Isolation and identification of diazotrophic bacteria from internal tissues ofPinus contortaandThuja plicata

Abstract

<jats:p>Lodgepole pine ( Pinus contorta var. latifolia Engelm. ex S. Watson) and western red cedar ( Thuja plicata Donn ex D. Don) are capable of growing in soils that are severely nitrogen-limited. A rarely evaluated but possible nitrogen source for gymnosperms growing at nutrient poor sites is nitrogen fixation by endophytic bacteria. To investigate this possibility, we looked for nitrogen-fixing bacteria in surface-sterilized needle, stem, and root tissues of naturally regenerating lodgepole pine and western red cedar seedlings (2–4 years old) and trees (&gt;20 years old) growing near Williams Lake, Chilliwack Lake, and Boston Bar, British Columbia, Canada. Ninety-nine bacterial isolates were obtained by plating surface-sterilized plant tissue extracts on tryptic soy agar (TSA) and combined carbon medium agar (CCMA), a nitrogen-free medium. Bacterial isolates were identified using gas chromatographic fatty acid methyl ester (GC-FAME) and 16S rRNA gene analyses and were evaluated for nitrogenase activity using an acetylene reduction assay. Representatives of the genera Bacillus and Paenibacillus dominated the culturable endophytic bacterial community in tissues of both tree species, with three Paenibacillus isolates and one Dyadobacter fermentans strain demonstrating consistently high acetylene-reduction activities. Our results raise the possibility that endophytic nitrogen fixation provides a supplementary nitrogen source for naturally regenerated lodgepole pine and western red cedar.</jats:p>

Substrate utilization by endophytic bacteria Paenibacillus polymyxa P2b-2R that may facilitate bacterial entrance and survival inside diverse plant hosts

“…Paenibacillus polymyxa strain P2b was isolated from surface-sterilized stem tissue of a lodgepole pine seedling that was naturally regenerating near Williams Lake, British Columbia, Canada (latitude: 52°05″N; longitude: 122°54″W; elevation: 1300 m; Sub-Boreal Pine Spruce (SBPSdc) Zone; Bal et al 2012). Paenibacillus polymyxa P2b-2R is a spontaneous antibiotic-resistant mutant that was derived from strain P2b (Bal et al 2012).…”

“…Paenibacillus polymyxa P2b-2R is a spontaneous antibiotic-resistant mutant that was derived from strain P2b (Bal et al 2012). Paenibacillus polymyxa P2b-2R is resistant to 200 mg/L rifamycin and was stored at −80°C on combined carbon medium (CCM; Rennie 1981) amended with 20% glycerol (Bal et al 2012).…”

“…Paenibacillus polymyxa P2b-2R is a spontaneous antibiotic-resistant mutant that was derived from strain P2b (Bal et al 2012). Paenibacillus polymyxa P2b-2R is resistant to 200 mg/L rifamycin and was stored at −80°C on combined carbon medium (CCM; Rennie 1981) amended with 20% glycerol (Bal et al 2012). To evaluate the ability of P2b-2R to degrade plant cell wall components, 50 μL of P2b-2R (∼10 7 cfu/mL) was spot-inoculated on Luria-Bertani (LB) agar amended with one of the following substrates (5 g/L): sodium CMC, beechwood xylan, or sodium polypectate, and incubated for two days at 30°C.…”

“…Bal et al (2012) successfully isolated several Paenibacillus strains that possess significant acetylene-reduction activity (N-fixing capacity) from the extracts of surface-sterilized lodgepole pine seedling and tree tissues. When one of the strains, P. polymyxa P2b-2R, was re-inoculated in lodgepole pine and grown in an N-limited soil for 13 months, the bacterial strain successfully colonized internal stem and root tissues of lodgepole pine seedlings, fixed a significant amount of N from the atmosphere (79%), and promoted the overall growth of seedlings (Anand et al 2013;Yang et al 2016).…”

Nitrogen Fixation Outside and Inside Plant Tissues

“…Based on earlier work with lodgepole pine suggesting that rhizospheric BNF contributed only small amounts of N to seedlings (Chanway & Holl, 1991) as well as reports that BNF in sugarcane was endophytic, we searched for endophytic diazotrophs in lodgepole pine as a possible explanation for the ability of this species to grow on N-deficient substrates. We successfully isolated several Paenibacillus strains that possessed significant acetylene reduction activity from extracts of surface-sterilized lodgepole pine seedling and tree tissues (Bal et al, 2012) and tested them for BNF with lodgepole pine and western red cedar seedlings in a 15 N soil dilution assay (Bal & Chanway, 2012a,b). When pine was reintroduced to one of the strains, Paenibacillus polymyxa strain P2b-2R, and grown in a very N-limited soil, seedlings were found to derive more than half (66%) of their foliar N from BNF, but their growth was inhibited compared to non-inoculated controls 9 months after planting (Bal & Chanway, 2012a).…”