificity in PGPR-plant cooperation. 3. Materials & methods. To evaluate transcriptomic responses of each partner during the Azospirillum-rice cooperation, RNA samples obtained from Azospirillum root-associated cells and rice roots (cultivars Nipponbare and Cigalon) were analyzed on microarrays. 4. Results. Transcriptomic analyses evidence the regulation of 453 genes in root-associated Azospirillum cells and 7384 genes in rice roots. Whereas none of the Azospirillum properties involved in the modification of plant hormonal balance are significantly regulated under the experimental conditions, several genes of the plant partner implicated in phytohormone signaling are induced or repressed during the interaction. The induction of plant and bacterial genes involved in ROS detoxification suggest that defense response of the host plant play a key role in Azospirillum-rice cooperation. In addition, many genes display expression profile that depends on the strain/cultivar combination. 5. Conclusion. Combination specific responses observed at the transcriptomic level are consistent with metabolomic observations previously reported for Azospirillum-rice cooperation (2), suggesting that evolutionary processes have led to a preferential interaction between a strain and its original host cultivar. (Résumé d'auteur

Molecular characterization of arbuscular mycorrhizal fungi in an agroforestry system reveals the predominance of Funneliformis spp. associated with Colocasia esculenta and Pterocarpus officinalis adult trees and seedlings

Pterocarpus officinalis (Jacq.) is a leguminous forestry tree species endemic to Caribbean swamp forests. In Guadeloupe, smallholder farmers traditionally cultivate flooded taro (Colocasia esculenta) cultures under the canopy of P. officinalis stands. The role of arbuscular mycorrhizal (AM) fungi in the sustainability of this traditional agroforestry system has been suggested but the composition and distribution of AM fungi colonizing the leguminous tree and/or taro are poorly characterized. An in-depth characterization of root-associated AM fungal communities from P. officinalis adult trees and seedlings and taro cultures, sampled in two localities of Guadeloupe, was performed by pyrosequencing (GS FLX+) of partial 18S rRNA gene. The AM fungal community was composed of 215 operational taxonomic units (OTUs), belonging to eight fungal families dominated by Glomeraceae, Acaulosporaceae, and Gigasporaceae. Results revealed a low AM fungal community membership between P. officinalis and C. esculenta. However, certain AM fungal community taxa (10% of total community) overlapped between P. officinalis and C. esculenta, notably predominant Funneliformis OTUs. These findings provide new perspectives in deciphering the significance of Funneliformis in nutrient exchange between P. officinalis and C. esculenta by forming a potential mycorrhizal network. (Résumé d'auteur

Increasing the diversity of leguminous plant improves soil functionalities and wheat growth in a P-deficient soil

A study was conducted in glasshouse conditions to assess the influences of the leguminous plant diversity on the soil microbial functions and their consequences on the wheat growth. Three legume species were targeted: faba bean, alfalfa and pea. All the combinations of one, two or three species were performed in pots filled with a P deficient soil collected from a field located near Marrakech (Morocco). After 3 months culture, the plants were harvested and the shoot and root parts were dried, weighed and analyzed for their N and P contents. The soil catabolic functions were measured using the SIR (Substrate Induced Respiration) method. The mycorrhizal soil infectivity was assessed according to Kisa et al. (2007) and the arbuscular mycorrhizal (AM) diversity was estimated by PCR/Sequencing. Fluorescent Pseudomonads (FP), known to have PGPR capacities have been enumerated, identified (PCR/sequencing) and characterized for their inorganic phosphate solubilizing activities and for their effect on wheat growth. The results showed that an increase of legume diversity involved: (i) significant differences between the microbial functions within the treatments, (ii) changes in the abundance and diversity of the AM communities, (iii) an increase of the FP abundance, most of them solubilizing inorganic phosphate and promoting the wheat growth. These results suggest that the management of the legume plant cover diversity can optimize the positive impact of legume on the agrosystem productivity resulting from an increase in soil microbial functions, soil microbial diversity, AM symbiosis efficiency in sustainable agricultural practices (crop rotation, intercropping systems, etc). Acknowlegments: This work was funded by the project Fabatropimed supported by Agropolis Foundation under the reference ID 1001-009. (Texte intégral

Genome wide profiling of Azospirillum lipoferum 4B gene expression during interaction with rice roots

Azospirillum-plant cooperation has been mainly studied from an agronomic point of view leading to a wide description of mechanisms implicated in plant growth-promoting effects. However, little is known about genetic determinants implicated in bacterial adaptation to the host plant during the transition from free-living to root-associated lifestyles. This study aims at characterizing global gene expression of Azospirillum lipoferum 4B following a 7-day-old interaction with two cultivars of Oryza sativa L. japonica (cv. Cigalon from which it was originally isolated, and cv. Nipponbare). The analysis was done on a whole genome expression array with RNA samples obtained from planktonic cells, sessile cells, and root-adhering cells. Root-associated Azospirillum cells grow in an active sessile-like state and gene expression is tightly adjusted to the host plant. Adaptation to rice seems to involve genes related to reactive oxygen species (ROS) detoxification and multidrug efflux, as well as complex regulatory networks. As revealed by the induction of genes encoding transposases, interaction with root may drive bacterial genome rearrangements. Several genes related to ABC transporters and ROS detoxification display cultivar-specific expression profiles, suggesting host specific adaptation and raising the question of A. lipoferum 4B/rice cv. Cigalon co-adaptation. (Résumé d'auteur

Impacto de la simbiosis micorrítica arbuscular en el crecimiento temprano del cultivo de tara (Caesalpinia spinosa (Molina) Kuntze)

Tara (Caesalpinia spinosa) ha sido cultivada durante muchos años en bosques naturales, principalmente para la recolección de sus vainas y sus semillas dado su alto valor por sus múltiples usos desde tiempos antiguos. Sin embargo, poco se conoce sobre la ecología y el estado de conservación de los bosques de tara. La simbiosis micorrítica puede desempeñar un rol central en el crecimiento temprano de esta leguminosa y constituye un componente clave para la mejora de las prácticas de manejo. El objetivo de este estudio fue describir el estado micorrítico de tara en plantaciones localizadas en Perú y evaluar el impacto micorrítico sobre el crecimiento temprano de plántulas de Tara en condiciones de invernadero. Los resultados mostraron que la tara se asoció principalmente con hongos micorríticos arbusculares de la familia Glomeraceae, notablemente Rhizophagus spp. La micorrización controlada con esporas de R. irregularis mejoró significativamente el crecimiento de tara en invernadero, así como la absorción de nutrientes como el fósforo (P) y el nitrógeno (N). Por lo tanto, C. spinosa podría ser considerada como “altamente dependiente de la micorriza”. Estos resultados destacan la necesidad de considerar la simbiosis micorrítica arbuscular para mantener en forma sostenible la productividad y estabilidad de las plantaciones de tara

A quest for microbial indicators of the Tuber melanosporum production using an environmental genomics approach

The "brûlé" - the area where the truffles tend to grow to a mature stage- is commonly characterized by a drastic drop in the plant diversity and biomass around those trees, which have been mycorrhized by Tuber melanosporum. Despite its presence, which indicates the development as well as the activity of the mycelium network of this symbiont, the "brûlé" is not systematically associated with the production of ascocarps. To date, understanding how truffle producing ecosystems function has been a puzzling task for researchers, particularly when attempting to explain what elements are involved in the spatio-temporal dynamics of the colonization process, in the intensity of the "brûlés" and in what influences the yield. In the frame of the ANR SYSTRUF project (2010-2014), and within a partnership with the professional bodies (FFT, CETEF) and some volunteer truffle growers, we have aimed to identify which bacterial and mycorrhizal (Glomeromycetes) markers are intrinsically associated with the productive status of the "brûlés", in relation to the non-producing "brûlés" and to the "brûlés" - deprived oaks. Two "natural"-spontaneous and slightly managed-truffle orchards (truffières) and two "planted" ones were sampled during November 2010 in the Languedoc-Roussillon region (at the ascocarp maturation stage). These truffières are composed of the evergreen holm oak (Quercus ilex) and are managed without mechanical soil laboring; in addition, they possess a spontaneous plant cover, which is kept to a minimal extent by the owners. Soil and root samples from plant species persisting on the "brûlé" have been collected around the oak trees (classified as: with a producing "brûlé", with a non-producing "brûlé", and "brûlé"-deprived ones) in order to (1) determine by pyrosequencing the genetic diversity of the total soil bacterial community (16S rDNA gene) and of the arbuscular mycorrhizae (18S rDNA gene) associated to the oak companion plants, and to (2) potentially identify specific taxa linked to the producing status of the "brûlé", at the level of a single truffière or along the four sampling sites. Eventually, the approach of relating sequence data with the producing status and the floral diversity of the studied ecosystems opens up new avenues for the study of the interaction networks established between Tuber melanosporum and certain components of the microbial and floral compartments, characterizing the truffières managed with low anthropic input. (Résumé d'auteur