Claude Plassard

Strategies and methods for studying the rhizosphere—the plant science toolbox

This review summarizes and discusses methodological approaches for studies on the impact of plant roots on the surrounding rhizosphere and for elucidation of the related mechanisms, covering a range from simple model experiments up to the field scale. A section on rhizosphere sampling describes tools and culture systems employed for analysis of root growth, root morphology, vitality testing and for monitoring of root activity with respect to nutrient uptake, water, ion and carbon flows in the rhizosphere. The second section on rhizosphere probing covers techniques to detect physicochemical changes in the rhizosphere as a consequence of root activity. This comprises compartment systems to obtain rhizosphere samples, visualisation techniques, reporter gene approaches and remote sensing technologies for monitoring the conditions in the rhizosphere. Approaches for the experimental manipulation of the rhizosphere by use of molecular and genetic methods as tools to study rhizosphere processes are discussed in a third section. Finally it is concluded that in spite of a wide array of methodological approaches developed in the recent past for studying processes and interactions in the rhizosphere mainly under simplified conditions in model experiments, there is still an obvious lack of methods to test the relevance of these findings under real field conditions or even on the scale of ecosystems. This also limits reliable data input and validation in current rhizosphere modelling approaches. Possible interactions between different environmental factors or plant-microbial interactions (e.g. mycorrhizae) are frequently not considered in model experiments. Moreover, most of the available knowledge arises from investigations with a very limited number of plant species, mainly crops and studies considering also intraspecific genotypic differences or the variability within wild plant species are just emerging.

Two differentially regulated phosphate transporters from the symbiotic fungus Hebeloma cylindrosporum and phosphorus acquisition by ectomycorrhizal Pinus pinaster.

Ectomycorrhizal symbiosis markedly improves plant phosphate uptake, but the molecular mechanisms underlying this benefit are still poorly understood. We identified two ESTs in a cDNA library prepared from the ectomycorrhizal basidiomycete Hebeloma cylindrosporum with significant similarities to phosphate transporters from the endomycorrhizal fungus Glomus versiforme and from non-mycorrhizal fungi. The full-length cDNAs corresponding to these two ESTs complemented a yeast phosphate transport mutant (Deltapho84). Measurements of (33)P-phosphate influx into yeast expressing either cDNA demonstrated that the encoded proteins, named HcPT1 and HcPT2, were able to mediate Pi:H(+) symport with different affinities for Pi (K(m) values of 55 and 4 mum, respectively). Real-time RT-PCR showed that Pi starvation increased the levels of HcPT1 transcripts in H. cylindrosporum hyphae grown in pure culture. Transcript levels of HcPT2 were less dependent on Pi availability. The two transporters were expressed in H. cylindrosporum associated with its natural host plant, Pinus pinaster, grown under low or high P conditions. The presence of ectomycorrhizae increased net Pi uptake rates into intact Pinus pinaster roots at low or high soil P levels. The expression patterns of HcPT1 and HcPT2 indicate that the two fungal phosphate transporters may be involved in uptake of phosphate from the soil solution under the two soil P availability conditions used.

Diversity in phosphorus mobilisation and uptake in ectomycorrhizal fungi

Abstract• IntroductionPhosphorus (P) is often the first or second element limiting aboveground net primary productivity of forests. Besides low available inorganic orthophosphate (Pi) concentrations, soil may contain high total P contents, as insoluble mineral P or as organic P. Most plants form mycorrhizal associations that improve their P nutrition. Three main hypotheses have been proposed to explain this positive effect through an increase of (1) P mobilisation from mineral P, (2) P mobilisation from organic P and (3) soil exploration and P uptake. However, the positive effect of mycorrhizal symbiosis may be variable with the fungal species forming the association. This could be due to the different abilities of mycorrhizal fungi to mobilise P and/or to take up Pi from the soil.• ObjectivesThe aim of this review was to examine our current knowledge about the capacity of ectomycorrhizal fungi to release organic compounds as low-molecular-weight organic anions and phosphatases thought to have a role for mineral and organic P mobilisation, respectively. The diversity of Pi transporters among mycorrhizal species is also examined.• ResultsThe main conclusion is that the study of the functional diversity of ectomycorrhizal fungi in situ is still a challenging question and could be addressed by combining different tools now available to make large-scale studies.

Efficiency of acid phosphatases secreted from the ectomycorrhizal fungus Hebeloma cylindrosporum to hydrolyse organic phosphorus in podzols

Ectomycorrhizal fungi may improve the phosphate nutrition of their host plants by secreting, into the soil solution, acid phosphatases (AcPases) able to release orthophosphate (Pi) from soil organic phosphorus (Po). Using cation-exchange chromatography, we separated four fractions with AcPase activity secreted by the ectomycorrhizal fungus Hebeloma cylindrosporum grown in a pure culture under P-starved conditions. Each AcPase active fraction displayed strong ability in vitro to hydrolyse a wide range of phosphate monoesters, but none of them efficiently hydrolysed phytate. Their efficiency to release Pi from soil NaHCO(3)-extractable Po was studied in a sandy podzol used intact or autoclaved. Soils were collected in a 15-year-old Pinus pinaster stand, receiving regular fertilization or not. Autoclaving increased the NaHCO(3)-extractable Po concentrations by 55% in unfertilized and by 32-43% in fertilized soils. The efficiency of each AcPase fraction was affected significantly by the soil fertilization regime and the soil treatment (intact vs. autoclaved). The proportion of labile Po enzyme ranged from 0% to 11% and 14% to 48% after 1 h of incubation in bicarbonate solutions extracted from intact and autoclaved soils, respectively. This work suggests that AcPases secreted from H. cylindrosporum could be efficient in recycling Po pools from soil microorganisms that may be delivered by soil autoclaving.

Pinus pinaster seedlings and their fungal symbionts show high plasticity in phosphorus acquisition in acidic soils.

Young seedlings of maritime pine (Pinus pinaster Soland in Aït.) were grown in rhizoboxes using intact spodosol soil samples from the southwest of France, in Landes of Gascogne, presenting a large variation of phosphorus (P) availability. Soils were collected from a 93-year-old unfertilized stand and a 13-year-old P. pinaster stand with regular annual fertilization of either only P or P and nitrogen (N). After 6 months of culture in controlled conditions, different morphotypes of ectomycorrhiza (ECM) were used for the measurements of acid phosphatase activity and molecular identification of fungal species using amplification of the ITS region. Total biomass, N and P contents were measured in roots and shoots of plants. Bicarbonate- and NaOH-available inorganic P (Pi), organic P (Po) and ergosterol concentrations were measured in bulk and rhizosphere soil. The results showed that bulk soil from the 93-year-old forest stand presented the highest Po levels, but relatively higher bicarbonate-extractable Pi levels compared to 13-year-old unfertilized stand. Fertilizers significantly increased the concentrations of inorganic P fractions in bulk soil. Ergosterol contents in rhizosphere soil were increased by fertilizer application. The dominant fungal species was Rhizopogon luteolus forming 66.6% of analysed ECM tips. Acid phosphatase activity was highly variable and varied inversely with bicarbonate-extractable Pi levels in the rhizosphere soil. Total P or total N in plants was linearly correlated with total plant biomass, but the slope was steep only between total P and biomass in fertilized soil samples. In spite of high phosphatase activity in ECM tips, P availability remained a limiting nutrient in soil samples from unfertilized stands. Nevertheless young P. pinaster seedlings showed a high plasticity for biomass production at low P availability in soils.

Do pH changes in the leaf apoplast contribute to rapid inhibition of leaf elongation rate by water stress? Comparison of stress responses induced by polyethylene glycol and down-regulation of root hydraulic conductivity

We have dissected the influences of apoplastic pH and cell turgor on short-term responses of leaf growth to plant water status, by using a combination of a double-barrelled pH-selective microelectrodes and a cell pressure probe. These techniques were used, together with continuous measurements of leaf elongation rate (LER), in the (hidden) elongating zone of the leaves of intact maize plants while exposing roots to various treatments. Polyethylene glycol (PEG) reduced water availability to roots, while acid load and anoxia decreased root hydraulic conductivity. During the first 30 min, acid load and anoxia induced moderate reductions in leaf growth and turgor, with no effect on leaf apoplastic pH. PEG stopped leaf growth, while turgor was only partially reduced. Rapid alkalinization of the apoplast, from pH 4.9 ± 0.3 to pH 5.8 ± 0.2 within 30 min, may have participated to this rapid growth reduction. After 60 min, leaf growth inhibition correlated well with turgor reduction across all treatments, supporting a growth limitation by hydraulics. We conclude that apoplastic alkalinization may transiently impair the control of leaf growth by cell turgor upon abrupt water stress, whereas direct hydraulic control of growth predominates under moderate conditions and after a 30-60 min delay following imposition of water stress.

The Beneficial Effect of Mycorrhizae on N Utilization by the Host-Plant: Myth or Reality?

Nitrogen represents the major element in plants. This element is present in most of the basic constituents: purines, pyrimidines, and amino acids, and is important for the autotrophic capacity of the plants, being part of the light harvesting molecules, e.g., chlorophyll. De facto, N deficiency in plants leads rapidly to chlorosis and stunted growth. Plants being sessile organisms, they are completely dependent on the N availability in the soil solution for their growth and productivity. In soil, a harsh competition exists for N acquisition between the microorganisms (mainly bacteria and fungi) and the plants. However, plant species have developed various strategies of N acquisition. These strategies are including microorganism partners into a symbiotic association. First, legumes are associated with bacteria to assimilate aerial N. A second form of mutualism is the association between plants and mycorrhizal fungi. The fungus and the plant are associated underground forming a close bound between the hyphae and the root cells, the so-called mycorrhizal root. These mycorrhizal roots are characterized by the presence of an extensive network of mycelium developing out of the root and exploring the soil. Therefore, the fungal partner considerably increases the volume of soil that is exploited compared to the nonmycorrhizal root system (Rousseau et al. 19994). The mycelium also develops between the root cells inside the root cortex. All mineral and nutrient exchanges between the host cell (carbohydrate release) and the fungal cells (phosphorus and nitrogen release) are thought to take place in this fungal‐plant interface (Smith and Read 1997). Melin and Nilsson (1952) were the first to demonstrate the N uptake by the extra radical mycelium and its transfer to the host plant. Since this pioneering study, a considerable amount of work has been devoted to the capacities of mycorrhizal symbiosis to modify the access to the various N-forms from the soil, including the uptake and assimilation of N by the partners. The main purpose of

HcPT1.2 participates in Pi acquisition in Hebeloma cylindrosporum external hyphae of ectomycorrhizas under high and low phosphate conditions

ABSTRACT Ectomycorrhizal fungi improve tree phosphorus nutrition through transporters specifically localized at soil-hyphae and symbiotic interfaces. In the model symbiosis between the fungus Hebeloma cylindrosporum and the maritime pine (Pinus pinaster), several transporters possibly involved in phosphate fluxes were identified, including three H+:Pi transporters. Among these three, we recently unraveled the function of one of them, named HcPT2, in both pure culture and symbiotic interaction with P. pinaster. Here we investigated the transporter named HcPT1.2, by analyzing inorganic phosphate transport ability in a yeast complementation assay, assessing its expression in the fungus associated or not with the plant, and immunolocalizing the proteins in ectomycorrhizas. We also evaluated the effect of external Pi concentration on expression and localization of HcPT1.2. Our results revealed that HcPT1.2 is involved in Pi acquisition by H. cylindrosporum mycelium, irrespective of the external Pi concentrations.