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 《-》

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 《-》

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 ... -  《-》

Association mapping of ectomycorrhizal traits in loblolly pine (Pinus taeda L.).

To understand how diverse mutualisms coevolve and how species adapt to complex environments, a description of the underlying genetic basis of the traits involved must be provided. For example, in diverse coevolving mutualisms, such as the interaction of host plants with a suite of symbiotic mycorrhizal fungi, a key question is whether host plants can coevolve independently with multiple species of symbionts, which depends on whether those interactions are governed independently by separate genes or pleiotropically by shared genes. To provide insight into this question, we employed an association mapping approach in a clonally replicated field experiment of loblolly pine (Pinus taeda L.) to identify genetic components of host traits governing ectomycorrhizal (EM) symbioses (mycorrhizal traits). The relative abundances of different EM fungi as well as the total number of root tips per cm root colonized by EM fungi were analyzed as separate mycorrhizal traits of loblolly pine. Single-nucleotide polymorphisms (SNPs) within candidate genes of loblolly pine were associated with loblolly pine mycorrhizal traits, mapped to the loblolly pine genome, and their putative protein function obtained when available. The results support the hypothesis that ectomycorrhiza formation is governed by host genes of large effect that apparently have independent influences on host interactions with different symbiont species.

Piculell BJ ，José Martínez-García P ，Nelson CD ，Hoeksema JD ... -  《-》

Reducing airborne ectomycorrhizal fungi and growing non-mycorrhizal loblolly pine (Pinus taeda L.) seedlings in a greenhouse.

Stottlemyer AD ，Wang GG ，Wells CE ，Stottlemyer DW ，Waldrop TA ... -  《-》