... Chris Chanway: Faculty of Land and Food Systems, Faculty of Forestry, Univer-sity of British ... more ... Chris Chanway: Faculty of Land and Food Systems, Faculty of Forestry, Univer-sity of British Columbia, Vancouver, British Columbia, Canada V6T ... The primary mechanisms thought to lead to beneficial effects for the plant are nitrogen fixation (Boddey and Döbereiner 1995) and ...
We inoculated lodgepole pine (Pinus contorta var. latifolia (Dougl.) Engelm.) with Paenibacillus ... more 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 NO 3 ) 2 to facilitate detection of N 2 -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 N 2fixation 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.
Lodgepole pine ( Pinus contorta var. latifolia Engelm. ex S. Watson) and western red cedar ( Thuj... more 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 (>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 nitr...
ABSTRACT Paenibacillus polymyxa P2b-2R is a bacterium that originated from internal lodgepole pin... more ABSTRACT 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.
Inoculation of western red cedar with Paenibacillus polymyxa P2b-2R, an endophytic diazotroph of ... more 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 Nlimited soil mix containing 0.7 mM of available N labeled as Ca(15 NO 3) 2. P2b-2R developed a persistent endophytic population comprising 10 2-10 6 cfu g −1 plant tissue inside pine roots, stems, and needles. At the end of the growth period, P2b-2R had reduced 15 N 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 Nlimited 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.
We characterized nif gene structure of Paenibacillus polymyxa strain P2b-2R, an endophytic diazot... more We characterized nif gene structure of Paenibacillus polymyxa strain P2b-2R, an endophytic diazotroph that can provide up to 79 % of foliar N in lodgepole pine through biological nitrogen fixation. We amplified a 388-bp internal nifH gene fragment from P2b-2R using the single specific primer-polymerase chain reaction (SSP-PCR) and performed a Southern blot analysis of Pst I/HindIII-digested genomic DNA to evaluate the sequence, copy number, and location in the nif gene operon. This strain was found to possess a single copy of the nifH gene with nifB located directly upstream of nifH and D. Phylogenetic analyses of the full nifH, partial nifB, and nifD, and 16s rDNA (rrs) gene sequences indicated that P2b-2R was part of a monophyletic cluster with other members of the genus Paenibacillus and suggest that nifH is transmitted horizontally while nifD is transmitted vertically. Our results provide the first full nifH sequence from a P. polymyxa strain and indicate that arrangement of genes in the P2b-2R nif operon is consistent with those observed in other species of the genus Paenibacillus.
We inoculated lodgepole pine (Pinus contorta var. latifolia (Dougl.) Engelm.) with Paenibacillus ... more 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 NO 3 ) 2 to facilitate detection of N 2 -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 N 2fixation 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.
Lodgepole pine (Pinus contorta var. latifolia Engelm. ex S. Watson) and western red cedar (Thuja ... more 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 (>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.
We characterized nif gene structure of Paenibacillus polymyxa strain P2b-2R, an endophytic diazot... more We characterized nif gene structure of Paenibacillus polymyxa strain P2b-2R, an endophytic diazotroph that can provide up to 79 % of foliar N in lodgepole pine through biological nitrogen fixation. We amplified a 388-bp internal nifH gene fragment from P2b-2R using the single specific primer-polymerase chain reaction (SSP-PCR) and performed a Southern blot analysis of Pst I/HindIII-digested genomic DNA to evaluate the sequence, copy number, and location in the nif gene operon. This strain was found to possess a single copy of the nifH gene with nifB located directly upstream of nifH and D. Phylogenetic analyses of the full nifH, partial nifB, and nifD, and 16s rDNA (rrs) gene sequences indicated that P2b-2R was part of a monophyletic cluster with other members of the genus Paenibacillus and suggest that nifH is transmitted horizontally while nifD is transmitted vertically. Our results provide the first full nifH sequence from a P. polymyxa strain and indicate that arrangement of genes in the P2b-2R nif operon is consistent with those observed in other species of the genus Paenibacillus.
... Chris Chanway: Faculty of Land and Food Systems, Faculty of Forestry, Univer-sity of British ... more ... Chris Chanway: Faculty of Land and Food Systems, Faculty of Forestry, Univer-sity of British Columbia, Vancouver, British Columbia, Canada V6T ... The primary mechanisms thought to lead to beneficial effects for the plant are nitrogen fixation (Boddey and Döbereiner 1995) and ...
We inoculated lodgepole pine (Pinus contorta var. latifolia (Dougl.) Engelm.) with Paenibacillus ... more 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 NO 3 ) 2 to facilitate detection of N 2 -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 N 2fixation 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.
Lodgepole pine ( Pinus contorta var. latifolia Engelm. ex S. Watson) and western red cedar ( Thuj... more 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 (>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 nitr...
ABSTRACT Paenibacillus polymyxa P2b-2R is a bacterium that originated from internal lodgepole pin... more ABSTRACT 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.
Inoculation of western red cedar with Paenibacillus polymyxa P2b-2R, an endophytic diazotroph of ... more 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 Nlimited soil mix containing 0.7 mM of available N labeled as Ca(15 NO 3) 2. P2b-2R developed a persistent endophytic population comprising 10 2-10 6 cfu g −1 plant tissue inside pine roots, stems, and needles. At the end of the growth period, P2b-2R had reduced 15 N 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 Nlimited 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.
We characterized nif gene structure of Paenibacillus polymyxa strain P2b-2R, an endophytic diazot... more We characterized nif gene structure of Paenibacillus polymyxa strain P2b-2R, an endophytic diazotroph that can provide up to 79 % of foliar N in lodgepole pine through biological nitrogen fixation. We amplified a 388-bp internal nifH gene fragment from P2b-2R using the single specific primer-polymerase chain reaction (SSP-PCR) and performed a Southern blot analysis of Pst I/HindIII-digested genomic DNA to evaluate the sequence, copy number, and location in the nif gene operon. This strain was found to possess a single copy of the nifH gene with nifB located directly upstream of nifH and D. Phylogenetic analyses of the full nifH, partial nifB, and nifD, and 16s rDNA (rrs) gene sequences indicated that P2b-2R was part of a monophyletic cluster with other members of the genus Paenibacillus and suggest that nifH is transmitted horizontally while nifD is transmitted vertically. Our results provide the first full nifH sequence from a P. polymyxa strain and indicate that arrangement of genes in the P2b-2R nif operon is consistent with those observed in other species of the genus Paenibacillus.
We inoculated lodgepole pine (Pinus contorta var. latifolia (Dougl.) Engelm.) with Paenibacillus ... more 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 NO 3 ) 2 to facilitate detection of N 2 -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 N 2fixation 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.
Lodgepole pine (Pinus contorta var. latifolia Engelm. ex S. Watson) and western red cedar (Thuja ... more 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 (>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.
We characterized nif gene structure of Paenibacillus polymyxa strain P2b-2R, an endophytic diazot... more We characterized nif gene structure of Paenibacillus polymyxa strain P2b-2R, an endophytic diazotroph that can provide up to 79 % of foliar N in lodgepole pine through biological nitrogen fixation. We amplified a 388-bp internal nifH gene fragment from P2b-2R using the single specific primer-polymerase chain reaction (SSP-PCR) and performed a Southern blot analysis of Pst I/HindIII-digested genomic DNA to evaluate the sequence, copy number, and location in the nif gene operon. This strain was found to possess a single copy of the nifH gene with nifB located directly upstream of nifH and D. Phylogenetic analyses of the full nifH, partial nifB, and nifD, and 16s rDNA (rrs) gene sequences indicated that P2b-2R was part of a monophyletic cluster with other members of the genus Paenibacillus and suggest that nifH is transmitted horizontally while nifD is transmitted vertically. Our results provide the first full nifH sequence from a P. polymyxa strain and indicate that arrangement of genes in the P2b-2R nif operon is consistent with those observed in other species of the genus Paenibacillus.
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