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.

The evolution of sour taste.

The evolutionary history of sour taste has been little studied. Through a combination of literature review and trait mapping on the vertebrate phylogenetic tree, we consider the origin of sour taste, potential cases of the loss of sour taste, and those factors that might have favoured changes in the valence of sour taste-from aversive to appealing. We reconstruct sour taste as having evolved in ancient fish. By contrast to other tastes, sour taste does not appear to have been lost in any major vertebrate taxa. For most species, sour taste is aversive. Animals, including humans, that enjoy the sour taste triggered by acidic foods are exceptional. We conclude by considering why sour taste evolved, why it might have persisted as vertebrates made the transition to land and what factors might have favoured the preference for sour-tasting, acidic foods, particularly in hominins, such as humans.

Split down the middle: studying arbuscular mycorrhizal and ectomycorrhizal symbioses using split-root assays.

Most land plants symbiotically interact with soil-borne fungi to ensure nutrient acquisition and tolerance to various environmental stressors. Among these symbioses, arbuscular mycorrhizal and ectomycorrhizal associations can be found in a large proportion of plants, including many crops. Split-root assays are widely used in plant research to study local and systemic signaling responses triggered by local treatments, including nutrient availability, interaction with soil microbes, or abiotic stresses. However, split-root approaches have only been occasionally used to tackle these questions with regard to mycorrhizal symbioses. This review compiles and discusses split-root assays developed to study arbuscular mycorrhizal and ectomycorrhizal symbioses, with a particular emphasis on colonization by multiple beneficial symbionts, systemic resistance induced by mycorrhizal fungi, water and nutrient transport from fungi to colonized plants, and host photosynthate allocation from the host to fungal symbionts. In addition, we highlight how the use of split-root assays could result in a better understanding of mycorrhizal symbioses, particularly for a broader range of essential nutrients, and for multipartite interactions.

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.

Fungal Shaker-like channels beyond cellular K+ homeostasis: A role in ectomycorrhizal symbiosis between Hebeloma cylindrosporum and Pinus pinaster.

Potassium (K+) acquisition, translocation and cellular homeostasis are mediated by various membrane transport systems in all organisms. We identified and described an ion channel in the ectomycorrhizal fungus Hebeloma cylindrosporum (HcSKC) that harbors features of animal voltage-dependent Shaker-like K+ channels, and investigated its role in both free-living hyphae and symbiotic conditions. RNAi lines affected in the expression of HcSKC were produced and used for in vitro mycorrhizal assays with the maritime pine as host plant, under standard or low K+ conditions. The adaptation of H. cylindrosporum to the downregulation of HcSKC was analyzed by qRT-PCR analyses for other K+-related transport proteins: the transporters HcTrk1, HcTrk2, and HcHAK, and the ion channels HcTOK1, HcTOK2.1, and HcTOK2.2. Downregulated HcSKC transformants displayed greater K+ contents at standard K+ only. In such conditions, plants inoculated with these transgenic lines were impaired in K+ nutrition. Taken together, these results support the hypothesis that the reduced expression of HcSKC modifies the pool of fungal K+ available for the plant and/or affects its symbiotic transfer to the roots. Our study reveals that the maintenance of K+ transport in H. cylindrosporum, through the regulation of HcSKC expression, is required for the K+ nutrition of the host plant.