N2-Fixation and Seedling Growth Promotion of Lodgepole Pine by Endophytic Paenibacillus polymyxa

Abstract

We inoculated lodgepole pine (Pinus contorta var. latifolia (Dougl.) Engelm.) with Paenibacillus polymyxa P2b-2R, a diazotrophic bacterium previously isolated from internal stem tissue of a naturally regenerating pine seedling to evaluate biological nitrogen fixation and seedling growth promotion by this microorganism. Seedlings generated from pine seed inoculated with strain P2b-2R were grown for up to 13 months in a N-limited soil mix containing 0.7 mM available N labeled as Ca((15)NO3)2 to facilitate detection of N2-fixation. Strain P2b-2R developed a persistent endophytic population comprising 10(2)-10(6) cfu g(-1) plant tissue inside pine roots, stems, and needles during the experiment. At the end of the growth period, P2b-2R had reduced seedling mortality by 14 % and (15)N foliar N abundance 79 % and doubled foliar N concentration and seedling biomass compared to controls. Our results suggest that N2-fixation by P. polymyxa enhanced growth of pine seedlings and support the hypothesis that plant-associated diazotrophs capable of endophytic colonization can satisfy a significant proportion of the N required by tree seedlings growing under N-limited conditions.

Nitrogen Fixation Outside and Inside Plant Tissues

“…After a 13-month growth period, pine seedlings treated with P2b-2R were observed to derive most of their foliar N (79%) from BNF (Anand et al, 2013), which was confirmation of the ability of lodgepole pine to fix N after colonization by P. polymyxa. When compared with previous results, we found that inoculated seedlings grown for 7, 9 and 13 months derived 30%, 66% (Bal & Chanway, 2012a) and 79% (Anand et al, 2013), respectively, of their foliar N from the atmosphere.…”

“…The comparatively early onset of BNF and delayed seedling growth response suggest that development of a fully effective N 2 -fixing bacterial population, able to enhance foliar N concentration in pine, is not a rapid process. Establishment of fully effective BNF in pine does not appear to depend on the population size of endophytic diazotrophs because P2b-2R colonization of root, stem and needle tissues in older seedlings did not differ significantly from younger seedlings (Anand et al, 2013), though it is possible that external root colonization was quantitatively related to BNF. However, if BNF is endophytic and requires physiological modifications of bacteria, e.g., differentiation of Paenibacillus into a N 2 -fixing form similar to Rhizobium bacteroids (Postgate, 1998), or plants, e.g., the establishment of specialized sites of BNF within plant tissues or cells, to be fully effective, some time may be required for them to complete.…”

“…While P. polymyxa possesses several characteristics that can result in plant growth promotion (Chanway, 2002), the enhanced performance of 13-month old seedlings was likely caused by an increase in the amount of N derived from BNF because the proportion of foliar N from BNF (68%-79%) (Anand et al, 2013) in older seedlings was only marginally higher than that of ninemonth old seedlings (64%-66%) (Bal & Chanway, 2012a), but the concentration of foliar N (2.35%) (Anand et al, 2013) was 5-fold greater than nine-month old seedlings. The difference in total foliar N content, i.e., foliar N concentration x foliar biomass, was even greater and represented an elevation of foliar N concentration from a level considered to be very severely N deficient in all nine-month old seedlings (Bal & Chanway, 2012a) and the 13-month old controls (Anand et al, 2013) to one that is adequate for healthy lodgepole pine (Ballard & Carter, 1986). The comparatively early onset of BNF and delayed seedling growth response suggest that development of a fully effective N 2 -fixing bacterial population, able to enhance foliar N concentration in pine, is not a rapid process.…”

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

“…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). Furthermore, a green fluorescent protein-labeled derivative of P2b-2R was shown to colonize internal tissues of lodgepole pine seedlings (Anand and Chanway 2013a).…”

Mining into interspecific bacterial interactions

“…Many paenibacilli are known to act as PGPR in agricultural systems (Anand et al, 2013). Bacteria of this genus are able to colonize diverse habitats like water, soil and insects (Berge et al, 2002;Bosshard et al, 2002;Daane et al, 2002;Peters et al, 2006;Timmusk et al, 2009).…”