Annals of Botany 79: 3 9, 1997 Plant Growth-promoting Rhizobacteria and Soybean [Glycine max (L.) Merr.] Growth and Physiology at Suboptimal Root Zone Temperatures FENG ZHANG*, NARJES DASHTI*, R. K. HYNES and DONALD L. SMITH* * Department of Plant Science, Macdonald Campus of McGill Uni ersity, 1,111 Lakeshore Road, Ste. Anne de Belle ue, PQ, H9X 3V9 and Agrium Inc., 15 Inno ation Boule ard, Saskatoon, SK, S7N X8, Canada Received: 9 March 199 Accepted: 8 August 199 Application of plant growth-promoting rhizobacteria (PGPR) has been shown to increase legume growth and development under optimal temperature conditions, and specifically to increase nodulation and nitrogen fixation of soybean [Glycine max (L.) Merr.] over a range of root zone temperatures (RZTs). Nine rhizobacteria applied into soybean rooting media were tested for their ability to reduce the negative effects of low RZT on soybean growth and development by improving the physiological status of the plant. Three RZTs were tested: 5, 17 5, and 15 C. At each temperature some PGPR strains increased plant growth and development, but the stimulatory strains varied with temperature. The strains that were most stimulatory at each temperatures were as follows: 15 C Serratia proteamaculans 1 1; 17 5 C Aeromonas hydrophila P73, and 5 C Serratia liquefaciens 8. Because enhancement of plant physiological activities were detected before the onset of nitrogen fixation, these stimulatory effects can be attributed to direct stimulation of the plant by the PGPR rather than stimulation of plant growth via improvement of the nitrogen fixation symbiosis. 1997 Annals of Botany Company Key words: Legume, nitrogen fixation, nodulation, root zone temperature, PGPR. INTRODUCTION Co-inoculation studies with plant growth promoting rhizobacteria (PGPR) and (Brady)rhizobium have shown increased plant nodulation and nitrogen fixation under normal growth conditions (Verma et al., 198; Li and Alexander, 1988). Recently, Zhang et al. (199) investigated the effects of root zone temperature (RZT) on the tripartite association of nitrogen-fixing bacteria, PGPR and soybean plants. These studies showed that the co-inoculation of B. japonicum with some PGPR strains increased soybean nodulation and nitrogen fixation at both optimal and suboptimal RZTs. For example, nodule number and dry weight of plants receiving Serratia proteamaculans 1 1 were increased by 33 and 5%, respectively at 15 C RZT, while for plants receiving Aeromonas hydrophila P73 they were increased by 7 and 31% at 17 5 C RZT. Nodule number and weight of plants receiving Serratia liquefaciens 8 at 5 C RZT were and % higher than plants not inoculated with this PGPR. To elucidate whether this occurs through direct effects on nodulation or through improved plant growth and plant physiological activities, we tested whether PGPR can increase soybean plant growth, and physiological activities such as photosynthesis and transpiration, which may lead to improved soybean growth at a range of RZTs. MATERIALS AND METHODS Two experiments were conducted; both were arranged in completely randomized split-plot designs with three replications. The main-plot units consisted of three RZTs, 5, 17 5, and 15 C (Zhang, Lynch and Smith, 1995). Various PGPR strains (Zhang et al., 199) formed the sub-plot units. There were a total of six and eight sub-plot units within each mainplot of expts 1 and, respectively (Zhang et al., 199). Although three of the strains were common to both experiments, all strains in expt were rifampicin resistant as a result of selection in the presence of this antibiotic. A control (no PGPR inoculation) was included in each experiment. Otherwise, experimental conditions, plant material management, production of PGPR inocula, and statistical analyses were the same as reported by Zhang et al. (199). Plants from both experiments were harvested 5 d after inoculation (DAI) and leaf area (Delta-T area meter, Delta- T Devices Ltd., Cambridge, UK), leaf number, plant shoot weight, and root weight were recorded. Leaf photosynthetic rate, plant transpiration and stomatal conductance (LI- portable photosynthesis system, LI-COR Inc., Lincoln, NE, USA) were measured four times during the course of both expts 1 and. The first measurement was taken at 17 DAI, with subsequent measurements taken every 1 days. For correspondence. 35-73 97 33 7 $5. bo933 1997 Annals of Botany Company
Zhang et al. PGPR and Soybean Growth and Physiology All measurements were made between 1 and 17 h. Chlorophyll fluorescence was investigated at the same time measuring Fv Fm [photochemical efficiency of photosystem II (CF-1 chlorophyll fluorescence, Morgan Scientific Inc., Andover, MA, USA)]. RESULTS Plant leaf de elopment and dry weight The effects of the nine PGPR strains on leaf and plant dry weight varied with RZT. At optimal RZT (5 C), S. liquefaciens 8 increased plant total dry weight in both experiments and leaf number and area in the second experiment, when compared with non-pgpr-inoculated controls (Table 1). Pseudomonas fluorescens 31 1, P. putida G11 3, and S. proteamaculans 1 1 (in expt 1) decreased plant total dry weight at 5 C RZT. At 17 5 C RZT A. hydrophila P73 increased total plant dry weight and pod number in the first experiment (Table ), while at the lowest (15 C) RZT, S. proteamaculans 1 1 increased plant leaf area and total plant dry weight in both experiments and leaf number, as compared with the non-pgpr-inoculated control, in expt 1 (Table 3). Photosynthesis, transpiration and stomatal conductance Plant photosynthetic rates averaged across the four measurements taken at 17 7 DAI were affected by PGPR strains in a way that varied with RZT. At optimal RZT (5 C) the photosynthetic rates of plants receiving S. liquefaciens 8 increased and 5 % over the non- PGPR-inoculated plants in expts 1 and, respectively (Fig. 1). In expt, P. putida 31 3 and P. putida 1 1 also increased plant photosynthetic rates at 5 C RZT compared to non-pgpr-inoculated plants. All strains that increased plant photosynthesis at optimal growth RZT also caused TABLE 1. Effect of plant growth promoting rhizobacteria (PGPR) on soybean growth at root zone temperature of 5 C Plant leaf Plant dry weight (mg) Pod number* PGPR strains Number Area (cm ) Root Shoot Total (per plant) Expt 1 1 1 8 918 97 5 51 8 5 8 5 95 19 73 7913 13 5 G11 3 9 3 97 89 11 511 9 8 P73 55 5 95 973 5159 3 9 3 31 1 51 5 97 9 113 5188 8 5 Non-PGPR 9 5 979 11 555 751 1 5 9 8 7 78 5 18 9 175 1 8 Expt 1 1 1 17 88 1 8 7 1151 17 5311 735 G11 3 5 1 1193 99 3 31 3 5 13 13 58 91 3 3 7 987 13 55 897 3 9 9 3 113 13 51 998 1 1 3 1 1 515 8 Non-PGPR 53 91 89 38 195 1 11 18 9 8 757 7 891 5 Means within the same column and experiment were analysed by an ANOVA protected test. * Pod number was only measured in the first experiment. numerical increases in plant transpiration and stomatal conductance, however, these increases did not reach the P 5 level of significance. At 17 5 C RZT, P. putida G11 3 (expt ), and S. liquefaciens 8 (expts 1 and ) stimulated plant photosynthesis. S. liquefaciens 8 increased photosynthetic rates by 3 8 and 5 9% in expts 1 and, respectively (Fig. ). Plant photosynthetic rate was increased 9 5% by inoculating with S. proteamaculans 1 1 in the first experiment, while a 3 % increase resulted from inoculation with P. putida G11 3 in the second experiment. In expt 1, plant photosynthetic rate was also increased by P. fluorescens 31 1 and A. hydrophila P73, whereas it was inhibited by P. putida 31 3 in expt (Fig. ). Leaf transpiration and stomatal conductance were only affected by P. putida G11 3 which increased leaf transpiration when compared to non-pgpr-inoculated plants in the second experiment. At the lowest RZT (15 C), S. proteamaculans 1 1 increased leaf photosynthetic rate in both experiments, while P. putida 31 3 and P. fluorescens 3 9 increased this variable in the second expt (Fig. 3). Plant photosynthetic rates were increased 37 and 3 5% by inoculating with S. proteamaculans 1 1 compared to the non-pgprinoculated controls in expts 1 and, respectively. Although photosynthetic rates of plants receiving S. liquefaciens 8 did not increase compared to those of the non-pgprinoculated plants, plant transpiration and stomatal conductance were increased by inoculating with S. liquefaciens 8 in the first experiment (Fig. 3). The changes in plant photosynthetic rates across the four measurement times were calculated for the most stimulatory PGPR for each RZT. At 15 C RZT photosynthetic rates of plants receiving S. proteamaculans 1 1 were higher than those of non-pgpr-inoculated plants for the last two measurements in expt 1 and the last three measurements in
Zhang et al. PGPR and Soybean Growth and Physiology 5 TABLE. Effects of plant growth promoting rhizobacteria (PGPR) on soybean growth at root zone temperature of 17 5 C Plant leaf Plant dry weight (mg) Pod number* PGPR strains Number Area (cm ) Root Shoot Total (per plant) Expt 1 1 1 9 35 317 151 19 3 8 3 7 353 391 188 13 8 G11 3 9 3 357 3 153 3 7 P73 9 355 389 31 785 3 8 31 1 9 5 357 33 198 31 3 Non-PGPR 8 7 33 335 159 197 1 7 1 1 8 3 37 39 1 1 Expt 1 1 8 371 5 177 8 8 1 5 33 8 177 53 G11 3 8 3 35 38 159 1951 31 3 8 5 378 31 1355 1775 3 3 7 3 371 7 11 17 3 9 1 5 58 3 181 17 1 1 31 3 387 19 31 Non-PGPR 3 3 393 3 1553 1 5 8 93 1 7 78 7 Means within the same column and experiment were analysed by an ANOVA protected test. * Pod number was only measured in the first experiment. TABLE 3. The effect of plant growth promoting rhizobacteria (PGPR) on soybean growth at root zone temperature of 15 C Plant leaf Plant dry weight (mg) Pod number* PGPR strains Number Area (cm ) Root Shoot Total (per plant) Expt 1 1 1 17 3 8 391 553 98 1 8 8 1 37 338 81 1 G11 3 1 39 353 98 1 P73 11 5 373 9 83 1 31 1 1 39 31 1 Non-PGPR 1 5 315 373 78 1 1 9 5 5 1 13 7 183 3 Expt 1 1 1 3 9 379 111 8 7 3 8 59 G11 3 9 7 315 37 717 31 3 1 8 313 55 9 3 3 9 8 311 397 777 3 9 11 37 7 9 581 1 1 9 7 51 75 3 9 Non-PGPR 1 7 3 37 381 751 3 17 8 158 7 Means within the same column and experiment were analysed by an ANOVA protected test. * Pod number was only measured in the first experiment. expt (Figs and 5), whereas other measurements were not different. Photosynthetic rates of plants receiving A. hydrophila P73 in expt 1 and P. putida G11 3 in expt at 17 5 C RZT were increased at the first three measurements when compared to non-pgpr-inoculated plants (Figs and 5). The plants receiving S. liquefaciens 8 at 5 C RZT showed the same pattern of response as those maintained at 17 5 C RZT in either expt 1 or. The photochemical efficiency of photosystem II was monitored through fluorescence measurements made at the same time as photosynthesis measurements; however, there was no effect of PGPR applications on this variable (data not shown). DISCUSSION Inoculation of soybean plants with PGPR strains produced a wide range of effects which varied among strains of PGPR and over RZTs. Some PGPR strains stimulated plant growth, development, and some plant physiological events, and a few strains inhibited these processes. At the optimal
Zhang et al. PGPR and Soybean Growth and Physiology Photosynthetic rate (µmol m s 1 ) Photosynthetic rate (µmol m s 1 ) Transpiration (mmol m s 1 ) 3 1 Transpiration (mmol m s 1 ) 3 1 Stomatal conductance (cm s )..3..1 Stomatal conductance (cm s )..3..1 G11-3 -8 1-1 31-1 P73 Experiment 1 G11-3 -8 1-1 31-3 3-3 3-9 1-1 Experiment FIG. 1. Effects of plant growth promoting rhizobacteria on plant photosynthesis, transpiration, and stomatal conductance for plants grown at 5 C root zone temperature. Vertical lines on top of each bar indicate one standard error unit (n ). G11-3 -8 1-1 31-1 P73 Experiment 1 G11-3 -8 1-1 31-3 3-3 3-9 1-1 Experiment FIG.. Effects of plant growth promoting rhizobacteria on plant photosynthesis, transpiration, and stomatal conductance for plants grown at 17 5 C root zone temperature. Vertical lines on top of each bar indicate one standard error unit (n ). RZT (5 C), S. liquefaciens 8 consistently increased plant leaf development and dry matter accumulation, whereas P. fluorescens 31 1 and P. putida G11 3 decreased these variables (Table 1). At 17 5 C RZT, A. hydrophila P73 and P. putida G11 3 had a positive effect on plant growth and development (Table ), while at 15 C RZT S. proteamaculans 1 1 significantly increased plant dry matter accumulation (Table 3). Some PGPR strains stimulated plant growth at one RZT, but inhibited it at another. For example, S. proteamaculans 1 1 increased all plant growth variables tested in this study at 15 C, but decreased plant root and shoot dry matter at optimal RZT (5 C). These results indicate that the physiological effects of PGPR on soybean plants are affected by soil temperature, and agree with our concurrent findings, which indicate that the effects of a mixed inoculum of B. japonicum and PGPR on soybean nodulation and nitrogen fixation varied with RZT (Zhang et al., 199). The most effective strains, within each temperature, for stimulating both the nitrogen fixation symbiosis and plant growth were consistent. Current soybean production in eastern Canada is at the northernmost North American limit of the crop. Soybean production practices involve seeding in May and harvesting in September. Measurements taken at the Macdonald Campus of McGill University meteorology facility indicate a mean soil temperature at a depth of 1 cm of 1 C in May, 15 C in June, 18 C in July, 19 C in August, and 13 C in September (Lynch and Smith, 1993). Because low temperature is considered a major limiting factor for soybean nodulation in the short season area, PGPR intended
Zhang et al. PGPR and Soybean Growth and Physiology 7 Photosynthetic rate (µmol m s 1 ) Transpiration (mmol m s 1 ) Stomatal conductance (cm s ) 3 1..3..1 G11-3 -8 1-1 31-1 P73 Experiment 1 G11-3 -8 1-1 31-3 3-3 3-9 1-1 Experiment FIG. 3. Effects of plant growth promoting rhizobacteria on plant photosynthesis, transpiration, and stomatal conductance for plants grown at 15 C root zone temperature. Vertical lines on top of each bar indicate one standard error unit (n ). for use as plant nodulation and growth stimulators in eastern Canada should be screened and selected for at a range of RZTs extending from 15 to 18 C (nodulation processes usually end in late June in eastern Canada). Photosynthesis was more sensitive to the application of PGPR at both the optimal and suboptimal RZTs than transpiration and stomatal conductance (Figs 1, and 3). Within each RZT, comparison of the photosynthetic rate increases by the best stimulatory strains common to both experiments showed that photosynthetic rates of plants receiving S. liquefaciens 8 at 17 5 C RZT and S. proteamaculans 1 1 at 15 C RZT had increases over the control plants of 3 1, and 33 8%, respectively. These percentage increases were higher than those of plants receiving S. liquefaciens 8 at the optimal 5 C RZT ( 7%). Photosynthetic rate (µmol m s 1 ) 8 17 7 37 Days after inoculation 15 C 17.5 C 5 C FIG.. Photosynthetic rate of PGPR (plant growth promoting rhizobacteria)-treated and non-pgpr-inoculated plants over time at three temperatures (results from the first experiment). ( ), Non- PGPR-inoculated plants; ( ), ( ), and ( ) Indicate plants receiving Serratia proteamaculans 1 1, Aeromonas hydrophila P73, and Serratia liquefaciens 8, respectively. Each point represents the mean ( s.e.) value of six observations. The change in photosynthetic rate over time showed that plant photosynthesis was increased by PGPR application over a wide range of plant growth stages (Figs and 5). Uppermost leaf regreening was used as an indicator of the time of the onset of nitrogen fixation (Zhang et al., 1995) and indicated that plants grown at 5, 17 5, and 15 C RZTs started to fix atmospheric nitrogen at 15, 37, and 9 DAI, respectively. Since photosynthesis (Figs and 5), transpiration, and stomatal conductance (data not shown) were increased by stimulatory strains before the onset of nitrogen fixation at 17 5 and 15 C (the first measurement was taken after the onset of nitrogen fixation by plants at 5 C RZT), 7
8 Zhang et al. PGPR and Soybean Growth and Physiology Photosynthetic rate (µmol m s 1 ) 15 C 17.5 C 5 C legume crops have also been demonstrated to affect symbiotic nitrogen fixation positively by enhancing root nodule number or mass (Grimes and Mount, 198; Yahalom, Okon and Dovrat, 1987; Zhang et al., 199) and increase nitrogenase activity (Iruthayathas, Gunasekaran and Vlassak, 1983; Alagawadi and Gaur, 1988). Therefore, the observed increases in plant growth and development due to PGPR addition in this experiment may come about in one of two ways: (a) improvement of photosynthesis and plant growth, leading to improved nodulation and nitrogen fixation, or (b) improvement of plant nodulation and nitrogen fixation leading to improved plant growth. Since the PGPR strains which resulted in the greatest stimulation of growth caused increases in plant photosynthetic rates prior to the onset of nitrogen fixation, the first of these two possibilities seems likely to be the cause. In summary, the results of this study indicated that: (a) some of the PGPR strains tested stimulated aspects of soybean growth and development, whereas several strains were inhibitory; (b) some of the PGPR strains which inhibited plant growth at one temperature, stimulated it at another; (c) the effects of the stimulatory strains varied with RZT in a way that was unique to each strain; (d) the effects of PGPR on legume plant growth and development were apparent before the onset of nitrogen fixation (for 17 5 and 15 C RZTs) and can therefore be attributed to improved overall plant photosynthesis and growth, whereas after commencement of nitrogen fixation they may have been due to improvement of plant physiological activities, as well as plant nodulation and nitrogen fixation. Overall the findings indicated that some PGPR stimulate soybean growth and development, but the exact nature of these effects varied among PGPR and RZT. 17 7 37 Days after inoculation FIG. 5. Photosynthetic rate of PGPR (plant growth promoting rhizobacteria)-treated and non-pgpr-inoculated plants over time at three temperatures (results from the second experiment). ( ) Non- PGPR-inoculated plants; ( ), ( ), and ( ) Indicate plants receiving Serratia proteamaculans 1 1, Pseudomonas putida G11 3, and Serratia liquefaciens 8, respectively. Each point represents the mean ( s.e.) value of six observations. the improvement of plant growth, development and physiological activities must have been due to direct effects of PGPR on overall physiology rather than specific effects on nitrogen fixation. These results indicated that at least some PGPR directly affect the overall physiology of soybean plants. The mechanisms of growth promotion by PGPR are not well understood; however, a wide range of possibilities have been postulated, including an increase in mobilization of insoluble nutrients and subsequent enhancement of uptake by the plants (Lifshitz et al., 1987), and production of plant growth regulators that stimulate plant growth (Gaskins, Albrecht and Hubbel, 1985). PGPR applied to 7 ACKNOWLEDGEMENTS We thank Mr S. Liebovitch and Mrs B. Schultz for their expert assistance and wish to acknowledge the Natural Sciences and Engineering Research Council of Canada and the Conseil de la recherche en pe cherie et en agroalimentaire du Que bec for funds pertaining to parts of this research. LITERATURE CITED Alagawadi AR, Gaur AC. 1988. Associative effect of Rhizobium and phosphate-solubilising bacteria on the yield and nutrient uptake of chickpea. Plant and Soil 15: 1. Gaskins MH, Albrecht SL, Hubbel DH. 1985. Rhizosphere bacteria and their use to increase productivity: A review. Agriculture, Ecosystem and En ironment 1: 99 11. Grimes HD, Mount MS. 198. Influence of Pseudomonas putida on nodulation of Phaseolus ulgaris. Soil Biology and Biochemistry : 7 3. Iruthayathas EE, Gunasekaran S, Vlassak K. 1983. Effect of combined inoculation of Azospirillum and Rhizobium on nodulation and nitrogen fixation of winged bean and soybean. Scientific Horticuture : 31. Li DM, Alexander A. 1988. Co-inoculation with antibiotic-producing bacteria to increase colonization and nodulation by rhizobia. Plant and Soil 18: 11 19. Lifshitz R, Kloepper JW, Kozlowshi M, Simonson C, Carlson J, Tipping
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