Agronomy Journal 94:925-929 (2002)
© 2002 American Society of Agronomy
RICE
Influence of Rhizobial Inoculation on Photosynthesis and Grain Yield of Rice
Shaobing Peng*,
Jatish C. Biswas,
Jagdish K. Ladha,
Prasad Gyaneshwar and
Yizhu Chen
Crop, Soil, and Water Sci. Div., International Rice Research Institute (IRRI), DAPO Box 7777, Metro Manila, Philippines
* Corresponding author (s.peng{at}cgiar.org)
Received for publication August 9, 2001.
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ABSTRACT
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Rhizobial inoculation increases grain yield in rice (Oryza sativa L.), a nonlegume plant, but little is known about the mechanism(s) involved. This study was conducted to determine whether inoculation with rhizobia could influence leaf photosynthesis of rice plants under greenhouse conditions. Rice seeds and pot soil were inoculated with three rhizobial strains with or without added N fertilizer. Single-leaf net photosynthetic rates were measured with portable photosynthesis systems (LI-6200 and LI-6400) at several growth stages. Stomatal conductance, chlorophyll fluorescence, specific leaf weight, and leaf N content were also measured. Grain yield and yield components were determined at maturity. A significant increase in single-leaf net photosynthetic rate by rhizobial inoculation was observed in all three independent experiments. The effect of rhizobial inoculation on photosynthesis was greater in zero-N than in 90 kg N ha-1 treatment. The increase in photosynthetic rate by rhizobial inoculation was 12% averaged across all treatments in the three experiments. The effects of rhizobial inoculation on stomatal conductance, specific leaf weight, and leaf N content were relatively small and less consistent than photosynthetic rate. Chlorophyll fluorescence data suggest that the increase in photosynthetic rate following rhizobial inoculation was not associated with conversion efficiency of light energy in photosystem II. Rhizobial inoculation increased grain yield by 16%. The increase in grain yield was due to an increase in total biomass production rather than harvest index. These results suggest that certain strains of rhizobia can promote rice growth and yield through mechanisms that improve single-leaf net photosynthetic rate.
Abbreviations: DAE, days after emergence • PBS, phosphate-buffered saline
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INTRODUCTION
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PLANT GROWTH–PROMOTING RHIZOBACTERIA have been reported in rice, a nonlegume plant (Yanni et al., 1997; Biswas et al., 2000a, 2000b). Two endophytic strains of Rhizobium leguminosarum bv. trifolii (E11 and E12) increased grain yield of rice by 10 to 45% over a wide range of N supply in a field inoculation experiment (Yanni et al., 1997). Biswas et al. (2000a) reported that rhizobial inoculation increased rice grain yield by 8 to 22% at different N rates under greenhouse conditions. Plant growth–promoting rhizobacteria, including free-living and symbiotic diazotrophs, influence crop growth and development by various mechanisms. For example, symbiotic and nonsymbiotic diazotrophs fix atmospheric N2, part of which is taken up by associated plants for assimilation (Bashan and Holguin, 1997; Yoneyama et al., 1997). Nitrogen 15–based studies indicated that the growth promotion in rice plants by rhizobial inoculation was not associated with biological N2 fixation (Biswas et al., 2000a).
Some reports have demonstrated the role of rhizobia in promoting growth of nonlegumes following inoculation (Fyson and Oaks, 1990; Haque and Ghaffar, 1993; Chabot et al., 1996; Noel et al., 1996; Yanni et al., 1997), but little is known about the mechanism(s) involved. Probable mechanisms were increased root growth that favored higher nutrient uptake (Chabot et al., 1996; Yanni et al., 1997), disease control (Haque and Ghaffar, 1993), and production of a phytohormone (Chabot et al., 1996). Recently, Volpin and Phillips (1998) reported that inoculated rhizobia influence the physiological status of inoculated plants by increasing root respiration. Biswas et al. (2000a) reported that rhizobial inoculation significantly increased uptake of N, P, K, and Fe by rice plants compared with the uninoculated control. In another study, Biswas et al. (2000b) observed a significant increase in vigor of rice seedlings following rhizobial inoculation. This benefit of early seedling development could carry over to significantly increase grain yield at maturity.
At least 90% of the biomass of higher plants is derived from CO2 assimilated through photosynthesis (Zelitch, 1982). The single-leaf net photosynthetic rate of rice plants is largely affected by stomatal conductance, leaf N content, and specific leaf weight (Peng, 2000). The growth promotion by rhizobial inoculation could be attributed to the increase in single-leaf and/or whole-plant photosynthetic rate. To our knowledge, no research has been published to determine the effect of inoculation with growth-promoting rhizobia on photosynthesis in rice plants. Therefore, this study was undertaken to investigate whether inoculation with rhizobia can influence leaf photosynthesis of rice plants under greenhouse conditions.
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MATERIALS AND METHODS
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Experimental Sites and Soil
Three greenhouse experiments were conducted in 1997, 1998, and 1999 at the International Rice Research Institute (IRRI), Los Baños, Philippines. A lowland soil from IRRI's experimental farm, commonly known as Maahas clay loam (Aquandic Epiaquoll), was used. The major soil properties were pH 6.23 (1:1 w/v water), 12 g kg-1 organic C, 35 mg kg-1 Olsen P, 1.5 cmolc kg-1 exchangeable K, 0.46 dS m-1 electrical conductivity (1:1), 26.7 mg kg-1 NH4–N, 1.5 g kg-1 total N, and 20.28 cmolc kg-1 cation exchange capacity.
In Exp. I, air-dried potted soil (6 kg pot-1) received N, P, and K from urea [(NH2)2CO], sodium phosphate (Na3PO4), and KCl at 90, 26, and 33 kg ha-1 N, P and K, respectively. One-third of N and full doses of P and K fertilizers were applied before sowing. Supplemental N fertilizer was applied in two equal doses, the first at 34 d after emergence (DAE) and the second at the panicle initiation stage. A completely randomized design with seven replicates was utilized. Experiments II and III were managed in the same way as Exp. I, except for N rate, cultivar, and rhizobial strains (Table 1).
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Table 1. Bacterial strains for rhizobial inoculation, cultivars, and the rate of N application in three pot experiments conducted from 1997 through 1999.
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Inocula Preparation and Inoculation
Rhizobium leguminosarum bv. trifolii E12 and Rhizobium sp. IRBG74 were grown in yeast mannitol (YM) broth (Somasegaran and Hoben, 1985) and Bradyrhizobium sp. IRBG271 in half-strength tryptone glucose yeast extract broth (BBL, Cockeysville, MD). Exponentially growing cells in shaken broth culture were collected by centrifugation for 10 min at 4°C and washed with sterile PO4–buffered saline (PBS; pH 7.0). Cell pellets were suspended in 1 mL of PBS, transferred to 1.5-mL Eppendorf tubes, and recentrifuged at 10000 x g for 1 min. The pelleted cells were suspended in 0.5% (w/v) xanthan gum.
In Exp. I, ‘Oking seroni’ (a rice cultivar from Indonesia) seeds of similar size were sorted and coated with strains E12, IRBG74, or IRBG271 in the proportion of 3.5 x 105 cells seed-1, and four to five seeds were sown at equal depth in potted soil. Pots were thinned at 5 DAE, and one healthy plant was allowed to grow in each pot. The pot soil was again inoculated at 20 DAE with a 1-mL culture (106 colony-forming units mL-1) in PBS. Two strains (E12 and IRBG74) were used in Exp. II and III to confirm earlier results. Each experiment had a control (uninoculated) treatment that included heat-killed rhizobial cells.
Photosynthesis and Related Parameters
Single-leaf net photosynthetic rates and stomatal conductance were measured with a LI-6400 portable photosynthesis system (LI-COR, Lincoln, NE) at 25 and 40 DAE in Exp. I and II. Single-leaf net photosynthetic rates were also measured at the flowering stage in Exp. II because photosynthetic rates at this stage have a positive correlation with grain yield (Cook and Evans, 1983). The LI-6400 was operated in the open mode. External air was scrubbed of CO2 and mixed with a supply of pure CO2 to result in a reference concentration of 350 µL L-1. Flow rate was 500 µmol s-1, and external humidity was 50 to 60%. A light-emitting diode light source was placed at the upper half of the leaf chamber (2 by 3 cm). The middle portion of the uppermost fully expanded leaf was chosen for the measurement under photosynthetically active radiation (PAR) of 1500 µmol m-2 s-1 provided by the light source. The light saturation point for rice single-leaf photosynthesis was 880 to 1170 µmol m-2 s-1 (Yoshida, 1981). All measurements were done between 0930 and 1200 h.
In Exp. III, single-leaf net photosynthetic rates and stomatal conductance were measured with a LI-6200 portable photosynthesis system (LI-COR, Lincoln, NE) at the early grain-filling stage to confirm the results of Exp. I and II with a different instrument. Measurements were done under natural sunlight with photosynthetically active radiation >1500 µmol m-2 s-1. A 0.25-L chamber was used to cover the middle portion of leaves. The gas exchange system was operated as a closed system to measure photosynthetic rates over a 20-s period. A flow rate of about 230 µmol s-1 was maintained to stabilize humidity inside the chamber during the course of each measurement. The 20-s measurement duration resulted in a temperature increase <1°C inside the chamber.
After measurement of photosynthetic rate, leaf area was determined by a leaf area meter (AAM-8 Hayashi Denkoh Co., Kashiwa, Japan), followed by drying at 70°C for 72 h in Exp. II. Specific leaf weight was calculated as the ratio of dry weight to leaf area. Leaf chlorophyll fluorescence was measured with a PAM-2000 portable chlorophyll fluorometer (Walz, Effeltrich, Germany) at 30 DAE in Exp. II. Leaf N content was determined by micro Kjeldahl digestion and distillation of the entire leaf blade in Exp. III (Bremner and Mulvaney, 1982).
Agronomic Parameters
Grain yield and yield components were measured at maturity in Exp. II. Panicles per pot and spikelets per panicle were counted. Moisture content of grains was measured using a Satake HM3A Moistex Meter (Satake Eng. Co., Tokyo, Japan), and grain yield per pot was calculated as [(100 - moisture content of the sample) x fresh grain weight]/86 to convert the sample to 14% moisture content.
Statistical Analyses
Data were analyzed following analysis of variance (SAS Inst., 1987), and means were compared by Duncan's Multiple Range Test at the P = 0.05 level.
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RESULTS
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Inoculation with all three strains increased single-leaf net photosynthetic rates compared with the uninoculated control at 25 and 40 DAE in Exp. I, but the increase was statistically significant only for the strain IRBG74 (Table 2). At both 25 and 40 DAE, photosynthetic rate increased by 14% following inoculation with the strain IRBG74 compared with the control while the other two strains increased photosynthetic rates by 9 to 12%.
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Table 2. Net photosynthetic rate of rice leaves (cultivar Oking seroni) as influenced by rhizobial inoculation (Exp. I). Measurements were taken at 25 and 40 d after emergence (DAE).
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Enhancement in photosynthetic rate by rhizobial inoculation was also observed in Exp. II when more cultivars and N rates were used (Table 3). However, the effect of rhizobial inoculation on photosynthesis was not consistent across treatments and growth stages, ranging from -16 to 37% compared with the uninoculated control. In general, IRBG74 was more effective in promoting photosynthetic rate than E12. The effect of rhizobial inoculation on photosynthesis was greater in IR72 than in Oking seroni and greater in the zero-N than in the 90 kg N ha-1 treatment. The inoculation response was comparatively higher at the early stage of inoculation in both cultivars.
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Table 3. Net photosynthetic rate of rice leaves (cultivars Oking seroni and IR72) as influenced by rhizobial inoculation (Exp. II). Measurements were taken at 25 and 40 d after emergence (DAE) and flowering stage. Flowering date for IR72 and Oking seroni was 85 and 90 DAE, respectively.
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The effect of rhizobial inoculation on stomatal conductance was much smaller and less consistent than on photosynthesis in Exp. II (Table 4). For example, inoculation with E12 actually significantly decreased stomatal conductance compared with the uninoculated control in Oking seroni for the zero-N treatment. Within each measurement date, there was a poor correlation between photosynthetic rate and stomatal conductance (r = 0.41).
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Table 4. Stomatal conductance of rice leaves (cultivars Oking seroni and IR72) as influenced by rhizobial inoculation (Exp. II). Measurements were taken at 25 and 40 d after emergence (DAE).
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Rhizobial inoculation significantly increased specific leaf weight in 5 out of 16 comparisons (Table 5). The effect of rhizobial inoculation on specific leaf weight was greater at 40 than 25 DAE. Chlorophyll fluorescence data indicated that rhizobial inoculation did not consistently affect efficiency of primary conversion of light energy of photosystem II [Fv/Fm (the ratio of variable to maximal chlorophyll fluorescence)] (Table 6).
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Table 5. Specific leaf weight of rice leaves (cultivars Oking seroni and IR72) as influenced by rhizobial inoculation (Exp. II). Measurements were taken at 25 and 40 d after emergence (DAE).
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Table 6. Efficiency of primary conversion of light energy of photosystem II (Fv/Fm) in rice leaves (cultivars Oking seroni and IR72) as influenced by rhozobial inoculation (Exp. II). Measurements were taken at 30 d after emergence (DAE).
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The LI-6200 utilized in Exp. III detected 11 to 13% increase in photosynthetic rate by inoculation with E12 or IRBG74 compared with the uninoculated control (Table 7). Stomatal conductance increased by 7 to 12% following rhizobial inoculation, but the increase was not statistically significant. The effect of rhizobial inoculation on leaf N content was nonsignificant.
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Table 7. Net photosynthetic rate, stomatal conductance, and leaf N content of rice leaves (cultivar IR72) as influenced by rhizobial inoculation (Exp. III). Measurements were taken at early grain-filling stage.
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Rhizobial inoculation increased sink size by increasing either panicle number or spikelet number per panicle (Table 8). The increase in spikelet number per panicle was more pronounced in the 90 kg N ha-1 than in the zero-N treatment. Grain yield was significantly increased by rhizobial inoculation, except for Oking seroni grown under high N and inoculated with IRBG74 and IR72 grown under zero N and inoculated with E12. Greater increase in grain and straw yield by rhizobial inoculation was observed in IR72 than in Oking seroni. The increase in grain yield by rhizobial inoculation was 16% averaged across all treatments in Exp. II. Rhizobial inoculation did not increase harvest index in either cultivar (data not shown).
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Table 8. Yield components and grain yield of rice (cultivars Oking seroni and IR72) as influenced by rhizobial inoculation and N rate (Exp. II).
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DISCUSSION
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A significant increase in single-leaf net photosynthetic rate by rhizobial inoculation was observed in all three independent experiments. The increase in photosynthetic rate by rhizobial inoculation was 12% averaged across all treatments in the three experiments. The effect of rhizobial inoculation on stomatal conductance and specific leaf weight was relatively small and less consistent than on photosynthetic rate. Many studies have reported a close relationship between stomatal conductance and photosynthetic rate (e.g., Makino et al., 1984b). A positive correlation between photosynthetic rate and specific leaf weight was observed in rice plants (Murata, 1961). We cannot completely rule out the possibility that rhizobial inoculation enhanced photosynthetic rate by increasing stomatal conductance or specific leaf weight, and more detailed measurements on these parameters are necessary in future studies. Chlorophyll fluorescence data suggest that the increase in photosynthetic rate following rhizobial inoculation was not associated with conversion efficiency of light energy in photosystem II.
DeJong and Phillips (1981) reported higher leaf apparent photosynthesis and increased leaf N content in Alaska pea (Pisum sativum L.) following rhizobial inoculation. In that study, both leaf N content and photosynthetic rate increased linearly with symbiotically fixed N2. A close relationship between photosynthetic rate and leaf N content was reported for both greenhouse- and field-grown rice plants (Yoshida and Coronel, 1976; Peng et al., 1995). In this study, the effect of rhizobial inoculation on leaf N content was relatively small and statistically nonsignificant. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) is a key enzyme of CO2 photosynthetic fixation in green plants and is also the predominant leaf protein (Ellis, 1979). About 50% of total soluble protein and 25% of total N are associated with Rubisco protein in rice leaves (Makino et al., 1984a). Single-leaf net photosynthetic rate is closely correlated with Rubisco content (Makino et al., 1983). The effect of rhizobial inoculation on leaf N content and Rubisco content deserves further study.
Grain yield was significantly increased by rhizobial inoculation in both cultivars. Rhizobial inoculation increased sink size by increasing either panicle number or spikelet number per panicle. The increase in grain yield was due to an increase in total biomass production rather than harvest index. This increase was more prominent in IR72 at the higher N rate than in the Oking seroni cultivar. Yanni et al. (1997) also reported higher grain yield following inoculation with E12 in a field experiment in Egypt. In separate experiments, Biswas et al. (2000a)(2000b) reported that rhizobial inoculation increased rice grain yield at different N rates under greenhouse conditions.
With the same rhizobial strains, and under similar growing condition as in this study, Biswas et al. (2000a) found that the growth promotion in rice plants by rhizobial inoculation was not associated with biological N2 fixation based on 15N dilution. Furthermore, indole-3-acetic acid accumulated in the external root environment of rice plants when grown gnotobiotically with rhizobia (Biswas et al., 2000a). A positive correlation between grain yield and photosynthetic rate, especially at the zero-N level, was observed in this study. The data presented by Biswas et al. (2000a)(2000b), together with the data in the present study, suggest that certain strains of rhizobia can promote rice growth and yield though mechanisms that improve single-leaf net photosynthetic rate rather than biological N2 fixation.
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ABSTRACT
INTRODUCTION
