Paenibacillus Polymyxa P2b-2R Research Articles

Biological nitrogen fixation and plant growth promotion of lodgepole pine by an endophytic diazotroph Paenibacillus polymyxa and its GFP-tagged derivative

Paenibacillus polymyxa P2b-2R is an endophytic diazotroph originally isolated from lodgepole pine. It is reported to fix significant amounts of nitrogen (N) and promote plant growth. To evaluate the endophytic colonization sites, a green fluorescent protein (GFP) derivative of P2b-2R was generated (P2b–2Rgfp), but the effects of GFP modification on the functioning of P2b-2R have not been fully elucidated yet. In this study, we wanted to confirm and contrast the N-fixing and plant-growth-promoting abilities of P2b-2Rgfp with those of the wild-type P2b-2R in lodgepole pine. Pine seedlings were grown in an N-limited environment and harvested 2, 4, 8, and 12 months after inoculation to evaluate the endophytic and rhizospheric colonization by both strains, the amount of N-fixed, and seedling growth enhancement. Both P2b-2R and P2b-2Rgfp strains formed persistent rhizospheric and endophytic populations; fixed N; and enhanced seedling growth continuously after 4 months. P2b-2Rgfp-treated seedlings outperformed the P2b-2R-treated seedlings in terms of biomass only during the initial stages of plant development, but the differences decreased during the trial and were not significant towards the end. To the best of our knowledge, this is the first study that reports the effects of GFP-tagging of an endophyte when inoculated into a gymnosperm tree species.

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

Bacterial endophytes are thought to enter plants either through pre-existing openings in plant tissues or by creating openings by hydrolyzing major plant cell wall components. A lodgepole endophyte, Paenibacillus polymyxa P2b-2R, consistently formed endophytic colonies when inoculated in diverse plant hosts, viz., lodgepole pine, western red cedar, corn, canola, and tomato. We were interested to know, whether or not this bacterial strain possesses enzymes that can hydrolyze three major plant cell wall components namely cellulose, xylan, and pectin to facilitate entrance into the host plants. Using a BIOLOG assay, we also tested this bacterial strain’s ability to utilize carbon sources that might facilitate its entrance and hence its survival inside host plants. Paenibacillus polymyxa P2b-2R hydrolyzed sodium carboxymethylcellulose, beechwood xylan, and sodium polypectate and utilized 39 of the 95 carbon sources (41%) tested. Of the 39 carbon substrates oxidized by P2b-2R, the “carbohydrates” group represents the largest source of utilizable carbon (23 out of 39). Thus, it can be concluded that P. polymyxa P2b-2R is able to degrade major cell wall components (cellulose, xylan, and pectin) and utilize some of the available carbon substrates, possibly to gain entry and survive inside the plant and form endophytic colonies thereafter.

Plant growth promotion and nitrogen fixation in canola (Brassica napus) by an endophytic strain of Paenibacillus polymyxa and its GFP-tagged derivative in a long-term study

Paenibacillus polymyxa P2b-2R is an endophytic strain capable of fixing nitrogen (N) and promoting growth in a broad range of hosts. In this study, the ability of Paenibacillus polymyxa P2b-2R to fix N and promote the growth of canola (Brassica napus L.) was determined in a 3-month-long trial. We also compared growth promoting and N-fixing capability of wild-type P2b-2R and its GFP-tagged counterpart. Canola seedlings were inoculated with strain P2b-2Rgfp or P2b-2R. Non-inoculated seedlings were used as controls. Canola seedlings were grown for 3 months before evaluating plant growth promotion (length, biomass, and pod mass) and N fixation (using foliar 15N dilution assay). P2b-2Rgfp inoculation significantly promoted canola growth by increasing seedling length by 2-fold, biomass by 2.5-fold, and fixing 35% of its foliar N, respectively. P2b-2R inoculation also increased seedling length by 70%, biomass by 2-fold, and fixed 27% of N. Seedling biomass and length, and the amount of N fixed by P2b-2Rgfp-treated seedlings was significantly greater than the P2b-2R-treated seedlings. P2b-2R or P2b-2Rgfp inoculation also lead to an increase of pod mass (more than 50%), indicating that these strains might play a role in enhancing the yield of canola by acting as biofertilizer.

Effects of Paenibacillus polymyxa inoculation and different soil nitrogen treatments on lodgepole pine seedling growth

An endophytic diazotroph, Paenibacillus polymyxa P2b-2R, has been shown to colonize internal tissues of lodgepole pine (Pinus contorta var. latifolia Engelm. ex S. Watson) and stimulate seedling growth possibly through nitrogen (N) fixation in an N-limited environment. We evaluated the effects of different soil N concentrations on lodgepole pine seedling growth after inoculation with P2b-2R in a year-long greenhouse study. Seedlings were subjected to monthly application of a nutrient solution containing one of four soil N concentrations as Ca(NO3)2 (5% 15N label): 0.0029, 0.029, 0.29, and 2.9 μmol·L–1, referred to as very low, low, medium, and high soil N treatments, respectively. Foliar 15N atom percent excess and foliar N content values of P2b-2R inoculated seedlings were not significantly different from controls at any N treatment. Similarly, rhizospheric and endophytic population of P2b-2R did not vary significantly across N treatments. Inoculation with P2b-2R resulted in seedling growth inhibition during early stages of the experiment; however, by month 12, inoculated seedlings from the very low soil N treatment had accumulated 56.3% and 46.4% more root and shoot biomass than controls, respectively. These results suggest that pine biomass stimulation by P2b-2R may depend on soil N concentrations and that such growth stimulation may occur without providing significant amounts of fixed N.

Seedling growth promotion and nitrogen fixation by a bacterial endophyte Paenibacillus polymyxa P2b-2R and its GFP derivative in corn in a long-term trial

A plant growth promoting endophyte, Paenibacillus polymyxa P2b-2R, originally isolated form a lodgepole pine seedling and its green fluorescent protein (GFP) derivative, P2b-2Rgfp, were evaluated for their ability to survive, fix atmospheric nitrogen (N) and promote plant growth when inoculated into corn (Zea Mays L.) in a long-term trial. We were also interested to see the effects of GFP-tagging of P2b-2R on its ability to promote growth of corn seedlings in a long-term study. Corn seedlings were inoculated with either strain P2b-2R or P2b-2Rgfp and non-inoculated seedlings were treated as controls. Seedlings were harvested after 3 months and evaluated for plant growth promotion (length and biomass) and N fixation (15N foliar dilution assay). Colonization and survival of P2b-2R and P2b-2Rgfp outside (rhizosphere) and inside (internal tissues) the inoculated seedlings were also determined. Both strains survived inside and outside corn seedlings forming rhizospheric and endophytic colonies in stem and root tissues. Inoculation by P2b-2R strain promoted corn plant growth via enhancing seedling length and biomass by 52 % and 53 %, respectively. Similarly, P2b-2Rgfp inoculation enhanced seedling length by 68 % and biomass by 67 %. Corn seedlings inoculated with strain P2b-2R derived 30 % of foliar N from the atmosphere and seedlings inoculated with P2b-2Rgfp derived 32 % of foliar N from the atmosphere. But there was no statistically significant difference between P2b-2R and P2b-2Rgfp treated seedlings in terms of overall seedling length, biomass and amount of N fixed in this long-term trial. These results combined with the results from an earlier study suggest that P. polymyxa P2b-2R and its GFP-tagged derivative is capable of enhancing overall plant growth throughout the life cycle of corn plant.

Evidence of nitrogen fixation and growth promotion in canola (Brassica napus L.) by an endophytic diazotroph Paenibacillus polymyxa P2b-2R

An endophytic diazotroph, Paenibacillus polymyxa P2b-2R, previously isolated from internal tissues of lodgepole pine tree was tested for its ability to colonize, fix nitrogen, and promote growth of an important oilseed crop, canola. For this, we inoculated canola seeds with P. polymyxa P2b-2R and grew them for up to 60 days. Canola seedlings were harvested thrice, 20, 40, and 60 days after inoculation, to determine the rhizospheric and endophytic P2b-2R population (colony-forming units (cfu)) and quantify the biological nitrogen fixation (15N dilution assay) and growth promotion (height and biomass). The entire experiment was repeated to confirm the treatment effects. P2b-2R colonized canola rhizosphere with a population abundance of 1.41 × 108 cfu/g dry root in the first growth trial and 7.49 × 107 cfu/g dry root in the second growth trial. P2b-2R developed a persistent endophytic population in canola root, comprising 3.48 × 105 cfu/g fresh weight in the first growth trial and 2.36 × 103 cfu/g fresh weight in the second growth trial. In the first growth trial, P2b-2R inoculation enhanced seedling height by 25 % and biomass by 30 % after 60 days of inoculation. Similarly, in the second growth trial, P2b-2R inoculation promoted seedling height by 21 % and biomass by 24 %. P2b-2R inoculated seedlings fixed 22 and 19 % of nitrogen from the atmosphere in the first and second growth trials, respectively. These results clearly indicate that P. polymyxa P2b-2R, a lodgepole pine endophyte, is capable of fixing nitrogen and promoting growth of an important oilseed crop canola, which is an indication of its broad range host capability.

Can a diazotrophic endophyte originally isolated from lodgepole pine colonize an agricultural crop (corn) and promote its growth?

Several diazotrophic Paenibacillus strains were isolated from extracts of surface-sterilized lodgepole pine seedling and tree tissues. One strain, Paenibacillus polymyxa P2b-2R, was found to fix high amounts of nitrogen when reintroduced to the gymnosperms, lodgepole pine and western red cedar. We wanted to determine if P. polymyxa P2b-2R could colonize rhizosphere and internal tissues, fix N and promote growth of corn (Zea mays L), an important agricultural crop. We inoculated corn seeds with P. polymyxa strain P2b-2R and grew seedlings for 30 days. Corn seedlings were harvested 10, 20 and 30 days after inoculation for evaluation of endophytic and rhizospheric colonization as well as nitrogen fixation and growth promotion. P2b-2R successfully colonized the rhizosphere as well as internal root tissues of corn (i.e., endophytically) with population densities near 106 cfu. Corn seedling growth was promoted significantly by inoculation with P2b-2R with an increase of up to 35% in length and up to 30% in biomass after 30 days of inoculation. Seedlings inoculated with P2b-2R derived up to 20% of foliar nitrogen from atmosphere after 30 days of inoculation, which is significant considering the fact that this was a short growth trial. These results suggest that P. polymyxa P2b-2R might have a broad range of plant hosts and is able to fix N and promote the growth of at least one important agricultural crop i.e. Corn.

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

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.

Evidence of nitrogen fixation in lodgepole pine inoculated with diazotrophicPaenibacillus polymyxa

Diazotrophic bacteria previously isolated from internal tissues of naturally regenerating lodgepole pine ( Pinus contorta var. latifolia (Dougl.) Engelm.) seedlings were tested for their ability to colonize and fix nitrogen (N) in pine germinants in two experiments. Surface sterilized pine seed was sown in glass tubes containing an autoclaved sand – montmorillonite clay mixture that contained a N-limited nutrient solution labeled with15N as 0.35 mmol·L–1Ca(15NO3)2(5%15N label). Pine seed was inoculated with one of three of the following bacterial strains: Paenibacillus polymyxa P2b-2R, P. polymyxa P18b-2R, or Dyadobacter fermentans P19a-2R, and seedlings grew for either 27 or 35 weeks. At the end of each plant growth period, P. polymyxa strain P2b-2R was detected in the pine rhizosphere but not inside plant tissues. Pine foliar N concentrations were not affected by bacterial inoculation but significant foliar15N dilution was observed in seedlings treated with strain P2b-2R (30% and 66%, P < 0.05, in the first and second experiments, respectively). This strain also reduced seedling biomass in both experiments but effects were significant only in the second experiment (36%, P < 0.05). Notwithstanding the negative effect of bacterial inoculation on seedling growth, pine seedlings inoculated with strain P2b-2R derived 30% and 66%, respectively, of their foliar N from bacterial N fixation in two seedling growth experiments. These results demonstrate the possibility that some endophytic diazotrophs facilitate pine seedling growth in N-poor soils.

N2-fixation and growth promotion in cedar colonized by an endophytic strain of Paenibacillus polymyxa

Inoculation of western red cedar with Paenibacillus polymyxa P2b-2R, an endophytic diazotroph of a pine, was previously shown to result in biological nitrogen fixation (BNF) in seedlings grown under N-limited conditions, but biomass accumulation was reduced after a 9-month growth period. To determine if the seedling growth reduction was temporary, we inoculated cedar seed with strain P2b-2R and grew seedlings for up to 13 months in a N-limited soil mix containing 0.7 mM of available N labeled as Ca(15NO3)2. P2b-2R developed a persistent endophytic population comprising 102–106 cfu g−1 plant tissue inside pine roots, stems, and needles. At the end of the growth period, P2b-2R had reduced 15N foliar N abundance by 36 % and increased shoot biomass by 46 % compared to controls. Our results indicate that inoculated seedlings derived 36 % of foliar N from the atmosphere and suggest that BNF by P. polymyxa can significantly enhance growth of cedar in a N-limited soil if seedlings are grown for a sufficient amount of time. These findings support the hypothesis that endophytic diazotrophs may facilitate regeneration and growth of western red cedar at N-poor sites.

Detection of GFP-labeled Paenibacillus polymyxa in autofluorescing pine seedling tissues

Paenibacillus polymyxa P2b-2R is a bacterium that originated from internal lodgepole pine (Pinus contorta var. latifolia (Dougl.) Engelm.) seedling stem tissue and fixes nitrogen (N) in association with pine and western red cedar (Thuja plicata Donn.). To evaluate endophytic colonization by this microorganism, we generated P. polymyxa P2b-2Rgfp, a green fluorescent protein (GFP)-labeled derivative of P2b-2R, and grew pine seedlings that were inoculated with the marked strain in a N-limited soil. Tissue disintegration during sample preparation precluded examination of needles for the GFP-labeled endophyte but GFP was detected on roots and in stems of 2- to 14-week-old pine seedlings using confocal laser scanning microscopy. Due to excessive autofluorescence of seedling tissues, labeled bacteria were clearly discernible only in stem tissues of 4- and 6-week-old seedlings. P2b-2Rgfp colonized the root surface extensively and was detected inside the stem cortex, primarily intracellularly. Some labeled bacteria appeared to contain endospores and none were detected in vascular tissues. We conclude that P. polymyxa P2b-2R is capable of endophytic colonization of pine seedlings with specific colonization sites that include the stem cortex but that GFP labeling is of limited value for localization of endophytic bacteria in pine seedling tissues.

15N foliar dilution of western red cedar in response to seed inoculation with diazotrophic Paenibacillus polymyxa

Diazotrophic bacteria previously isolated from internal tissues of naturally regenerating lodgepole pine (Pinus contorta var. latifolia (Dougl.) Engelm.) seedlings were tested for their ability to fix N in association with western red cedar (Thuja plicata Donn.). Surface-sterilized cedar seed was sown in glass tubes containing an autoclaved sand/montmorillonite clay mixture that contained a N-free nutrient solution labeled with 15N as 0.35 mM Ca(15NO3)2 (5 % 15N label). After sowing, seed was inoculated with one of three bacterial strains: Paenibacillus polymyxa P2b-2R, P. polymyxa P18b-2R, or Dyadobacter fermentans P19a-2R. At the end of the 27-week plant growth period, P2b-2R was the only strain detected in the cedar rhizosphere. No bacteria were found inside plant tissues. Cedar foliar N concentrations were significantly enhanced (26–33 %, P < 0.05) in response to inoculation with all three bacterial strains but 15N dilution (23 %, P < 0.05) was observed only in seedlings treated with strain P2b-2R. This strain also reduced seedling dry weight (27 %, P < 0.05). We observed similar trends in a second experiment with slight modifications to the protocol, but the magnitude of foliar 15N dilution was greater (56 %, P < 0.05). Based on our results, we conclude that cedar seedlings inoculated with strain P2b-2R derived 23 and 56 % of their foliar N from bacterial N fixation in two seedling growth experiments.