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Effect of ammonium and nitrate on 15N2-fixation of Azospirillum spp. and Pantoea agglomerans in association with wheat plants
MicrobioL Res. (1997) 152,377-383
Microbiological Research
co Gustav Fischer Verlag
Effect of ammonium and nitrate on 15N2-fixation of Azospirillum spp. and Pantoea agglomerans in association with wheat plants Silke Ruppel, Wolfgang Merbach Institute of Vegetable and Ornamental Crop Production GroBbeerenJErfurt e.v., Theodor-Echtermeyer-Weg 1, D-14979 GroBbeeren, Germany Center for Agricultural Landscape and Land Use Research (ZALF), Institute of Rhizosphere Research and Plant Nutrition, Eberswalder StraBe 84, D-15374 Miincheberg Accepted: September 21, 1997
Abstract The dinitrogen fixing ability of two diazotrophic bacterial strains Pantoea agglomerans and Azospirillum spp. which are proved to express N2-fixing activity in presence of additional inorganic nitrogen sources was tested in association with wheat plants in hydroponic experiments using 15N2 incubation. The effect of 100 ppm nitrogen added as NH4CI or KN0 3 to wheat plants on dinitrogen fixing activity of native as well as inoculated bacteria was determined. Enrichment of 15N, that means fixed dinitrogen, was detected in plant growth media, in roots and shoots of wheat plants grown 26 days in 15Nz enriched atmosphere. Highest 15N amounts were found in wheat shoots. As well as the form of nitrogen applied and the bacterial strain inoculated effected plant growth, nitrogen uptake and the amount of biologically fixed dinitrogen. Ammonia or nitrate supply to plants did not repress 15N2 fixation. Distribution of 15N within the plant and media was mainly influenced by the inoculated bacterial strain. The detected dinitrogen fixing ability in presence of inorganic nitrogen of both bacterial strains in pure culture was confirmed even in association with wheat plants. That finding offers the possibility to select diazotrophic bacterial strains in pure culture which are able to fix dinitrogen in association with plants when additional inorganic nitrogen was fertilized. Key words: Diazotrophic bacteria - dinitrogen fixation plant bacteria association - 15N2-enrichment - ammonia and nitrate fertilization - Azospirillum spp. - Pan toea agglomerans
Corresponding author: 'S. Ruppel
Introduction Bacterial fixation of atmospheric nitrogen has been documented with different bacterial species in pure culture and in association with plants (Boddey and Dobereiner 1988; Haatela et at. 1988; Okon and Labandera-Gonzalez 1994; Pacovsky 1990). In several experiments the inoculation of diazo trophic bacteria increased the nitrogen uptake of mais, sugar cane and wheat. However, plant growth promoting effects were not always attributed to an inproved nitrogen nutrition of plants (Bashan et at. 1990; Freitas and Gennida 1990; Murty and Ladha 1988). It is still questionable if diazotrophic bacteria living in association with plants are able to cover a significant part of the nitrogen demand of the plant (Hurek et al. 1989; Zimmer et al. 1988). Certainly, the total nitrogen demand of the plant definitely can not be covered by dinitrogen fixing bacteria due to energetic limitations (Gutschik 1982). Therefore diazotrophic bacterial strains which are able to fix atmospheric nitrogen in presence of additional nitrogen sources were selected (Bali etal. 1992; Hartmann etal. 1988; Ruppel and Merbach 1995). Two of these strains Azospirillum spp. and Pantoea agglomerans, we isolated from wheat and Amophila arena ria, fixed in pure culture atmospheric nitrogen in presence of ammonia- and nitrate nitrogen (Ruppel and Merbach 1995). First results with rhizobium legume symbiosis (Doughton et al. 1995) and Azalla-Anabaena symbiosis (Okoronkwo etat. 1989) exist about N2-fixing activity of bacteria on plants in presence of additional mineral nitrogen. If the N2-fixing ability in presence of additional nitrogen sources of Microbiol. Res. 152 (1997) 4
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diazotrophic strains measured in pure culture also occurs in non symbiotic association with plants after nitrogen fertilization is still unknown. Unexplained is also which part of the fixed dinitrogen is used for bacterial cell growth and which part is delivered into the surrounding medium or for plants disposal. So the main object of these investigations was to test the nitrate and ammonium N tolerance of nitrogenase activity of two diazotrophic strains in association with wheat plants using 15N 2 incubation expeliments and to investigate the distribution of fixed ISN 2 within the plant.
Material and methods Bacterial strains. Pantoea agglomerans was isolated from the phyllosphere of winter wheat (Triticum aestivum L.). The strain is able to fix atmospheric nitrogen in presence of additional ammonium or nitrate nutrition (up to 4 mM N) in pure culture (Ruppel and Merbach 1995), to reduce nitrate to ammonia and to produce phytohormones (Auxin: Indole-3-acetic acid and Indole-3-lactic acid; Cytokinin compounds: N6-Isopenty ladenosine and N 6-Isopentyladenine) (Scholz-Seidel and Ruppel 1992). Azospirillum spp. was isolated from the rhizosphere of AmmophUa arena ria (L.). The strain also is able to fix atmospheric nitrogen in presence of additional ammonium or nitrate nutrition (up to 0.3 mM N) in pure culture (Ruppel and Merbach 1995), to produce phytohormones (Auxin), but is unable to reduce nitrate (ScholzSeidel and Ruppel 1992). To obtain starter cultures the bacterial strains were grown in liquid complex medium (Hirte 1961) on an orbital shaker at 28°C for 24 hours. The cells were washed twice in physiological sodium chloride solution and calibrated on a 'cell density of 109 cfu ml- I. Plant growth test using 15N 2 -incubation. The effect of bacterial inoculation and nitrogen supply on plant growth, nitrogen nuttition and biological dinitrogen fixation was investigated in an semisterile hydroponic pot experiment. Winter wheat seeds (variety 'Miras') were surface sterilized using saturated brom solution for 30 seconds, then washed six times in sterile 0.05 M NaCl solution and germinated in sterile plates. Ten seedlings were inserted into a 250 ml bottle containing 50 ml nutrient media (Bothe and Zimmer 1988) and inoculated with bactelia 107 cfu ml- I final concentration 24 h after planting. Treatments tested were without bacterial inoculation, inoculated with Pantoea agglomerans and inoculated with Azospirillum spp. The effect of additional inorganic nitrogen on bacterial N2-fixing ability at the plant was tested using 100 ppm N as potassium nitrate or ammonium chloride and without nitrogen in the nutrient med.~a. The bottles were sterile closed 378
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using cotton balls and incubated in a phytotron at 12 h day and night cycle at 16°C and 12 DC, respectively. After one week three replicates of each treatment were harvested to determine plant growth and nitrogen content in media, root and shoot. Simultaneously eight replicates were sealed up with rubber septa and five replicates were incubated with 19.76 atom % ISN 2 excess and three replicates were used as control without gas addition. ISNzgas was prepared from ('sNH4)2S04 (Ohyama and Kumazawa 1981). 26 days later plants were harvested, dry matter, nitrogen content and 15N content were determined in media, root and shoot samples. Determination of total nitrogen and 15N. Plants were divided into leaves and roots, dlied at 65°C, and ground after dry matter was determined. Total nitrogen was analysed by the method of Kjeldahl modified by Bremner and Mulvany (1982) and the NH4 CI originated from Kjeldahl extracts was used to determine ISN using an emission spectrometer N0I6 (Faust et al. 1981). The amount of N in plant or medium derived from the atmosphere (Ndfa) was determined as follows: Nfda = amount of 15N in plant or medium [mg pOt]- I* 100* (,sN abundance in incubation air [atom % ISN excess]tl. Statistics. Mean value were compared by Tukey 's HSD test at P~0.05 (STATISTICA 1995), different letters indicate significant differences.
Results Effect of nitrogen nutrition and bacterial inoculation on root and shoot growth and nitrogen yield
Bacterial inoculation as well as nitrogen supply effected nitrogen uptake of roots and shoots and shoot growth significantly. N supply (both form s) increased nitrogen yield of shoots and the total plants in all cases (Table 1). Total plant dry matter was only increased in treatments supplied with ammonia nitrogen. Without bacterial inoculation shoot growth and N uptake were highest in treatments supplied with ammonia nitrogen followed by treatments supplied with nitrate nitrogen and the lowest shoot growth was detectable without addition of inorganic nitrogen (Table 1). The bacterial inoculation effect on plant growth and uptake differed with the form of nitrogen applied. Only in the treatment without inorganic nitrogen bacterial inoculation increased shoot growth and nitrogen yield of the shoot slightly (Table 1). In treatments supplied with ammonia bacterial inoculation did not effect shoot dry matter production or total nitrogen yield. Root dry matter was un effected by both nitrogen supply and bacterial inoculation (Table 1). Nitrogen
Table 1. Effect of bacterial inoculation (Pantoea aggiomerans, Azospirillum spp.) and nitrogen supply (100 ppm NH/ -N or N0 3--N) on total shoot and root growth (dry matter documented as mg vessel- I) and N yield (mg vessel- I) after 33 days of growth (means of five replicates) N -fertilization
withollt nitrogen NH4+-N
N0 3--N
Bacterial
Root
inoculation
Dry matter
Nyield
Dry matter
N yield
Dry matter
N yield
without P aggiomerans Azospirillum spp. without P aggiomerans Azospirillum spp. without P agglomerans Azospirillum spp.
155.4 152.8 153.4 151.6 146.6 149.6 144.6 150.0 156.6 15.27
2.41 2.13 2.29 2.00 2.30 2.59 3.24* 3.26* 4.11 * 0.55
228.2 240.0 246.2 269.6*a 269.8* 265.6* 254.8* 233.4 222.6 18.2
7.37 7.89 7.55 11.55* 11.27* 11.13 10.48* 9.74* 8.45* 0.92
383.6 392.8 399.6 421.2* 416.4* 415.2* 399.4 383.4 379.2 18.1
9.78 10.02 9.84 13.55* 13.57* 13.72* 13.72* 13.00* 12.53* 0.51
LSD P::;0.05 a
Shoot
Plant total
(*) significant different to control without nitrogen and without bacterial inoculation at P::; 0.05. IS
4 JIB
exc .
vessel
IS
·1
c 3 2
b
Fig. 1. 15N content in plant growth media, roots and shoots of wheat plants after 26 days incubation in 15N2 enriched atmos'phere (19.76 at % 15N exc.)(mean values of all treatments), different letters indicate significant differences at P::; 0.05 .
Fig. 2. Effect of ammonia and nitrate supply on 15N distribution in media and shoots of wheat plants (main effect of N supply), different letters indicate significant differences at P::; 0.05. Further details see Tables 1 and 2.
yield of the root was increased after nitrate addition and inoculation with Azospirillum spp. intensified this effect significantly while shoot growth and N yield of the shoot were significantly decreased in treatments inoculated with Azospirillum spp. and supplied with nitrate.
inoculated strains had. Ammonia addition significantly reduced the 15N enrichment in the plant nutrient media while in shoots 15N enrichment was increased as main effect of all treatments (Fig. 2). That means ammonia addition caused a shift of biologically fixed dinitrogen from the plant growth media (where bacteria were inoculated) into plant shoots without enrichment in roots. Clearest was that effect after inoculation with the bacterial strain Azospirillum spp. (Fig. 3). In contrast to the 15N enrichment in shoots following Azospirillum spp. inoculation and ammonia addition, inoculation of P. agglomerans let to an enrichment of 15N in plant growth media when nitrate was added (Table 2).
Effect of nitrogen and bacteria on plant and media
15N enrichment
in
15N enrichment was detectable in media, root and shoot samples after 26 days incubation in 15N2 enriched atmosphere (Fig. 1). The highest amount of 15N was detected in shoots followed by media samples. The 15N amounts in root samples were near the detection limit. As well as form of applied nitrogen and the bacterial strain inoculated effected significantly the 15N enrichment in plants and media and the distribution of the biologically fixed dinitrogen (Table 2). Surprising was the 15N enrichment in non inoculated vessels. Probably there occured native diazotrophic bacteria within the spermosphere with the same dinitrogen fixing activities as the selected and
Effect of inorganic nitrogen on biologically dinitrogen fixation of P. agglomerans and Azospirillum spp. in association with wheat plants
The form of nitrogen applied to the plant (NH/-N, N0 3--N or without inorganic nitrogen) effected significantly the dinitrogen fixing activity of the inoculated Microbial. Res. 152 (1997) 4
379
15
-I
IS
vessel
7 ~g
5 ~~~------------------~~
~c vessel
-I
4 3
WIthOut
2
o ~~........:
Fig. 3. Effect of bacterial inoculation on I5N distribution in media and shoots of wheat plants after addition of ammonia (100 ppm N), different letters indicate significant differences at P:S; 0.05.
P, Ilggiomerans Azo 'Ptrillum spp_ Fig.4. Effect of nitrogen supply (100 ppm N as ammonia and nitrate or without inorganic nitrogen) on dinitrogen fixation (total I5N content per vessel after 26 days incubation in I5N2 enriched atmosphere) after inoculation with P. agglomerans and Azospirillum spp., different letters indicate significant differences at P:S; 0.05.
Table2. Effect of bacterial inoculation (Pantoea agglomerans, Azospirillum spp.) and nitrogen supply (100 ppm NH/-N or N0 3--N) on I5N enrichment in media, roots and shoots after 26 days incubation in I5N 2 enriched atmosphere (19.76 at % ISNexc.) (means of five replicates). N-fertilization Bacterial inoculation
Media (Ilg I5N vessel-I)
(% of total)
Root (Ilg I5Nexc. vessel-I)
(% of total)
Shoot (Ilg I5Nexc. vessel-I)
(% of total)
Total (Ilg I5Nexc. vessel-I)
(% of total)
without N
2.18 0.82 2.60 1.08 0.66 0.54 1.78 2.86* 1.90 0.80
38.3 21.4 42.3 27.8 14.3 9.5 26.7 44.1 36.8
1.12 0.58 0.78 0.52 0.34 0.50 1.42 0.36 0.72 0.61
19.6 15.1 12.7 13.4 7.4 8.8 21.3 5.6 14.0
2.40 2.44 2.76 2.28 3.62 4.66*a 3.46 3.26 2.54 1.30
42.1 63.5 45.0 58.8 78.3 81.8 52.0 50.3 49.2
5.70 3.84 6.14 3.88 4.62 5.70 6.66 6.48 5.16 2.03
100 100 100 100 100 100 100 100 100
without P. agglomerans
Azospiritlum spp. without P. agglomerans Azospirillum spp. without P. agglomerans Azospirillum spp.
NH4+-N NO 3--N LSDP:S;0.05 a
(*) significant different to control without nitrogen and without bacterial inoculation at P:S; 0.05.
bacteria (Fig. 4). The bacterial strain Pantoea agglomerans fixed increasing amounts of dinitrogen when the plant was supplied with additional inorganic nitrogen. The amount of 15N bound in media and shoot of plants was nearly doubled when plants were supplied with nitrate compared to plants grown without inorganic nitrogen. Addition of inorganic nitrogen did not effect the dinitrogen fixing activity (expressed as 15N enrichment in media and shoots) of Azospirillum spp. (Fig. 4). As well as in bacterial pure culture (Ruppel and Merbach 1995) and in association with wheat plants the strain Azospirillum spp. fixed higher amounts of dinitrogen than the strain P. agglomerans without addition of inorganic nitrogen. Both strains showed the same reaction in their N2-fixing ability in association with the plant as they showed in pure culture after addition of 380
Microbiol. Res. 152 (1997) 4
nitrate. Ammonia addition did not effect the N2-fixing ability of both strains living in association with the plant while in pure culture their activity was drastically reduced (Ruppel and Merbach 1995).
Discussion 15N2fixation in association with wheat plants The detected 15N amounts in media, roots and shoots of wheat plants after 26 days incubation in 15N2 enriched atmosphere demonstrated that dinitrogen fixation at wheat plants occured. The highest 15N amount was detected in shoots of plants supplied with ammonia and inoculated with Azospirillum spp. (Table 2). However,
Table 3 . .Portion of Ndfa (N delived from the atmosphere) at the nitrogen nutrition of wheat plants (calculated for shoot increase during 26 days of incubation in 15N2 enriched atmosphere)
N-fertilization without N NH4+-N N0 3--N
Bacterial inoculation P. agglomerans Azospirillum spp. P. agglomerans Azospirillum spp. P. agglomerans Azospirillum spp.
15Nexc.
N-increase (/lg vessel-I)
(/lg vessel-I)
Ndfa Part of Ndfa at N (/lg vessel-I) nutrition of shoots (%)
2070 1760 4418 4696 3553 1980
2.44 2.76 3.62 4.66 3.26 2.54
12.3 27.6 36.2 46.6 32.6 25.4
the portion of nitrogen derived from the atmosphere (Ndfa) at the nitrogen nutrition of the plant was very small compared to results of rhizobium legume symbiosis where the Ndfa amounted between 13 -96% (Androsoff et al. 1995; Boddey and Urquiaga 1992). Our best treatment reached a maximum of 0.9% Ndfa (Table 3). Probably the young wheat plants (6 weeks old) in our experiment used a big amount of nutrients from the spermosphere and the shoot increase during 26 days of incubation time was too small to calculate the general biological dinitrogen fixing ability of diazotrophic bacteria living in association with plants, while Haahtela etal. (1988) measured between -1.9 and 17.7% of Ndfa at wheat plants grown in greenhouse from June to August .and Malik and Bilal (1988) estimated 12-15% Ndfa in shoots of kallar grass. The latter authors discussed some problems with the methology by using a non fixing control plant due to a biological dinitrogen fixation even in that control. That problem was underlined by our measurements in non inoculated treatments, where a measurable amount of 15N was detected. Inoculation with diazotrophic bacteria induced a significant effect on the distribution of fixed 15N within the plant. While most 15N was detectable within shoots when plants were inoculated with Azospirillum spp., the 15N was nearly equal distributed in media and shoots when P. agglomerans was applied. Effect of ammonia and nitrate on biological dinitrogen fixation The diazotrophic bacterial strains Azospirillum spp. and P. agglomerans were selected for their ability to fix dinitrogen in presence of additional inorganic nitrogen sources (Ruppel and Merbach 1995). Both strains were completely repressed in their N2-fixing activity in pure culture when 100 or 50 ppm N as ammonia were added. Only at low concentrations of 14 mg Nl-l we measured a nitrogenase activity. In association with wheat plants no repression of 15N enrichment was detectable when ammonia was added (Fig. 4). On the contrary 15N enrichment in shoots. was highest after ammonia addi-
0.61 0.91 0.41 0.51 0.50 0.88
tion (Fig. 2). That increased nitrogen accumulation derived from biological dinitrogen fixation in shoots of inoculated wheat plants could be attributed to an increased root carbon exudation which stimulates bacterial growth in the rhizosphere. The close correlation of above ground plant growth and the amount of carbon root exudation (Liljeroth et al. 1994) underlines this assumption because plants supplied with ammonia showed the significantly best growth rate during the 26 days incubation period. Increasing C availability and changes in carbon composition can simultaneously lead to an increased bacterial nitrogenase activity (Hartmann etal. 1988; Moreno- Vivian etal. 1989) or other bacterial enzyme activities as Jiang and Sato (1994) documented with phosphate solubilizing bacteria. Nitrogenase activity of the bacterial strain Pan toea agglomerans was nearly doubled when nitrate was added to the medium in pure culture (Ruppel and Merbach 1995) and even in association with wheat plants (Fig. 4). That finding offers the possibility to select diazotrophic bacterial strains in pure culture which are able to fix dinitrogen in association with plants when additional inorganic nitrogen was supplied. An increased specific nitrogenase activity in bacterial pure culture resulted also in an increased molecular nitrogen fixation in association with plants. However, our results documented, that the specific nitrogenase activity in bacterial pure culture with most effective nutritional and environmental conditions for dinitrogen fixation (0.5% mannit and 0.5% saccharose in semisolid CC medium and without inorganic nitrogen at 29°C) was not reached in association with the plant. The strain Azospirillum spp. for example fixed 0.75 flgN mg protein- 1h- 1in bacterial pure culture. Assuming the initially inoculated bacteria (0.25 mg protein per vessel) survived during experimental time in association with wheat plants, and the bacteria expressed the same nitrogenase activity as in pure culture, 116 flg dinitrogen could be fixed within 26 days. However, our experimental results showed only a N 2 fixation of about 28 flg N fixed per vessel within 26 days (Table 3). These results indicate that more than only the nitrogen conMicrobiol. Res. 152 (1997) 4
381
centration in the medium regulate the expression of bacterial nitrogenase activity in association with plants. Varying nitrogen nutrition effects root and shoot growth of plants and consequently root exudation and composition of root exudates (Bowen 1969; Liljeroth et al. 1990 a). Variation in amount and composition of root exudates effects microbial biomass and microbial community composition in the rhizosphere (Liljeroth et al. 1990 b; Trolldenier and v. Rheinbaben 1981). To cause an effective dinitrogen fixing activity a close association between the inoculated bacterial strain and the plant is a prerequisite. Therefore, the bacterial strain must be able to compete with the native microbial population and replace it partly in order not to stress the plant additionally. How plant nutrition and amount and composition of root exudates may effect the bacterial community composition and their activity is still unknown.
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Schriftenreihe Anwendung von Isotopen und Kernstrahlungen in der Wissenschaft und Technik. Berlin 5. Freitas, J. Rd., Gerrnida, J. J. (1990): Plant growth promoting rhizobacteria for winter wheat. Can. J. Microbiol. 36, 265-272. Gutschik, V. P. (1982): Energetics of microbial fixation of dinitrogen. Microbes and engineering aspects, Akademie Verlag, Berlin: 109 -168. Haahtela, K. , Laakso, T , Nurmiaho-Lassila, E.-L. , Korhonen, T K. (1988) : Effects of inoculation of Poa pratensis and Triticum aestivum with root-associated, N 2-fixing Klebsiella, Enterobacter and Azospirillum. Plant and Soil 106,239-248. Hattmann, A., Fu, H., Burris, R. H. (1988): Influence of amino acids on nitrogen fixation ability and growth of Azospirillum spp. Appl. Environment. Microbiol. 54, 87 -93. Hirte, W. F. (1961): Glycerin-Pepton-Agar, ein vorteilhafter Nahrboden flir bodenbakteriologische Arbeiten. Zbl. Bakt., Abt. II, 114, 141-146. Hurek, T , Reinhold, B., Grimm, B. , Fendrik, I., Niemann, E. - G. (1989): Occurrence of effective nitrogen-scavenging bacteria in the rhizosphere of kallar grass. In: Nitrogen fixation with non-legumes (Eds : Skinner, F. A.). Kluwer Academic Publisher, 321-330. Jiang, H. - Y, Sato, K. (1994): Interrelationships between bacterial populations on the root surface of wheat and growth of plant. Soil Sci. Plant Nutr. 40, 683-689. Liljeroth, E., Kuikman, P., Van Veen , J. A. (1994): Carbon translocation to the rhizosphere of maize and wheat and influence on the turnover of native soil organic matter at different soil nitrogen levels. Plant and Soil 161, 233-240. Liljeroth, E., Baath, E., Mathiasson, I., Lundborg, T (l990a) : Root exudation and rhizoplane abundance of barley (Hordeum vulgare L.) in relation to nitrogen fertilization and root growth. Plant and Soil 127, 81- 89. Liljeroth, E., Van Veen, J. A., Miller, H. 1. (1990b): Assimilate translocation to the rhizosphere of wheat lines and subsequent utilization by rhizosphere microorganisms at two soil nitrogen concentrations. Soil BioI. Biochem. 22, 1015-102I. Malik, K. A. , Bilal, R (1988) : Survival and colonization of inoculated bacteria in kallar grass rhizosphere and quantification of N 2 -fixation. Plant and Soil 110, 329-338. Moreno- Vivian, c., Caballero, F. J., Cardenas, J. , Castillo, F. (1989): Effect of the CfN balance on the regulation of nitrogen fixation in Rhodobacter capsulatus El Fl. Biochim. Biophys. Acta 977, 297-300. Murty, M. G., Ladha, J. K. (1988) : Influence of Azospirillum inoculation on the mineral uptake and growth of rice under hydroponic conditions. Plant and Soil 108, 281 - 285. Ohyama, F., Kumazawa, K. (1981): A simple method for the preparation, purification and storage of ISN2 gas for biological nitrogen fixation studies. Soil Sci. Plant Nutr. 27, 263- 265. Okon, Y, Labandera-Gonzalez, C. A . (1994): Agronomic applications of Azospirillum. In: Improving plant productivity with rhizosphere bacteria (Eds: Ryder, M. H., Stephens, P. M., Bowen, G. D.) 4. CSIRO Division of Soils, Graphic services, Adelaide Australia, 274-278.
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Microbiological Research
co Gustav Fischer Verlag
Effect of ammonium and nitrate on 15N2-fixation of Azospirillum spp. and Pantoea agglomerans in association with wheat plants Silke Ruppel, Wolfgang Merbach Institute of Vegetable and Ornamental Crop Production GroBbeerenJErfurt e.v., Theodor-Echtermeyer-Weg 1, D-14979 GroBbeeren, Germany Center for Agricultural Landscape and Land Use Research (ZALF), Institute of Rhizosphere Research and Plant Nutrition, Eberswalder StraBe 84, D-15374 Miincheberg Accepted: September 21, 1997
Abstract The dinitrogen fixing ability of two diazotrophic bacterial strains Pantoea agglomerans and Azospirillum spp. which are proved to express N2-fixing activity in presence of additional inorganic nitrogen sources was tested in association with wheat plants in hydroponic experiments using 15N2 incubation. The effect of 100 ppm nitrogen added as NH4CI or KN0 3 to wheat plants on dinitrogen fixing activity of native as well as inoculated bacteria was determined. Enrichment of 15N, that means fixed dinitrogen, was detected in plant growth media, in roots and shoots of wheat plants grown 26 days in 15Nz enriched atmosphere. Highest 15N amounts were found in wheat shoots. As well as the form of nitrogen applied and the bacterial strain inoculated effected plant growth, nitrogen uptake and the amount of biologically fixed dinitrogen. Ammonia or nitrate supply to plants did not repress 15N2 fixation. Distribution of 15N within the plant and media was mainly influenced by the inoculated bacterial strain. The detected dinitrogen fixing ability in presence of inorganic nitrogen of both bacterial strains in pure culture was confirmed even in association with wheat plants. That finding offers the possibility to select diazotrophic bacterial strains in pure culture which are able to fix dinitrogen in association with plants when additional inorganic nitrogen was fertilized. Key words: Diazotrophic bacteria - dinitrogen fixation plant bacteria association - 15N2-enrichment - ammonia and nitrate fertilization - Azospirillum spp. - Pan toea agglomerans
Corresponding author: 'S. Ruppel
Introduction Bacterial fixation of atmospheric nitrogen has been documented with different bacterial species in pure culture and in association with plants (Boddey and Dobereiner 1988; Haatela et at. 1988; Okon and Labandera-Gonzalez 1994; Pacovsky 1990). In several experiments the inoculation of diazo trophic bacteria increased the nitrogen uptake of mais, sugar cane and wheat. However, plant growth promoting effects were not always attributed to an inproved nitrogen nutrition of plants (Bashan et at. 1990; Freitas and Gennida 1990; Murty and Ladha 1988). It is still questionable if diazotrophic bacteria living in association with plants are able to cover a significant part of the nitrogen demand of the plant (Hurek et al. 1989; Zimmer et al. 1988). Certainly, the total nitrogen demand of the plant definitely can not be covered by dinitrogen fixing bacteria due to energetic limitations (Gutschik 1982). Therefore diazotrophic bacterial strains which are able to fix atmospheric nitrogen in presence of additional nitrogen sources were selected (Bali etal. 1992; Hartmann etal. 1988; Ruppel and Merbach 1995). Two of these strains Azospirillum spp. and Pantoea agglomerans, we isolated from wheat and Amophila arena ria, fixed in pure culture atmospheric nitrogen in presence of ammonia- and nitrate nitrogen (Ruppel and Merbach 1995). First results with rhizobium legume symbiosis (Doughton et al. 1995) and Azalla-Anabaena symbiosis (Okoronkwo etat. 1989) exist about N2-fixing activity of bacteria on plants in presence of additional mineral nitrogen. If the N2-fixing ability in presence of additional nitrogen sources of Microbiol. Res. 152 (1997) 4
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diazotrophic strains measured in pure culture also occurs in non symbiotic association with plants after nitrogen fertilization is still unknown. Unexplained is also which part of the fixed dinitrogen is used for bacterial cell growth and which part is delivered into the surrounding medium or for plants disposal. So the main object of these investigations was to test the nitrate and ammonium N tolerance of nitrogenase activity of two diazotrophic strains in association with wheat plants using 15N 2 incubation expeliments and to investigate the distribution of fixed ISN 2 within the plant.
Material and methods Bacterial strains. Pantoea agglomerans was isolated from the phyllosphere of winter wheat (Triticum aestivum L.). The strain is able to fix atmospheric nitrogen in presence of additional ammonium or nitrate nutrition (up to 4 mM N) in pure culture (Ruppel and Merbach 1995), to reduce nitrate to ammonia and to produce phytohormones (Auxin: Indole-3-acetic acid and Indole-3-lactic acid; Cytokinin compounds: N6-Isopenty ladenosine and N 6-Isopentyladenine) (Scholz-Seidel and Ruppel 1992). Azospirillum spp. was isolated from the rhizosphere of AmmophUa arena ria (L.). The strain also is able to fix atmospheric nitrogen in presence of additional ammonium or nitrate nutrition (up to 0.3 mM N) in pure culture (Ruppel and Merbach 1995), to produce phytohormones (Auxin), but is unable to reduce nitrate (ScholzSeidel and Ruppel 1992). To obtain starter cultures the bacterial strains were grown in liquid complex medium (Hirte 1961) on an orbital shaker at 28°C for 24 hours. The cells were washed twice in physiological sodium chloride solution and calibrated on a 'cell density of 109 cfu ml- I. Plant growth test using 15N 2 -incubation. The effect of bacterial inoculation and nitrogen supply on plant growth, nitrogen nuttition and biological dinitrogen fixation was investigated in an semisterile hydroponic pot experiment. Winter wheat seeds (variety 'Miras') were surface sterilized using saturated brom solution for 30 seconds, then washed six times in sterile 0.05 M NaCl solution and germinated in sterile plates. Ten seedlings were inserted into a 250 ml bottle containing 50 ml nutrient media (Bothe and Zimmer 1988) and inoculated with bactelia 107 cfu ml- I final concentration 24 h after planting. Treatments tested were without bacterial inoculation, inoculated with Pantoea agglomerans and inoculated with Azospirillum spp. The effect of additional inorganic nitrogen on bacterial N2-fixing ability at the plant was tested using 100 ppm N as potassium nitrate or ammonium chloride and without nitrogen in the nutrient med.~a. The bottles were sterile closed 378
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using cotton balls and incubated in a phytotron at 12 h day and night cycle at 16°C and 12 DC, respectively. After one week three replicates of each treatment were harvested to determine plant growth and nitrogen content in media, root and shoot. Simultaneously eight replicates were sealed up with rubber septa and five replicates were incubated with 19.76 atom % ISN 2 excess and three replicates were used as control without gas addition. ISNzgas was prepared from ('sNH4)2S04 (Ohyama and Kumazawa 1981). 26 days later plants were harvested, dry matter, nitrogen content and 15N content were determined in media, root and shoot samples. Determination of total nitrogen and 15N. Plants were divided into leaves and roots, dlied at 65°C, and ground after dry matter was determined. Total nitrogen was analysed by the method of Kjeldahl modified by Bremner and Mulvany (1982) and the NH4 CI originated from Kjeldahl extracts was used to determine ISN using an emission spectrometer N0I6 (Faust et al. 1981). The amount of N in plant or medium derived from the atmosphere (Ndfa) was determined as follows: Nfda = amount of 15N in plant or medium [mg pOt]- I* 100* (,sN abundance in incubation air [atom % ISN excess]tl. Statistics. Mean value were compared by Tukey 's HSD test at P~0.05 (STATISTICA 1995), different letters indicate significant differences.
Results Effect of nitrogen nutrition and bacterial inoculation on root and shoot growth and nitrogen yield
Bacterial inoculation as well as nitrogen supply effected nitrogen uptake of roots and shoots and shoot growth significantly. N supply (both form s) increased nitrogen yield of shoots and the total plants in all cases (Table 1). Total plant dry matter was only increased in treatments supplied with ammonia nitrogen. Without bacterial inoculation shoot growth and N uptake were highest in treatments supplied with ammonia nitrogen followed by treatments supplied with nitrate nitrogen and the lowest shoot growth was detectable without addition of inorganic nitrogen (Table 1). The bacterial inoculation effect on plant growth and uptake differed with the form of nitrogen applied. Only in the treatment without inorganic nitrogen bacterial inoculation increased shoot growth and nitrogen yield of the shoot slightly (Table 1). In treatments supplied with ammonia bacterial inoculation did not effect shoot dry matter production or total nitrogen yield. Root dry matter was un effected by both nitrogen supply and bacterial inoculation (Table 1). Nitrogen
Table 1. Effect of bacterial inoculation (Pantoea aggiomerans, Azospirillum spp.) and nitrogen supply (100 ppm NH/ -N or N0 3--N) on total shoot and root growth (dry matter documented as mg vessel- I) and N yield (mg vessel- I) after 33 days of growth (means of five replicates) N -fertilization
withollt nitrogen NH4+-N
N0 3--N
Bacterial
Root
inoculation
Dry matter
Nyield
Dry matter
N yield
Dry matter
N yield
without P aggiomerans Azospirillum spp. without P aggiomerans Azospirillum spp. without P agglomerans Azospirillum spp.
155.4 152.8 153.4 151.6 146.6 149.6 144.6 150.0 156.6 15.27
2.41 2.13 2.29 2.00 2.30 2.59 3.24* 3.26* 4.11 * 0.55
228.2 240.0 246.2 269.6*a 269.8* 265.6* 254.8* 233.4 222.6 18.2
7.37 7.89 7.55 11.55* 11.27* 11.13 10.48* 9.74* 8.45* 0.92
383.6 392.8 399.6 421.2* 416.4* 415.2* 399.4 383.4 379.2 18.1
9.78 10.02 9.84 13.55* 13.57* 13.72* 13.72* 13.00* 12.53* 0.51
LSD P::;0.05 a
Shoot
Plant total
(*) significant different to control without nitrogen and without bacterial inoculation at P::; 0.05. IS
4 JIB
exc .
vessel
IS
·1
c 3 2
b
Fig. 1. 15N content in plant growth media, roots and shoots of wheat plants after 26 days incubation in 15N2 enriched atmos'phere (19.76 at % 15N exc.)(mean values of all treatments), different letters indicate significant differences at P::; 0.05 .
Fig. 2. Effect of ammonia and nitrate supply on 15N distribution in media and shoots of wheat plants (main effect of N supply), different letters indicate significant differences at P::; 0.05. Further details see Tables 1 and 2.
yield of the root was increased after nitrate addition and inoculation with Azospirillum spp. intensified this effect significantly while shoot growth and N yield of the shoot were significantly decreased in treatments inoculated with Azospirillum spp. and supplied with nitrate.
inoculated strains had. Ammonia addition significantly reduced the 15N enrichment in the plant nutrient media while in shoots 15N enrichment was increased as main effect of all treatments (Fig. 2). That means ammonia addition caused a shift of biologically fixed dinitrogen from the plant growth media (where bacteria were inoculated) into plant shoots without enrichment in roots. Clearest was that effect after inoculation with the bacterial strain Azospirillum spp. (Fig. 3). In contrast to the 15N enrichment in shoots following Azospirillum spp. inoculation and ammonia addition, inoculation of P. agglomerans let to an enrichment of 15N in plant growth media when nitrate was added (Table 2).
Effect of nitrogen and bacteria on plant and media
15N enrichment
in
15N enrichment was detectable in media, root and shoot samples after 26 days incubation in 15N2 enriched atmosphere (Fig. 1). The highest amount of 15N was detected in shoots followed by media samples. The 15N amounts in root samples were near the detection limit. As well as form of applied nitrogen and the bacterial strain inoculated effected significantly the 15N enrichment in plants and media and the distribution of the biologically fixed dinitrogen (Table 2). Surprising was the 15N enrichment in non inoculated vessels. Probably there occured native diazotrophic bacteria within the spermosphere with the same dinitrogen fixing activities as the selected and
Effect of inorganic nitrogen on biologically dinitrogen fixation of P. agglomerans and Azospirillum spp. in association with wheat plants
The form of nitrogen applied to the plant (NH/-N, N0 3--N or without inorganic nitrogen) effected significantly the dinitrogen fixing activity of the inoculated Microbial. Res. 152 (1997) 4
379
15
-I
IS
vessel
7 ~g
5 ~~~------------------~~
~c vessel
-I
4 3
WIthOut
2
o ~~........:
Fig. 3. Effect of bacterial inoculation on I5N distribution in media and shoots of wheat plants after addition of ammonia (100 ppm N), different letters indicate significant differences at P:S; 0.05.
P, Ilggiomerans Azo 'Ptrillum spp_ Fig.4. Effect of nitrogen supply (100 ppm N as ammonia and nitrate or without inorganic nitrogen) on dinitrogen fixation (total I5N content per vessel after 26 days incubation in I5N2 enriched atmosphere) after inoculation with P. agglomerans and Azospirillum spp., different letters indicate significant differences at P:S; 0.05.
Table2. Effect of bacterial inoculation (Pantoea agglomerans, Azospirillum spp.) and nitrogen supply (100 ppm NH/-N or N0 3--N) on I5N enrichment in media, roots and shoots after 26 days incubation in I5N 2 enriched atmosphere (19.76 at % ISNexc.) (means of five replicates). N-fertilization Bacterial inoculation
Media (Ilg I5N vessel-I)
(% of total)
Root (Ilg I5Nexc. vessel-I)
(% of total)
Shoot (Ilg I5Nexc. vessel-I)
(% of total)
Total (Ilg I5Nexc. vessel-I)
(% of total)
without N
2.18 0.82 2.60 1.08 0.66 0.54 1.78 2.86* 1.90 0.80
38.3 21.4 42.3 27.8 14.3 9.5 26.7 44.1 36.8
1.12 0.58 0.78 0.52 0.34 0.50 1.42 0.36 0.72 0.61
19.6 15.1 12.7 13.4 7.4 8.8 21.3 5.6 14.0
2.40 2.44 2.76 2.28 3.62 4.66*a 3.46 3.26 2.54 1.30
42.1 63.5 45.0 58.8 78.3 81.8 52.0 50.3 49.2
5.70 3.84 6.14 3.88 4.62 5.70 6.66 6.48 5.16 2.03
100 100 100 100 100 100 100 100 100
without P. agglomerans
Azospiritlum spp. without P. agglomerans Azospirillum spp. without P. agglomerans Azospirillum spp.
NH4+-N NO 3--N LSDP:S;0.05 a
(*) significant different to control without nitrogen and without bacterial inoculation at P:S; 0.05.
bacteria (Fig. 4). The bacterial strain Pantoea agglomerans fixed increasing amounts of dinitrogen when the plant was supplied with additional inorganic nitrogen. The amount of 15N bound in media and shoot of plants was nearly doubled when plants were supplied with nitrate compared to plants grown without inorganic nitrogen. Addition of inorganic nitrogen did not effect the dinitrogen fixing activity (expressed as 15N enrichment in media and shoots) of Azospirillum spp. (Fig. 4). As well as in bacterial pure culture (Ruppel and Merbach 1995) and in association with wheat plants the strain Azospirillum spp. fixed higher amounts of dinitrogen than the strain P. agglomerans without addition of inorganic nitrogen. Both strains showed the same reaction in their N2-fixing ability in association with the plant as they showed in pure culture after addition of 380
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nitrate. Ammonia addition did not effect the N2-fixing ability of both strains living in association with the plant while in pure culture their activity was drastically reduced (Ruppel and Merbach 1995).
Discussion 15N2fixation in association with wheat plants The detected 15N amounts in media, roots and shoots of wheat plants after 26 days incubation in 15N2 enriched atmosphere demonstrated that dinitrogen fixation at wheat plants occured. The highest 15N amount was detected in shoots of plants supplied with ammonia and inoculated with Azospirillum spp. (Table 2). However,
Table 3 . .Portion of Ndfa (N delived from the atmosphere) at the nitrogen nutrition of wheat plants (calculated for shoot increase during 26 days of incubation in 15N2 enriched atmosphere)
N-fertilization without N NH4+-N N0 3--N
Bacterial inoculation P. agglomerans Azospirillum spp. P. agglomerans Azospirillum spp. P. agglomerans Azospirillum spp.
15Nexc.
N-increase (/lg vessel-I)
(/lg vessel-I)
Ndfa Part of Ndfa at N (/lg vessel-I) nutrition of shoots (%)
2070 1760 4418 4696 3553 1980
2.44 2.76 3.62 4.66 3.26 2.54
12.3 27.6 36.2 46.6 32.6 25.4
the portion of nitrogen derived from the atmosphere (Ndfa) at the nitrogen nutrition of the plant was very small compared to results of rhizobium legume symbiosis where the Ndfa amounted between 13 -96% (Androsoff et al. 1995; Boddey and Urquiaga 1992). Our best treatment reached a maximum of 0.9% Ndfa (Table 3). Probably the young wheat plants (6 weeks old) in our experiment used a big amount of nutrients from the spermosphere and the shoot increase during 26 days of incubation time was too small to calculate the general biological dinitrogen fixing ability of diazotrophic bacteria living in association with plants, while Haahtela etal. (1988) measured between -1.9 and 17.7% of Ndfa at wheat plants grown in greenhouse from June to August .and Malik and Bilal (1988) estimated 12-15% Ndfa in shoots of kallar grass. The latter authors discussed some problems with the methology by using a non fixing control plant due to a biological dinitrogen fixation even in that control. That problem was underlined by our measurements in non inoculated treatments, where a measurable amount of 15N was detected. Inoculation with diazotrophic bacteria induced a significant effect on the distribution of fixed 15N within the plant. While most 15N was detectable within shoots when plants were inoculated with Azospirillum spp., the 15N was nearly equal distributed in media and shoots when P. agglomerans was applied. Effect of ammonia and nitrate on biological dinitrogen fixation The diazotrophic bacterial strains Azospirillum spp. and P. agglomerans were selected for their ability to fix dinitrogen in presence of additional inorganic nitrogen sources (Ruppel and Merbach 1995). Both strains were completely repressed in their N2-fixing activity in pure culture when 100 or 50 ppm N as ammonia were added. Only at low concentrations of 14 mg Nl-l we measured a nitrogenase activity. In association with wheat plants no repression of 15N enrichment was detectable when ammonia was added (Fig. 4). On the contrary 15N enrichment in shoots. was highest after ammonia addi-
0.61 0.91 0.41 0.51 0.50 0.88
tion (Fig. 2). That increased nitrogen accumulation derived from biological dinitrogen fixation in shoots of inoculated wheat plants could be attributed to an increased root carbon exudation which stimulates bacterial growth in the rhizosphere. The close correlation of above ground plant growth and the amount of carbon root exudation (Liljeroth et al. 1994) underlines this assumption because plants supplied with ammonia showed the significantly best growth rate during the 26 days incubation period. Increasing C availability and changes in carbon composition can simultaneously lead to an increased bacterial nitrogenase activity (Hartmann etal. 1988; Moreno- Vivian etal. 1989) or other bacterial enzyme activities as Jiang and Sato (1994) documented with phosphate solubilizing bacteria. Nitrogenase activity of the bacterial strain Pan toea agglomerans was nearly doubled when nitrate was added to the medium in pure culture (Ruppel and Merbach 1995) and even in association with wheat plants (Fig. 4). That finding offers the possibility to select diazotrophic bacterial strains in pure culture which are able to fix dinitrogen in association with plants when additional inorganic nitrogen was supplied. An increased specific nitrogenase activity in bacterial pure culture resulted also in an increased molecular nitrogen fixation in association with plants. However, our results documented, that the specific nitrogenase activity in bacterial pure culture with most effective nutritional and environmental conditions for dinitrogen fixation (0.5% mannit and 0.5% saccharose in semisolid CC medium and without inorganic nitrogen at 29°C) was not reached in association with the plant. The strain Azospirillum spp. for example fixed 0.75 flgN mg protein- 1h- 1in bacterial pure culture. Assuming the initially inoculated bacteria (0.25 mg protein per vessel) survived during experimental time in association with wheat plants, and the bacteria expressed the same nitrogenase activity as in pure culture, 116 flg dinitrogen could be fixed within 26 days. However, our experimental results showed only a N 2 fixation of about 28 flg N fixed per vessel within 26 days (Table 3). These results indicate that more than only the nitrogen conMicrobiol. Res. 152 (1997) 4
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centration in the medium regulate the expression of bacterial nitrogenase activity in association with plants. Varying nitrogen nutrition effects root and shoot growth of plants and consequently root exudation and composition of root exudates (Bowen 1969; Liljeroth et al. 1990 a). Variation in amount and composition of root exudates effects microbial biomass and microbial community composition in the rhizosphere (Liljeroth et al. 1990 b; Trolldenier and v. Rheinbaben 1981). To cause an effective dinitrogen fixing activity a close association between the inoculated bacterial strain and the plant is a prerequisite. Therefore, the bacterial strain must be able to compete with the native microbial population and replace it partly in order not to stress the plant additionally. How plant nutrition and amount and composition of root exudates may effect the bacterial community composition and their activity is still unknown.
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