Microb Ecol (2013) 66:369–374 DOI 10.1007/s00248-013-0196-1
PLANT MICROBE INTERACTIONS
N2-Fixation and Seedling Growth Promotion of Lodgepole Pine by Endophytic Paenibacillus polymyxa Richa Anand & Susan Grayston & Christopher Chanway
Received: 11 December 2012 / Accepted: 4 February 2013 / Published online: 19 February 2013 # Springer Science+Business Media New York 2013
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(15NO3)2 to facilitate detection of N2-fixation. Strain P2b-2R developed a persistent endophytic population comprising 102–106 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 15N foliar N abundance 79 % and doubled foliar N concentration and seedling biomass compared to controls. Our results suggest that N2fixation 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.
R. Anand IL-7, BC Children’s Hospital, 4480 Oak Street, Vancouver, BC V6H 3V4, Canada e-mail:
[email protected] C. Chanway Faculty of Land and Food Systems, University of British Columbia, 248-2357 Main Mall, Vancouver, BC V6T 1Z4, Canada R. Anand : S. Grayston : C. Chanway (*) Department of Forest Sciences, University of British Columbia, 3041-2424 Main Mall, Vancouver, BC V6T 1Z4, Canada e-mail:
[email protected] S. Grayston e-mail:
[email protected]
Introduction Lodgepole pine (Pinus contorta var. latifolia (Dougl.) Engelm.) is a commercially important gymnosperm species indigenous to western North America that grows from Alaska to California [1]. This species is capable of growing in very rocky substrates and is notable for its ability to thrive on nutrient poor, N-limited soils [2]. Chapman and Paul [3] recently provided compelling evidence of biological nitrogen fixation (BNF) in lodgepole pine based on tissue N content and δ15N values of trees growing in severely Ndeficient gravel. However, terrestrial plants capable of obtaining almost all N from BNF usually form root or stem nodules that contain N2-fixing diazotrophs [4]. Nodule-like tuberculate ectomycorrhizae with nitrogenase activity have been found on lodgepole pine roots in some forests [5], but their ecological role is not fully understood. The only known non-nodulating plants of commercial importance that can obtain most (up to 80 %) total plant N from BNF comprise a few cultivars of sugarcane (Saccharum officinarum L.) [6, 7]. The causative agent of BNF in sugarcane was initially thought to be an endophytic strain of Gluconoacetobacter diazotrophicus, but the involvement of at least two Herbaspirillum species was suggested later [8]. More recent studies indicate that the primary source of diazotrophy may involve a consortium of bacteria that live on or inside plant tissues [6, 7]. Based on earlier work with lodgepole pine suggesting that rhizospheric BNF contributed only small amounts of N to seedlings [9] as well as initial reports that BNF in sugarcane was endophytic [8], 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 [10] and tested them for BNF with lodgepole pine and western red cedar seedlings [11, 12]. When pine was
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reintroduced to one of the strains, Paenibacillus polymyxa strain P2b-2R, and grown in a very N-limited soil, seedlings derived 66 % of foliar N from BNF, but their growth was inhibited compared to non-inoculated controls 9 months after planting [11]. Similar effects, i.e., BNF and seedling growth inhibition, were observed with western red cedar [12], but cedar seedling growth promotion by P. polymyxa was observed when a longer growth period was used [13]. In our current study, we test the working hypothesis that the pine seedling growth reduction we observed after a 9month growth period [11] resulted from the additional demand on N2-fixing seedling resources by the energetically expensive process of BNF. If this is true, we predict that the seedling growth reduction is temporary because soil N depletion will eventually restrict the growth rate of control seedlings below that of N2-fixing seedlings. The specific objectives of this study were to evaluate pine seedling growth, N2-fixation, and endophytic colonization by P2b2R after inoculation with P. polymyxa P2b-2R using a longterm plant growth assay.
Materials and Methods Seed and Microorganisms Lodgepole pine seed was obtained from the British Columbia Ministry of Forests Tree Seed Centre, Surrey, British Columbia, Canada, and originated from a provenance located near Williams Lake, British Columbia, Canada (52°05′ N lat., 122°54′W long., elevation 1,300 m, Sub-Boreal Pine Spruce, SBPSdc Zone). P. polymyxa P2b-2R [8] is resistant to 200 mg L−1 rifamycin and was stored at −80 °C on combined carbon medium (CCM) [14] amended with 20 % glycerol. Seed inoculation and plant growth Seedling growth assays were performed in glass tubes (150 mm×25 mm in diameter) filled to 67 % capacity with a sand–Turface (montmorillonite clay, Applied Industrial Materials Corporation, Deerfield, IL) mixture (69 % w/w silica sand; 29 % w/w Turface; 2 % w/w CaCO3). The sand–Turface mixture was washed with 6 % NaOCl at room temperature with continuous shaking (60 rpm) for 6 h to remove organic nitrogen [15] and then washed three times with distilled water prior to drying and filling tubes. Each tube was fertilized to saturation with 17 mL of a nutrient solution which was modified by replacing KNO3 and Ca(NO3)-4H2O with Ca(15NO3)2 (5 % 15N label) (0.0576 gL−1) [9] and Sequestrene 330 Fe (CIBA-GEIGY, Mississauga, Ontario) with Na2FeEDTA (0.02 gL−1). Other nutrients in the nutrient solution included (in grams per liter) the following: KH2PO4, 0.14; MgSO4, 0.49; H3BO3, 0.001; MnCI2-4H2O, 0.001; ZnSO4-7H2O, 0.001;
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CuSO4-5H2O, 0.0001; and NaMoO4-2H2O, 0.001. Fertilized plant tubes were autoclaved for 1 h before use in experiments. Pine seed was surface-sterilized by immersion in 30 % hydrogen peroxide (H2O2) for 1.5 min, followed by three 30-s rinses in fresh sterile distilled water. The effectiveness of the surface sterilization procedure was confirmed by imprinting sterilized seed on tryptic soy agar (TSA) and checking for microbial contamination 2 days later. Seeds found to be free of surface contamination were placed in sterile cheese cloth bags containing moist autoclaved sand. These were placed in a loosely tied autoclavable plastic bag and stored at 4 °C for 5 weeks for stratification. Stratified seeds were again imprinted on TSA plates for 48 h before sowing to confirm the absence of surface contaminants. Ten randomly selected surface-sterilized seeds were crushed and imprinted on TSA plates supplemented with 200 mgL−1 rifamycin for 48 h to confirm the absence of internal seed contamination with P. polymyxa strain P2b-2R. Three surface-sterilized seeds were then aseptically sown in each glass tube and covered with 5 mm of autoclaved silica sand. Bacterial inoculum was prepared by thawing a frozen culture of strain P2b-2R and streaking onto CCM plates amended with 200 mgL−1 rifamycin. After the colonies grew, a loopful of the strain was inoculated into 1-L flasks containing 500 mL of fresh CCM broth amended with rifamycin. Flasks were then secured on a rotary shaker (150 rpm) and agitated for 24 h at room temperature. Bacteria were harvested by centrifugation (5,700×g, 30 min) and resuspended in sterile phosphate buffer (SPB) to a density of 106 cfumL−1. Heat-killed P2b-2R was prepared by autoclaving broth containing the strain P2b-2R for 1 h before centrifugation and resuspension in SPB. A sample of heat-killed P2b-2R was streaked on TSA plates and evaluated 1 week later to confirm the absence of live cells. For the seedling growth assay, 5 mL of the P2b-2R-SPB suspension were pipetted directly into each of the 70 replicate tubes containing lodgepole pine seed immediately after sowing. This process was repeated using heat-killed P2b-2R in SPB. Seeds in the 70 control tubes received 5.0 mL of SPB. Tubes were then placed in a growth chamber (Conviron CMP3244, Conviron Products Company, Winnipeg, MB) with photosynthetically active radiation at a canopy level of 300 μmol photons (400–700-nm wavelengths)m−2 s−1, an 18-h photoperiod, and a 20/14 °C day/ night temperature cycle. Seedlings were thinned to the largest single germinant per tube 2 weeks after sowing and were watered daily with sterile distilled water to constant weight and with nutrient solution without Ca( NO3)2 once a month. Seedling mortality was monitored throughout the 13-month growth period. Tubes containing dead seedlings were replaced as required from a separate pool of replicate tubes prepared for each treatment when the experiment was set up.
N2-Fixation and Seedling Growth Promotion of Lodgepole Pine
Quantification of Endophytic Colonization Three randomly selected seedlings from each treatment were harvested destructively 2, 4, 8, and 12 months after sowing to evaluate endophytic colonization. At each harvest, seedlings were washed thoroughly under running water for 3 h, surface-sterilized in 1.3 % sodium hypochlorite for 5 min, and washed three times with sterile distilled water. Seedlings were imprinted on TSA plates for 24 h to check for surface contamination. Root, stem, and needle tissue samples (20 mg fresh weight) were then triturated separately in 1 mL SPB using a mortar and pestle. Triturated tissue suspensions were then diluted serially, and 0.1 mL of each dilution was plated onto TSA supplemented with cycloheximide (100 mgL−1) to suppress fungal growth as well as TSA plus cycloheximide (100 mgL−1) and rifamycin (200 mgL−1). The number of colony forming units (cfu) was evaluated 72 h after incubation at 30 °C. Nitrogen Analysis and Seedling Growth Response After 13 months, the remaining seedlings (38–45 per treatment) were removed from tubes, separated into roots and shoots, and the roots were washed. After measuring the root and shoot length, needles were separated from stems and root, stem and needle tissues were dried for 2 days at 65 °C. Needles from each treatment were pooled into seven samples, each comprising seven seedlings, and then ground to a particle size
