Benefits provided by four ectomycorrhizal fungi to Pinus taeda under different external potassium availabilities.

Ectomycorrhizal fungi contribute to the nutrition of many woody plants, including those in the Pinaceae family. Loblolly pine (Pinus taeda L.), a native species of the Southeastern USA, can be colonized by multiple species of ectomycorrhizal fungi. The role of these symbionts in P. taeda potassium (K+) nutrition has not been previously investigated. Here, we assessed the contribution of four ectomycorrhizal fungi, Hebeloma cylindrosporum, Paxillus ammoniavirescens, Laccaria bicolor, and Suillus cothurnatus, in P. taeda K+ acquisition under different external K+ availabilities. Using a custom-made two-compartment system, P. taeda seedlings were inoculated with one of the four fungi, or kept non-colonized, and grown under K+-limited or -sufficient conditions for 8 weeks. Only the fungi had access to separate compartments in which rubidium, an analog tracer for K+, was supplied before harvest. Resulting effects of the fungi were recorded, including root colonization, biomass, and nutrient concentrations. We also analyzed the fungal performance in axenic conditions under varying supply of K+ and sodium. Our study revealed that these four ectomycorrhizal fungi are differentially affected by external K+ and sodium variations, that they are not able to provide similar benefits to the host P. taeda in our growing conditions, and that rubidium may be used with some limitations to estimate K+ transport from ectomycorrhizal fungi to colonized plants.

Frank HER ，Garcia K 《-》

Development of split-root assays for loblolly pine (Pinus taeda L.) seedlings to study ectomycorrhizal symbioses.

Split-root techniques are valuable to investigate systemic vs. local plant responses to biotic and abiotic environmental factors, including interactions with soil microbes. Loblolly pine (Pinus taeda L.) is an economically important tree species that associates with many ectomycorrhizal fungi. However, a protocol for the establishment of split-roots experiments with loblolly pine has not been described so far. This method successfully establishes a split-root system in eight weeks following germination of loblolly pine seedlings. Rapid lateral root elongation is promoted by cutting the primary root tip and growing the seedlings in a hydroponic medium. Lateral roots can then be divided into two separated compartments and inoculated with ectomycorrhizal fungi. The method was validated by growth of split roots with or without inoculation. Root dry biomass was not significantly different between separated non-inoculated roots. Ectomycorrhizal colonization was not detected on the non-inoculated side of roots that were inoculated only on one side, demonstrating the success of the technique as a valuable method for split-root experiments in P. taeda. In addition to ectomycorrhizal fungi, researchers can use this method with loblolly pine to study systemic and local responses to a variety of other biotic or abiotic factors in the root environment.•We describe a protocol to produce split-roots in loblolly pine (Pinus taeda L.) in eight weeks.•This protocol uses hydroponics to promote the elongation of loblolly pine roots.•We validated this protocol by determining split-root biomass and inoculating the seedlings with the ectomycorrhizal fungi Paxillus ammoniavirescens or Hebeloma cylindrosporum.

Rose BD ，Frank HER ，Garcia K 《-》

The ectomycorrhizal fungus Paxillus ammoniavirescens influences the effects of salinity on loblolly pine in response to potassium availability.

Salinity is an increasing problem in coastal areas affected by saltwater intrusion, with deleterious effects on tree health and forest growth. Ectomycorrhizal (ECM) fungi may improve the salinity tolerance of host trees, but the impact of external potassium (K+ ) availability on these effects is still unclear. Here, we performed several experiments with the ECM fungus Paxillus ammoniavirescens and loblolly pine (Pinus taeda L.) in axenic and symbiotic conditions at limited or sufficient K+ and increasing sodium (Na+ ) concentrations. Growth rate, biomass, nutrient content, and K+ transporter expression levels were recorded for the fungus, and the colonization rate, root development parameters, biomass, and shoot nutrient accumulation were determined for mycorrhizal and non-mycorrhizal plants. P. ammoniavirescens was tolerant to high salinity, although growth and nutrient concentrations varied with K+ availability and increasing Na+ exposure. While loblolly pine root growth and development decreased with increasing salinity, ECM colonization was unaffected by pine response to salinity. The mycorrhizal influence on loblolly pine salinity response was strongly dependent on external K+ availability. This study reveals that P. ammoniavirescens can reduce Na+ accumulation of salt-exposed loblolly pine, but this effect depends on external K+ availability.

Rose BD ，Dellinger MA ，Larmour CP ，Polishook MI ，Higuita-Aguirre MI ，Dutta S ，Cook RL ，Zimmermann SD ，Garcia K ... -  《-》

Potassium nutrition of ectomycorrhizal Pinus pinaster: overexpression of the Hebeloma cylindrosporum HcTrk1 transporter affects the translocation of both K(+) and phosphorus in the host plant.

Mycorrhizal associations are known to improve the hydro-mineral nutrition of their host plants. However, the importance of mycorrhizal symbiosis for plant potassium nutrition has so far been poorly studied. We therefore investigated the impact of the ectomycorrhizal fungus Hebeloma cylindrosporum on the potassium nutrition of Pinus pinaster and examined the involvement of the fungal potassium transporter HcTrk1. HcTrk1 transcripts and proteins were localized in ectomycorrhizas using in situ hybridization and EGFP translational fusion constructs. Importantly, an overexpression strategy was performed on a H. cylindrosporum endogenous gene in order to dissect the role of this transporter. The potassium nutrition of mycorrhizal pine plants was significantly improved under potassium-limiting conditions. Fungal strains overexpressing HcTrk1 reduced the translocation of potassium and phosphorus from the roots to the shoots of inoculated plants in mycorrhizal experiments. Furthermore, expression of HcTrk1 and the phosphate transporter HcPT1.1 were reciprocally linked to the external inorganic phosphate and potassium availability. The development of these approaches provides a deeper insight into the role of ectomycorrhizal symbiosis on host plant K(+) nutrition and in particular, the K(+) transporter HcTrk1. The work augments our knowledge of the link between potassium and phosphorus nutrition via the mycorrhizal pathway.

Garcia K ，Delteil A ，Conéjéro G ，Becquer A ，Plassard C ，Sentenac H ，Zimmermann S ... -  《-》

Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum.

Ectomycorrhizal fungi play an essential role in the ecology of boreal and temperate forests through the improvement of tree mineral nutrition. Potassium (K+ ) is an essential nutrient for plants and is needed in high amounts. We recently demonstrated that the ectomycorrhizal fungus Hebeloma cylindrosporum improves the K+ nutrition of Pinus pinaster under shortage conditions. Part of the transport systems involved in K+ uptake by the fungus has been deciphered, while the molecular players responsible for the transfer of this cation towards the plant remain totally unknown. Analysis of the genome of H. cylindrosporum revealed the presence of three putative tandem-pore outward-rectifying K+ (TOK) channels that could contribute to this transfer. Here, we report the functional characterization of these three channels through two-electrode voltage-clamp experiments in oocytes and yeast complementation assays. The expression pattern and physiological role of these channels were analysed in symbiotic interaction with P. pinaster. Pine seedlings colonized by fungal transformants overexpressing two of them displayed a larger accumulation of K+ in shoots. This study revealed that TOK channels have distinctive properties and functions in axenic and symbiotic conditions and suggested that HcTOK2.2 is implicated in the symbiotic transfer of K+ from the fungus towards the plant.

Guerrero-Galán C ，Delteil A ，Garcia K ，Houdinet G ，Conéjéro G ，Gaillard I ，Sentenac H ，Zimmermann SD ... -  《-》