Isolation and identification of endophytic diazotrophs from lodgepole pine trees growing at unreclaimed gravel mining pits in central interior British Columbia, Canada

Abstract

Unreclaimed gravel mining pits located in the central interior of British Columbia have very limited soil N-levels due to gravelly-textured soils with weak profile development, no organic forest floor, and low atmospheric nitrogen (N) inputs through precipitation. However, lodgepole pine (Pinus contorta var. latifolia) trees can be found growing well at these pits with tissue Ncontent and growth rate unaffected by extremely low soil N-levels, indicating that pine trees are able to meet their N-requirements from an unknown source. We hypothesized that biological Nfixation by bacteria living in the tree tissues (known as endophytic diazotrophs) could be a potential N source for these trees. To test this hypothesis, we isolated 77 potential endophytic diazotrophs from needle, stem and root tissues of pine trees on N-free culture media. Of these, 32 bacteria showed positive N-fixing ability when tested for nitrogenase enzyme activity using the acetylene reduction assay. These endophytic N-fixing bacteria were identified as mainly belonging to the genera Pseudomonas, Bacillus, Paenibacillus, and Rhizobium, which are well-known for their plant-beneficial traits including N-fixation. Therefore, it can be concluded that pine trees growing at these severely N-limited gravel pits naturally harbour endophytic diazotrophs, which could be involved in sustaining their vigorous growth, possibly through biological nitrogen fixation.

Sustaining the growth of Pinaceae trees under nutrient-limited edaphic conditions via plant-beneficial bacteria

“…This impressive suite of in vitro PGP capabilities could be related to the significant enhancement of pine and spruce length and biomass observed for these strains in the greenhouse growth trial. Bacterial strains belonging to Caballeronia and Paraburkholderia genera possess multiple PGP abilities as reported in previous studies using lab-based enzyme assays, genomic analyses, and in planta assays [9,33,[103][104][105]. Intriguingly, these genera were previously assigned to the plant-beneficial group of the Burkholderia genus [106, 107], which is rich in potent plant-probiotics [21,108].…”

“…Lodgepole pine has possibly the widest range of environmental tolerance among any coniferous species in North America [6]. It has been reported to thrive under severe soil, moisture and topographical conditions including road cuts, mining sites, fire-affected regions and extremely dry, nutrient-poor sites [7][8][9][10]. For instance, lodgepole pine trees were observed to naturally regenerate on a bare gravel substrate without topsoil at highly disturbed mining sites with growth rates similar to lodgepole pine trees growing at nearby undisturbed sites with intact topsoil [8,9].…”

“…It has been reported to thrive under severe soil, moisture and topographical conditions including road cuts, mining sites, fire-affected regions and extremely dry, nutrient-poor sites [7][8][9][10]. For instance, lodgepole pine trees were observed to naturally regenerate on a bare gravel substrate without topsoil at highly disturbed mining sites with growth rates similar to lodgepole pine trees growing at nearby undisturbed sites with intact topsoil [8,9]. Due to the ability of lodgepole pine trees to grow under such stressful environments, some studies have suggested that lodgepole pine trees may form close associations with beneficial microbes including plant-growth-promoting bacteria (PGPB) for their survival and growth [11,12].…”

Activation of the SA-Associated Plant Defense Pathway Alters the Composition of Soil Bacterial Communities

“…The Caballeronia genus is often reported to play an important role in xing nitrogen and promoting plant growth. Species in this genus are predominantly endophytic diazotrophic bacteria and N-xing bacteria (Padda et al 2018;Puri et al 2018;Puri et al 2020). This suggests that activation of SA signaling pathways in J. vulgaris plants bene ted bacteria that were more bene cial to plant growth, but further studies are needed to con rm this.…”

Simple methods to remove microbes from leaf surfaces

“…However, several methodological obstacles arise when studying endophytes, including the complete removal of leaf surface microorganisms (i.e., leaf epiphytes). Current methods used to confirm leaf surface sterility generally include polymerase chain reaction (PCR) and leaf prints on nutrient media [10][11][12][13][14][15][16][17][18]. We included scanning electronic microscopy (SEM) to improve the validation of leaf surface sterility and to evaluate the potential damage by these treatments on the integrity of leaf tissue.…”