Abstract: The effect of Plant Growth-Promoting Rhizobacteria (PGPR) on seed germination, seedling growth and yield of field grown maize were evaluated in three experiments. In these experiments six bacterial strains include P. putida strain R-168, P. fluorescens strain R-93, P. fluorescens DSM 50090, P. putida DSM291, A. lipoferum DSM 1691 and A. brasilense DSM 1690 were used. Results of first study showed seed inoculation significantly enhanced seed germination and seedling vigour of maize. In second experiment, leaf and shoot dry weight and also leaf surface area significantly were increased by bacterial inoculation in both sterile and non-sterile soil. The results showed that inoculation with bacterial treatments had a more stimulating effect on growth and development of plants in nonsterile than sterile soil. In the third experiment, Inoculation of maize seeds with all bacterial strains significantly increased plant height, 100 seed weight, number of seed per ear and leaf area. The results also showed significant increase in ear and shoot dry weight of maize.
INTRODUCTION
During the last couple of decades given the negative environmental impact of chemical fertilizers and their increasing costs, the use of soil microorganisms for sustainable agriculture has increased in various parts of the world. Plant Growth Promoting Rhizobacteria (PGPR) are a group of bacteria that actively colonize plant roots and increase plant growth and yield (Wu et al., 2005). The main active strains of PGPR belong to a range of genera, including Azospirillum, Azotobacter, Bacillus, Pseudomonas and Serratia (Bashan et al., 2004; Ramamoorthy et al., 2002; Zhang et al., 1996). The mechanisms by which PGPRs promote plant growth are not fully understood, but are thought to include: the ability to produce phytohormones (Egamberdiyeva, 2007; Shaharoona, 2006), asymbiotic N2 fixation (Mrkovacki and Milic, 2001; Salantur et al., 2006), against phytopathogenic microorganisms by production of siderophores, the synthesis of antibiotics, enzymes and/or fungicidal compounds (Ahmad et al., 2006; Bharathi et al., 2004; Jeun et al., 2004) and also solubilisation of mineral phosphates and other nutrients (Cattelan et al., 1999).
Significant increases in growth and yield of agronomical important crops in response to inoculation with PGPR have been reported by Asghar et al. (2002), Bashan et al. (2004) and Biswas et al. (2000). Azospirillum, Pseudomonas and Azotobacter strains could affect seed germination and seedling growth (Shaukat et al., 2006). Kloepper et al. (1992) has been shown that wheat yield increased up to 30% with Azotobacter inoculation and up to 43% with Bacillus inoculation. Strains of Pseudomonas putida and Pseudomonas fluorescens could increase root and shoot elongation in canola, (Glick et al., 1997) as well as wheat and potato (De Freitas and Germida, 1992; Frommel et al., 1993). Inoculation of plants with Azospirillum could result in significant changes in various growth parameters, such as increase in plant biomass, nutrient uptake, tissue N content, plant height, leaf size and root length of cereals (Bashan et al., 2004). Thus it has been shown that Azospirillum and Pseudomonas had the potential for agricultural exploitation and could use as natural fertilizers (Bashan et al., 1989; Cakmakc et al., 2006).
However, the effects of these bacteria on growth and yield of some crop plants studied in earlier studies. But the effects of PGPR on growth parameter from germination to yield were not evaluated simultaneously. The main objective of this research was to determine if PGPR strains could affects on seed germination, growth parameters of maize seedling in greenhouse and also grain yield of field grown maize.
MATERIALS AND METHODS
PGPR strains and inoculum’s preparation: Six bacterial strains include P. putida strain R-168, P. fluorescens strain R-93, P. fluorescens DSM 50090, P. putida DSM291, A. lipoferum DSM 1691 and A. brasilense DSM 1690 were obtained from the Soil Microbiology Department, Soil and Water Research, Tehran and were used in this study that conducted at 2005-2006.
Mode of application: Bacterial strains were used as maize seed treatments. Seeds of maize (SC 647) were surface-sterilized with 0.02% sodium hypochlorite for 2 min and rinsed thoroughly in sterile distilled water. For inoculation seeds were coated with 20% gum arabic as an adhesive and rolled into the suspension of bacteria (108 cfu mL-1) with perlit until uniformly coated. (Salanture et al., 2006). Seeds treated with sterile distilled water amended with gum arabic served as the nontreated control.
Seed germination bioassay: Germination tests were carried out by the paper towel method (Niranjan et al., 2004). Twenty five seeds for each treatment with three replications in completely randomized design and incubated in growth chamber at 28C. After 7 days the number of germinated seeds was counted. Root and shoot length of individual seedling was measured to determine the vigor index with following formula (Abdul Baki and Anderson, 1973):
|
Vigor index = (Mean root length+Mean shoot length)xGermination (%) |
Pot culture experiment: For the evaluation of maize seedling growth promotion with PGPRs, above bacterial strains were tested in both non-sterile and sterile soils at 2005. The plastic pots had 15 cm diameter and capacity to hold 2 kg of soil. For preparation of sterile soil, field soil was autoclaved twice for 20 min at 120C with a 24 h interval. All treatments (bacterial inoculationxsoil condition) arranged in 48 pots i.e., 3 replicates with 14 pots per replication and a double seed per pot. Treatments were arranged in a factorial experiment based on completely randomized design. Seedlings were watered daily and no artificial fertilization was used. After 30 days, fresh weight was determined and dry weight calculated by drying plants in an oven at 75C until the weight remained constant. For leaf area determination, the area of each expanded leaf was calculated as:
K x length x width |
Where:
k = 0.75 (Ruget et al., 1996).
Field experiment: Field experiment was conducted at research farm of Shahroud University of technology (latitude of 36 25 ’N and longitude of 54 57’ E with an elevation of 1345 m) in the period of May-September, 2006. The field soil was silty clay loam in texture, having pH, 7.8; EC, 3.9 dS m-1; 0.75% of organic carbon; 0.04% N, 6.4 and 320 ppm of available P and K, respectively. Seeds of maize were washed with distilled water then inoculation was performed by a suspension of any bacteria (108 cfu mL-1) with perlit mixture. Treatments were arranged as randomized complete block design with three replications. There were four rows in each plot. Which the row width and length was 0.7 and 9 m, respectively. Before sowing, the soil was fertilized with N, P and K at rate of 300, 150 and 50 kg ha-1 as urea, single super phosphate and potassium sulphate, respectively. Half of nitrogen was applied at sowing time and residue at the start of reproductive stage. Seeds were placed at 5 cm depth. At the third leaf stage, plants were thinned to 1 plant hill-1 for the appropriate final stand of 75000 plants ha-1. The soil was subsequently irrigated 10 days interval at the first and shortened to 7 days interval to summer. Ten mature maize plants were sampled from each treatment for final measurements in October (120 days after sowing). Leaf surface area was measured as describe above. In laboratory, samples were separated into different components and oven-dried at 75C until reached to constant weight.
Data analysis: Data were statistically treated by ANOVA, Least Significant Difference (LSD) test at probability level 0.05 was used to separate the means when the ANOVA F-test indicated a significant effect of the treatments.
RESULTS
Seed germination: Seed inoculation significantly enhanced seed germination and seedling vigour of maize. However, the rate of enhancement varied with bacterial strains. All bacteria except A. lipoferum DSM1691, increased seed germination up to 18.5% over nontreated control (Fig. 1). The highest enhancement of vigor indexes were obtained from A. brasilense DSM 1690 and P. putida strain R-168, which recorded 975 and 873 vigor index, respectively.
Effects of inoculation with bacteria on maize plant growth and development:
The results of pot study showed that inoculation of maize seeds with bacterial
strains did not affect leaf fresh weight and stem dry weight (Table 1). In contrast,
stem and total fresh weight significantly increased by inoculation in sterile
and non-sterile soil.
| Table 1: | Effect of bacterial inoculation on growth characteristics of maize seedlings at 30 days after sowing in different soil condition |
| *In a column, means followed by a common letter(s) are not significantly different at 5% level by LSD | |
| Fig. 1: | Effects of bacterial inoculations on seed germination and vigor index of maize 7 days after germination in vitro conditions |
The highest stem and total fresh weight were recorded from A. lipoferum DSM 1691 in non sterile soil and from A. brasilense DSM 1690 in sterile soil. Leaf and shoot dry weight were significantly enhanced by bacterial inoculation and soil type (p<0.05). Application of A. lipoferum DSM 1691 in non sterile soil and A. brasilense DSM 1690 in sterile soil, respectively had the best effect on leaf dry weight (0.52 g plant-1) and shoot dry weight (1.30 g plant-1). Furthermore, inoculation with bacterial strains had significant effect on leaf surface area under both soil conditions. The results revealed that in non sterile soil, A. lipoferum DSM 1691 caused an increase of 78.3% in leaf area while in sterile soil, leaf area was increased up to 65% in response to inoculation with A. brasilense DSM 1690 compared to control.
The results showed that inoculation with bacterial treatments had a more stimulating effect on growth and development of plants in nonsterile soil than sterile condition. P. putida strain R-168 and A. lipoferum DSM 1691 performed better than other strains in stimulating plant growth in pot experiment especially at nonsterile soil.
Inoculation of maize seeds with all bacteria strains significantly increased the plant height (14.3-21.7%) and leaf area (Table 2). However, seed dry weight increased with bacterial inoculation, but there were no significant differences between all treatments and control. 100 seed weight was significantly affected due to treatments with Azospirillum and Pseudomonas strains over the control. The most effective strain was P. fluorescens DSM50090 which increased 100 seed weight up to 44% over control. Effect of inoculation on number of seed per ear was also significant. Application of P. fluorescens strain R-93 and A. lipoferum DSM1691 gave the maximum number of seed per ear.
The results showed a clear and significant increase in ear and shoot dry weight
of maize (p<0.05). The increase in these parameters, with different inoculation
ranged from P. putida DSM291 (up to 70%) to A. lipoferum DSM1691
(up to 100 %) when compared with control (Table 2).
| Table 2: | Effect of seed treatment with PGPR on growth parameters of maize under field conditions |
| *In a column, means followed by a common letter(s) are not significantly different at 5% level by LSD | |
DISCUSSION
Plant growth promoting effects of PGPR strains in different crops were clearly demonstrated (El-Hawary et al., 2002; Wu et al., 2005). Bacterial inoculants are able to increase plant growth and germination rate, improve seedling emergence, responses to external stress factors and protect plants from disease (Lugtenberg et al., 2002).
This present investigation confirms the earlier studies. It revealed that under in vitro conditions, seed treatment with PGPR strains improved seed germination, seedling vigor, seedling emergence and seedling stand over the control. Similar improvement of seed germination parameters by rhizobacteria has been reported in other cereals such as sorghum (Raju et al., 1999) and pearl millet (Niranjan et al., 2003, 2004). The improvement in seed germination by PGPR was also found in work with wheat and sunflower (Shaukat et al., 2006a, b), where it was found that some PGPR induced increases in seed emergence, in some cases achieving increases up to 100% greater than controls. These findings may be due to the increased synthesis of hormones like gibberellins, which would have triggered the activity of specific enzymes that promoted early germination, such as a-amylase, which have brought an increase in availability of starch assimilation. Beside, significant increase in seedling vigor would have occurred by better synthesis of auxins (Bharathi et al., 2004).
In pot experiment, it was observed that inoculation with PGPR strains significantly promoted growth of seedling maize under different soil conditions. In general, inoculation resulted in early seedling growth and development. These results are similar with the findings of Dobbelaere et al. (2001, 2002), who assessed the inoculation effect of PGPR Azospirillum brasilense on growth of spring wheat. They observed that inoculated plants resulted in better germination, early development and flowering and also increase in dry weight of both the root system and the upper plant parts. Similarly, promotion in growth parameters and yields of various crop plants in response to inoculation with PGPR were reported by Gravel et al. (2007), Kozdroja et al. (2004) and Shaharoona et al. (2006). Inoculation of maize seeds with Azospirillum strains compared with Pseudomonas strains under experiment conditions resulted in a more visible increase in shoot development, especially during the establishment of the plant. Khalid et al. (2004) showed that responses of wheat growth to inoculation with rhizobacteria depend on plant genotype and PGPR strains as well as environmental conditions.
Soil condition influenced growth promotion by bacterial strains. Azospirillum brasilense DSM 1690 had more effect on growth parameters in sterile soil compared with non sterile soil. Martinez-Toledo et al. (1988) showed that the numbers of Azotobacter decreased as plant growth continued in non-sterile agricultural soils, while the numbers of Azotobacter associated with maize roots grown in sterile agricultural soils remained similar to those of the original inoculums. In contrast, inoculation with other bacterial treatments had a more stimulating effect on growth of plants in non-sterile soil than sterile condition. Abbass and Okon (1993) hypothesized that IAA and other plant hormones were responsible for increased growth of canola, tomato (Lycopersicon esculentum Mill.) and wheat (Triticum turgidum L.) in non-sterile soil inoculated with Azotobacter paspali. Auxins produced by rhizobacteria can influence plants growth, including root development which improve uptake of essential nutrients thus increasing plant growth (Vikram et al., 2007). This may imply rhizobacteria had more competitive ability to survive and affect the growth of inoculated plants in the presence of indigenous micro flora (Khalid et al., 2004).
In the other hand, as suggested by Roesti et al. (2006) this result mean that inoculums of the PGPR strains on the seeds may have shifted the bacterial community equilibrium at early stages of plant growth and favoured for growth of beneficial populations.
In this study, inoculation of PGPR strains increased all parameters determined in field experiment. The positive effects of PGPRs on the yield and growth of crops such as wheat (Ozturk et al., 2003; Salanture et al., 2006) maize (Egamberdiyeva, 2007) soybean (Cattelan et al., 1999) and sugar beet (Cakmakc et al., 2006) were explained by N2 fixation ability, phosphate solubilizing capacity and phytohormons production.
The present experiment revealed that seed inoculation with all bacteria resulted in an increased plant height and leaf area (Table 2). Similar increases in plant height and leaf area were observed in different crops inoculated with Pseudomonas, Azospirillum and Azotobacter strains (Martinez-Toledo et al., 1988; Shaukat et al., 2006a, b; Siddiqui and Shaukat., 2002). Burd et al. (2000) reported that plant growth promoting rhizobacteria might enhance plant height and productivity by synthesizing phytohormones, increasing the local availability of nutrients, facilitating the uptake of nutrients by the plants decreasing heavy metal toxicity in the plants antagonizing plant pathogens.
Results have also showed that plants inoculated with PGPRs generally have higher seed dry weight than un-inoculated plants. The increases in seed dry weight were derived mainly from increase in 100 seed weight and number of seed per ear. This finding was supported by Yasari and Patwardhan (2007) reported that application of Azotobacter and Azospirillum strains increased canola yield (21.17%), pod per plant (16.05%), number of branches (11.78%) and weight of 1000 grain (2.92%).
The higher ear and shoot dry weight response to all inoculants compared to control clearly showed the beneficial role of these rhizobacteria. The enhancing effect of seed inoculation with rhizobacteria on shoot dry weight and yield of maize were reported by Pandy et al. (1998) and 69389_ja Such an improvement might be attributed to N2-fixing and phosphate solubilizing capacity of bacteria as well as the ability of these microorganisms to produce growth promoting substances (Bashan et al., 2004; Vessey, 2003). In conclusion the results of this study suggest that simultaneous screening of rhizobacteria for growth and yield promotion under pot and field experiment is a good tool to select effective PGPR for biofertilizer development biotechnology.
ACKNOWLEDGMENTS
The researchers are thankful to Shahrood University of Technology and the Soil Microbiology Department, Soil and Water Research, Tehran for helping to carry out this study.