THE EFFECTS OF SOIL TEMPERATURE ON PLANT GROWTH, NODULATION AND NITROGEN FIXATION IN CASUARINA CUNNINGHAMIANA MIQ.

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1 New Phytol. (1985) 11, 441^5 441 THE EFFECTS OF SOIL TEMPERATURE ON PLANT GROWTH, NODULATION AND NITROGEN FIXATION IN CASUARINA CUNNINGHAMIANA MIQ. BY PAUL REDDELLi'2, Q ^ BOWENi AND A. D. ROBSON^ 1 CSIRO, Division of Soils, Private Bag No. 2, Glen Osmond 564, South Australia and ^Department of Soil Science and Plant Nutrition, University of Western Australia, Nedlands 69, Western Australia {Accepted 3 July 1985) SUMMARY The effects of soil temperatures between 15 and 3 C on plant growth, nodulation and nitrogen fixation in seedlings of Casuarina cunninghamiana Miq. inoculated with Frankia from two different sources were examined. The optimum soil temperature for the growth of plants dependent on symbiotic nitrogen fixation was 25 C. Decreasing the soil temperature below 25 C markedly decreased plant growth that was reliant on symbiotically fixed nitrogen; effects on the growth of plants supplied with mineral nitrogen were much smaller. At 15 C there was no response in plant growth to inoculation after 148 d, whereas plants supplied with nitrogenous fertilizer were 1 times the weight of uninoculated plants. Nodulation was delayed at 15 and 2 C with nodules formed at 15 C fixing no nitrogen in these studies. The production of fewer nodules at 2 C than at 25 C was partly compensated by the production of larger nodules. Nodule growth at 2 to 3 C was a prime determinant of nitrogen fixed, with the exception of one Frankia at 2 C. The amount of nitrogen-fixed g"^ nodule was the same for the two Frankia sources at 25 and 3 C, differences in effectiveness being due to nodule development. However, differences in the effectiveness of the two Frankia sources at 2 C were related to differences both in nodule development and in nitrogen-fixing ability. The absence of nitrogen fixation at 15 C would be expected to limit the natural distribution of Casuarina species reliant on symbiotically fixed nitrogen to areas where soil temperatures exceed 15 C for a major part of the potential growing season. Keywords: Casuarina cunninghamiana, Frankia, nodulation, nitrogen fixation, soil temperature. INTRODUCTION The effect of soil temperature on nodulation and nitrogen fixation by actinorhizal plants has received little study. Soil temperature could affect the growth of the host plant and each of the other major stages of symbiotic Ng fixation: growth oi Frankia in the rhizosphere, infection and nodule development, the nitrogen fixation processes, and subsequent nitrogen transfer to the plant. Studies with pure cultures of Frankia on laboratory media (Burggraaf & Shipton, 1982; Shipton & Burggraaf, 1983; Moiroud, Faure-Raynaud & Simonet, 1984) have found little growth below 2 C, with optimum growth at ^around 3 C. If growth of Frankia is also poor in the rhizosphere below 2 C, this could delay infection and nodule development, a phenomenon that has been observed on Ceanothus velutinus (Wollum & Youngberg, 1969). In studies using natural, uninoculated soil, Wollum & Youngberg (1969) found nodules first X/85/ $3./ ^^^^ The New Phytologist

2 442 P. R E D D E L L et al. appeared after 43 d at a soil temperature of 22 C, but nodulation did not occur until 7 d at a soil temperature of 15 C. Of course, factors other than the growth of Frankia in the rhizosphere may also have been operating. The effect of temperature on nitrogen fixation by detached nodules of actinorhizal plants has been studied. The information, albeit fragmentary, tends to show differences in response to temperature between plant genera which occur predominantly in temperate climates (for example Alnus, Myrica, Hippophde) and species of Casuarina which occur mostly in warm temperate to tropical climates. Waughman (1977) found nodules of Myrica gale, Alnus glutinosa and Hippophde rhamnoides to have optimum acetylene reduction activities at 2 to 25 C, whereas the optimum for Casuarina equisetifolia nodules was 35 C. For C. equisetifolia, acetylene reduction activities at 2 and 3 C respectively were 2-5 and 6-8 times that at 15 C. However, as Wheeler & McLaughlin (1979) have pointed out, 'it is not clear what relevance data from short term studies with detached nodules has to the behaviour of plants under natural conditions'. None of the studies outlined above examines the possible effect of soil temperatures on nodulation and nitrogen fixation by different Frankia strains on the one host, nor the effects of soil temperature on growth of the host plant, either when supplied with combined nitrogen or depending solely on symbiotic nitrogen fixation. This study addresses these factors using Casuarina cunninghamiana Miq. (with a natural distribution from 37 S to 12 S latitude) and two isolates of Frankia, one from a subtropical soil and one from a temperate soil. M A T E R I A L S AND M E T H O D S Experimental design Experiment 1. This experiment distinguished the time-course effects of soil temperature on nodulation from those on nitrogen fixation and plant growth. The design was a complete factorial of the following treatments: (i) Soil temperatures 2 and 25 C. (ii) Nitrogen treatments - nil (uninoculated), nitrogen application (NH4NO3 at 15mgNkg""^ soil) and inoculation with Frankia from a temperate soil {cb, collected originally from C. cunninghamiana at the Murrumbidgee River in the Australian Capital Territory; latitude 35 16' S). Harvests of four replicates of each treatment combination were taken 15, 31, 45, 58, 77, 99 and 148 days after planting. Experiment 2. A second experiment, established at the same time as Experiment 1, examined differences in nodulation and nitrogen fixation by two Frankia sources on C. cunninghamiana over a range of soil temperatures. Four soil temperatures (15, 2, 25 and 3 C) and four nitrogen treatments (the three used in Experiment 1 and an additional inoculation treatment - Frankia tn, collected from C. cunninghamiana near Townsville in Queensland; latitude 19 16' S) were used in this experiment. The four replicates of each treatment combination were harvested 148 days after planting. Experimental procedures Plant growth. Seeds of C. cunninghamiana (collected from Uriarra Crossing in the Australian Capital Territory; CSIRO Division of Forest Research Seedlot number 13149) were sown into trays (35x3x5 cm deep) containing steam-

3 Nodulation and N2-fixation in Casuarina 443 sterilized, washed, coarse river sand. These trays were placed in an air-conditioned glasshouse and watered regularly. Germination occurred within 18 d. Six weeks after sowing, seedlings were removed carefully from the trays, inoculated, and transplanted (one per pot) into undrained white polypropylene pots containing 1 kg of the steam-sterilized coarse river sand. Pots were then placed in temperaturecontrolled water tanks to maintain soil temperatures at 15, 2, 25 and 3 C ( + 1 C). The ambient glasshouse temperature ranged from 18 to 35 C. All seedlings were supplied with a nitrogen-free nutrient solution of the composition: KH2PO4 5 g \-\ CaSO4.2H2O 5 g \-\ MgSO^.yH^O 5 g l-\ KCl 1 g l-\ Fe EDTA 2 /ig l~^ and micronutrients 1 ml \~^ (Reddell & Bowen, 1985). Initially, this nitrogen-free nutrient solution was applied weekly at a rate of 25 ml per pot, but it was increased to 5 ml per pot after 35 d. All pots were watered to weight regularly. For the mineral nitrogen treatment, six applications of ammonium nitrate (NH^NOg) were made: 7, 14, 35, 63, 9 and 15 d after transplanting, totalling 15mgNkg~^ soil. The first two applications were at a rate of 15 mg N kg~^ soil and the last four were at 3 mg N kg~^ soil. Inoculation. Nodules to be used as inocula in this study were produced in the glasshouse by inoculating C. cunninghamiana seedlings with single nodules collected from the two field locations. Crushed nodule inocula of Frankia were then prepared from the glasshouse-grown nodules (Reddell & Bowen, 1985). Nodule material (13 mg of tn, 115 mg of cb) was surface-sterilized in 1 to 2 % calcium hypochlorite for 2 min, rinsed three times in sterile distilled water and then ground to a paste in a mortar and pestle with 1 ml of sterile distilled water. A suspension of this nodule material was made by adding 1 ml of 1 % sucrose in sterile distilled water to the paste. The suspension was then transferred to a 2 ml beaker and the roots of approximately 8 seedlings immersed in the suspension for 4 to 45 min. These seedlings were then transplanted into the pots and 1 ml of the crushed nodule suspension pipetted on to each pot. Harvest. At each harvest shoots, roots and nodules were separted. Dry weights were determined and samples analyzed for nitrogen using a micro-kjeldahl procedure (McLeod, 1982). RESULTS Experiment 1. Effects of temperature over time Plant growth and nitrogen content. Total plant dry weight for sequential harvests of the various treatments are presented in Figure 1. As the data are plotted on a logarithmic scale, the slopes of the curves give relative growth rates. The effect of temperature on the growth of seedlings varied with the source of nitrogen (Fig. 1). For inoculated plants [Fig. l(a)], growth at 25 C was greater than at 2 C from the first harvest to the final harvest. At both temperatures inoculated plants had an initial rapid phase of growth which slowed considerably until 77 d, following which a rapid increase in relative growth rate occurred. For plants supplied with combined nitrogen [Fig. l(c)], growth at 25 was greater than at 2 C for the first 77 d. During this period there was rapid growth at both soil temperatures. However, after 77 d, relative growth rates declined at both soil temperatures (but more so at 25 C), and by the final harvest plants grown at 2 C had greater dry weight than plants at 25 C.

4 444 P. REDDELL et al. jr plan ft 1 $ -! 3nt dry a. -1 Whole / \ Time since planting (d ) I \ Fig. 1. Growth curves (dry weight basis) of Casuarina cunninghamiana seedlings with three nitrogen treatments at 2 and 25 C soil temperatures (Experiment 1). Bars indicate standard errors ofthe means, n, time for nodulation. # #, 25 C; # #, 2 C. (a) Frankia cb; (b) nil; (c) applied nitrogen. 1 (X c 8 o> o o.o 31 / \ / \ Time since planting (d) 31 I \ Fig. 2. Effect of 2 and 25 C soil temperature on total nitrogen content of Casuarina cunninghamiana seedlings with three nitrogen treatments (Experiment 1). Bars indicate standard errors of the means; *, no standard error, n, time for nodulation. # #, 25 C; # #, 2 C. (a) Frankia cb\ (b) nil; (c) applied nitrogen. 148 For uninoculated seedlings to which no nitrogen was applied [Fig. l(b)], growth at a soil temperature of 25 C was greater than that at 2 C until 77 d after planting. From this time, growth of plants at 25 C ceased, whereas growth at 2 C continued to day 99. Between 99 and 148 d after planting, dry weights of seedlings at 2 and 25 C were similar. Figure 2 presents the nitrogen contents ofthe whole plant for successive harvests (also on a logarithmic scale). The effects of temperature on nitrogen contents were essentially similar to those for plant growth, indicating that soil temperature had little effect on nitrogen concentrations in plants. It is notable that in the inoculated pots at 25 C, the curves for nitrogen content per plant diverged from the uninoculated at 58 days (i.e. 27 d after nodulation) and were significantly different at 77 to 148 d. At 2 C, a rapid increase in nitrogen content per plant did not occur until after 77 d, and this was significantly different from uninoculated controls at the 99-and 148-d harvests. The total nitrogen contents of inoculated pots at 25

5 Nodulation and N^^-fixation in Casuarina Time since planting (d) Fig. 3. Effect of soil temperature on numbers of nodules on Casuarina cunninghamiana seedlings inoculated with Frankia cb (Experiment 1). Bars indicate standard errors of the mean. # #, 25 C;, 2 C. 2. a. I m -1 - S " Time since planting (d) Fig. 4. Effect of soil temperature on nodule dry weight on Casuarina cunninghamiana seedlmgs inoculated with Frankia cb (Experiment 1). Bars indicate standard ^errors of the means; *, no standard error. # #, 25 C; 1, 2 C. and 2 C were converging rapidly at 148 d, but this may have been an artefact of the pot cultures. Nitrogen content and nodule dry weight of inoculated plants were closely related over all temperatures and times. Nodulation. Nodule formation was delayed at the lower temperature. At 25 C nodules were first observed 31 d after planting, whereas at 2 C nodules were first observed 45 d after planting (Fig. 3). At all times, the numbers of nodules were significantly greater at 25 C (Fig. 3); for example, from day 77 to day 148 there were 2 5 to 4 times as many nodules at 25 C. Total nodule dry weight increased exponentially with time (Fig. 4) and, although total nodule weight was always greater at 25 C, the effect of temperature on nodule weight per plant decreased with time (Fig. 4). Thus, while numbers of nodules were always greater at 25 C, nodules at 2 C compensated to some extent by growing larger.

6 446 P. REDDELL et al. Table 1. Effect of soil temperature on shoot, root and nodule dry weights of Casuarina cunninghamiana seedlings grown with four nitrogen treatments for 148 d{±seof the mean) {Experiment 2) Temperature Soil temperature Shoot dry weight (g Root dry weight Nodule dry weight (mg plant"*) Nil NH4NO3 Frankia cb Frankia tn ^ B 3.IS 2 a ± ± ±-24-4 ±-1 l-57± ±-18-8 ±-13-3 ±-1-34 ± ± ±-2-7 ±-1-8 ±-1-4 ± l-ll±-16-7 ±-7-76 ± ±-8-78 ±-4-21 ±-5-3 ±-1-13 ±-4-42 ± ±1 181± ±8 l±-4 34± Nil Applied nitrogen Frankia cb Frankia tn Soil temperature ( C) Fig. 5. Effects of four soil temperatures on nitrogen contents of Casuarina cunninghamiana seedlings grown with two Frankia sources, with applied nitrogen and with no nitrogen source (Experiment 1). Bars indicate standard errors ofthe mean. Experiment 2. Effect of Frankia source on temperature response Plant growth and nitrogen content. Dry weight (Table 1) and nitrogen content (Fig. 5) of inoculated plants with both Frankia sources increased with increasing soil temperature to 25 C and then declined significantly at 3 C. At 2 and 25 C, plants inoculated with cb produced more dry weight and had greater nitrogen contents than plants inoculated with tn, but at 3 C these apparent differences

7 Nodulation and N^-fixation in Casuarina 447 Table 2. Effect of soil temperature on the nitrogen concentration (%) m shoots, roots and nodules of Casuarina cunninghamiana seedlings grown with four nitrogen treatments for 148 d {Experiment 2) Nitrogen treatment Soil temperature ( C) Nil NH4NO3 Frankia cb Shoots Roots Shoots Roots Shoots Frankia tn Roots Nodules Shoots Roots Nodules SE = -2. Table 3. Effect of soil temperature on nitrogen fixation* by nodules formed by Casuarina cunninghamiana with two different Frankia inocula { + SE of the mean) (Experiment 2) Soil temperature ( C) Frankia source cb tn -35 ±-1 Oil ± ± ±-5 * Nitrogen fixation expressed as mg N fixed per mg nodule dry weight; calculated by subtracting nitrogen content of uninoculated plants from that of inoculated plants and dividing by dry weight of nodules at 148 d. between Frankia sources in effectiveness were eliminated. Frankia source had no effect on growth or nitrogen content of inoculated pots at 15 C. In contrast to inoculated plants, nitrogen-fertilized plants produced optimum growth at 2 C, growth at 15, 25 and 3 C being only some 6% of that at 2 C (Table 1). However, these plants at 2 C had lower shoot nitrogen concentrations (Table 2) and consequently there was no increase in total nitrogen content (Fig. 5) associated with this growth response. This 2 C optimum was not evident in the time-course study until the final harvest at 148 d (Fig. 1), and perhaps was an artefact indicating that the N-fertilized plants were becoming 'pot bound'. Uninoculated, unfertilized plants grew poorly and had low nitrogen contents at all soil temperatures (Table 1, Fig. 5). Nitrogen concentrations in shoots, roots and nodules. Low temperatures reduced nitrogen concentrations both in shoots and nodules of inoculated plants but had no effect on nitrogen concentrations in roots (Table 2). Source of inoculum influenced this response: shoot nitrogen concentrations were lower only at 15 C for plants inoculated with cb; whereas with tn shoot nitrogen concentrations were lower at both 15 and 2 C than at 25 and 3 C. Nitrogen concentrations in shoots and roots of uninoculated and nitrogen-fertilized plants were generally unaffected by soil temperature. The exception to this was the lower shoot nitrogen concentration found in the nitrogen-fertilized plants grown at 2 C. ANP 11

8 448 p. R E D D E L L et al Nodulation and nitrogen fixation. All plants inoculated with Frankia formed nodules at all four temperatures. Irrespective of the Frankia source, increasing soil temperature to 25 C increased dry weight of nodules, but further increasing temperature to 3 C reduced nodule dry weight (Table 1). This pattern of temperature response, showing a sharp increase between 15 and 2 C for cb but a much lesser one for tn, was identical to that observed for plant dry weight (Table 1) and nitrogen content (Fig. 5). It is also notable that the proportion of total plant dry weight in nodules increased only between 15 and 2 C and was unaffected by further increasing soil temperature (see Table 1). This corresponds with the trend of increasing nitrogen concentration in nodules only between 15 and 2 C (Table 2). No significant nitrogen fixation occurred with either Frankia source at 15 C (Table 3). At 2 C nitrogen fixed g~^ nodule was dependent on Frankia source. With cb, there was no difference in nitrogen fixed g~^ nodule at 2, 25 and 3 C (Table 3), hence differences in total nitrogen fixed were dominated by total nodule development. However, with tn the nitrogen fixed g~^ nodule at 2 C was only one-third that at 25 and 3 C (Table 3). At these two temperatures (25 and 3 C) nitrogen fixed g~^ nodule was similar with cb and tn. DISCUSSION Over a range of soil temperatures from 15 to 3 C, growth of C. cunninghamiana seedlings dependent on symbiotically fixed nitrogen was more sensitive to high (3 C) and low (15 C) temperatures than was growth of seedlings supplied with combined nitrogen. At 15 C soil temperature, nodulation and nitrogen fixation were more sensitive to temperature than was the host-plant growth component of the symbiosis. However, at 3 C soil temperature, effects on host-plant growth probably limited nodule development. Differences occurred between the Frankia from two sources in the nitrogen fixed at 2 and 25 C. These differences were related to nodule development at both temperatures and to the amount of nitrogen fixed g~^ nodule at 2 C. With added nitrogen, plant growth was reasonable at 15 C soil temperature, and nitrogen uptake was similar to that at the other temperatures. However, inoculated plants grew poorly at 15 C and, although some nodules were formed with both Frankia strains, no nitrogen fixation resulted. This sensitivity of inoculated plants could be due to effects on Frankia growth in the rhizosphere, on infection processes, nodule development and/or nitrogen-fixing phases of the symbiosis. Poor growth and nitrogen fixation by Frankia on laboratory media at 15 C (Burggraaf & Shipton, 1982; Shipton & Burggraaf, 1983; Moiroud et al, 1984; Tjepkema, Ormierod & Torrey, 1981) suggest that poor growth of Frankia in the rhizosphere may occur at this temperature. However, there is not enough evidence in our study to distinguish between effects on growth in the rhizosphere and effects on the infection process per se. Separation of effects of two soil temperatures (2 and 25 C) on nodulation and nitrogen fixation showed that less plant growth and nodule dry weight at 2 C were the result of (i) reduced early growth (Fig. 1) and nitrogen content of nitrogen-limited plants (both uninoculated and inoculated; Fig. 2) at 2 C, (ii) delayed nodule formation at 2 C and (iii) formation of fewer nodules at 2 C (Fig. 4). The different growth pattern of inoculated plants from those with nitrogen applied (Fig. 1) illustrates the need for caution in interpreting results of studies

9 Nodulation and N2-fixation in Casuarina 449 in which only nitrogen-fertilized (i.e. nitrogen-adequate) plants are used as controls. It also poses the question of what are useful controls for physiological studies of actinorhizal symbioses. This problem has previously been discussed for VA mycorrhizal symbioses (Abbott & Robson, 1984). The second effect of temperature, a delay in time for nodulation to occur, has been reported for another actinorhizal species, Ceanothus velutinus (Wollum & Youngberg, 1969). This delayed nodule formation was probably related to decreased pre-infection Frankia growth at the lower temperature. In our studies the formation of fewer nodules at 2 C was offset by a 4 % increase in mean weight per nodule (nodule size). This illustrates that, as with legume nodule systems, partial compensating factors may operate when the infection stages of the symbiosis are limited by an environmental variable (discussed by Bowen, 1978). The influence of soil temperature on nodule function is indicated in this study by nitrogen fixed g~^ of nodule for Frankia cb and tn (Table 3). These values are only indicative of cumulative nitrogen-fixing activity as they are derived from the final weight of nodules and hence do not account for differences in time for nodulation and rates of nodule growth. However, the following interpretation can be made from our data. Differences in effectiveness of nitrogen fixation between cb and tn at 2 and 25 C were due to (i) greater nodule development by cb at both temperatures and (ii) reduced nitrogen fixed g~^ nodule by tn at 2 C. Both these factors have been previously shown to be important in determining the relative effectiveness of Casuarina species-frankia source combinations (Reddell & Bowen, 1985). It is tempting to correlate the poor fixation g~^ nodule by tn at 2 C with its subtropical origin, and the high rates of fixation by cb at 2 C with its cool-temperate origin, but many more Frankia need to be examined before such a generalization could be made. The similarity of N fixed g~^ nodule tissue at 2, 25 and 3 C for cb and at 25 and 3 C for tn (Table 3) suggests that the major effect of temperature in this range is on nodule development (or more particularly the amount of Frankia development in the nodule), as governed by plant-environment interactions. (For tn at 2 C it is possible that the Frankia biomass in the nodule was reduced relative to nodule weight, but this was not investigated.) Bond & Mackintosh (1975) demonstrated that nitrogen fixation by detached nodules of C. cunninghamiana increased with temperature up to 4 C, but they indicated that factors such as supply of photosynthate may modify this response. Considering their data and that of Waughman (1977) on effects of temperature on acetylene reduction by detached nodules, our studies suggest plant factors (e.g. photosynthate supply) override temperature effects on nitrogenase activity above 2 C. It is hence probable that reduced effectiveness and lower nodule production by both Frankta strains at 3 C are the result of some plant factor (e.g. assimilate availability in roots) which limits nodule development. 1 t^u There are important applied and ecological implications of this study. First the sensitivity of the symbiotic nitrogen-fixing system to soil temperatures below 2 C may well define the southern limits to the distribution of this and other Casuarina species (C. glauca, C. obesa) in Australia, as these species are dependent on nitrogen fixation for survival and growth under natural conditions (P. Reddell, G. U. Bowen & A. D. Robson, unpublished data). Secondly, in any screening programme to select highly effective Casuarina species-frankia strain combinations for use in plantations, it will be necessary to perform these at soil temperatures relevant to the area which is to be planted. 16-2

10 45O P. REDDELL et al. ACKNOWLEDGEMENTS We thank staff of the Routine Analytical Services Laboratory, CSIRO Division of Soils, for nitrogen analyses of plant material. This research was supported by the Rural Credits Development Fund of the Reserve Bank of Australia. REFERENCES ABBOTT, L. K. & ROBSON, A. D. (1984). The effect of vesicular-arbuscular mycorrhizas on plant growth. In: VA Mycorrhizas (Ed. by C. L. Powell & J. Bagyaraj), pp CRC Uniscience Series, CRC Press, Florida. BOND, G. & MACKINTOSH, A. H. (1975). Diurnal changes in nitrogen fixation in the root nodules of Casuarina. Proceedings of the Royal Society of London, Series B, 192, BowEN, G. D. (1978). Dysfunction and shortfalls in symbiotic responses. In: Plant Disease - An Advanced Treatise (Ed. by J. G. Horsfall & E. B. Cowling), vol. 3, pp Academic Press, New York. BuRGGRAAF, A. J. P. & SHIPTON, W. A. (1982). Estimates of Frankia growth under various ph and temperature regimes. Plant and Soil, 69, MCLEOD, S. (1982). Routine analytical methods. Notes on Soil Techniques, 4, MoiROUD, A., FAURE-RAYAUD, M. & SiMONET, P. (1984). Influence de basses temperatures sur la croissance et la survie de souches pure de Frankia isolees de nodules d'aulnes. Plant and Soil, 78, REDDELL, P. & BOWEN, G. D. (1985). Frankia source affects growth, nodulation and nitrogen fixation by Casuarina species. New Phytologist, 1, SHIPTON, W. A. & BURGGRAAF, A. J. P. (1983). Aspects of the cultural behaviour of Frankia and possible ecological implications. Canadian Journal of Botany, 61, TjEPKEMA, J. D., ORMEROD, W. & ToRREY, J. G. (1981). Factors affecting vesicle formation and acetylene reduction (nitrogenase activity) in Frankia sp. Cpll. Canadian Journal of Microbiology, 27, WAUGHMAN, G. J. (1977). Theeffectof temperature on nitrogenase activity. J'oMrna/o/ '*^mmen<a/fiofany, 28, WHEELER, C. T. & MCLAUGHLIN, M. E. (1979). Environmental modulation of nitrogen fixation in actinomycete nodulated plants. In: Symbiotic Nitrogen Fixation in the Management of Temperate Forests (Ed. by J. C. Gordon, C. T. Wheeler & D. A. Perry), pp. \14~141. Forest Research Laboratory, Oregon State University, Corvallis. WoLLUM, A. G. &YOUNGBERG, C. T. (1969). Effects of soil temperature on nodulation of CeanofAus i)e/mfmt«dougl. Proceedings of the Soil Science Society of America, 33,

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