Mobilisation of soil phytate : what are the keys to unlock phosphorus from this treasure ? – UNLOCKP

Phosphorus (P) is of vital importance for the metabolic requirements for growth and replication and therefore for plant production. However, P is taken up by plants and microorganisms only as orthophosphate (Pi), represented by H2PO4- and HPO42-. Due to its strong capacity to make complexes with cations and to its low mobility, concentrations of free Pi in the soil solution are generally low and estimated between 1 to 10 µM, concentrations that are often limiting for growth and yield, leading to massive use of P fertilizer in agriculture. P fertilizers are produced only from mineral P and exhaustion of world reserves of high quality mineral P is planned to occur in 90 years. Remarkably, besides the low level of available free Pi, soils contain a high amount of P that is linked to C-containing compounds to form organic P (Po) composed mainly by myo-inositol hexakisphosphate (phytate). To be used by plants and soil microorganisms, the phosphate group of Po compounds must be released from the ester bond linking it to carbon (C-O-PO3H2) by enzymes that are phosphatases. The release of Pi from soil Po hydrolysis appears therefore as a good alternative to increase the actual Pi bioavailability for plant growth and to decrease the need for chemical P fertilizers. However, experiments using transgenic plants expressing the A. niger phytase PhyA were not always able to gain more P when phyate was supplied to a range of soil. Our hypothesis is that it is required to take into account the natural complexity of living organism in the root environment.
In this project, we propose to examine two new biological strategies to improve the use of soil phytate by plants. The first strategy (ecological strategy) is based on the specific functionnal activities and the interaction of different types of soil microorganisms and invertebrates associated with the roots: (i) the mycorrhizal fungi, which are already known to provide the host plant with phosphorus not directly available to plant roots, (ii) the free bacteria which are living in the mycosphere environment, especially those able to release phytase into their environment, together with (iii) the role of bacteriovorous nematodes in the accelerated turn-over of those bacterial communities by grazing, which will in turn accelerate the liberation of Pi resulting from the phytate mineralization. The second strategy (biotechnological strategy) is based upon the production of microbial phytases, whether from fungal or bacterial origin, by the mycorrhizal fungus itself using genetic engineering. We will produce three transgenic ectomycorrhizal fungal strains of Hebeloma cylindrosporum with (i) the PhyA gene of Aspergillus niger, (ii) the phytase gene of Debaryomyces castelli CB 2923, and (iii) the PhyC gene of Bacillus subtilis. H. cylindrosporum is very easily associated with Pinus pinaster, an economically important tree species in France. We will grow young maritime plants, whether associated or not with different fungal strains, wether or not inoculated with a phytate–degrading bacteria (B. subtilis) wich has been isolated from ectomycorrhizal roots of maritime pine collected in the field, whether or not inoculated with a bacterial feeding nematode that have been isolated from the same field as the B. subtilis.