The effect of plant growth-promoting rhizobacteria (PGPR) belonging to the genera Azospirillum and Azotobacter on the growth and nutrient uptake of maize (Zea mays) was investigated in the field as factorial experiment. Azospirillum strains were: Z0 = no inoculation, Z1 = Azospirillum sp. Strain 21, Z2 = Azospirillum lipoferum DSM 1691, Z3 = Azospirillum brasilense DSM 1690 and Azotobacter strains were: A0 = no inoculation, A1 = Azotobacter sp. Strain 5, A2 = Azotobacter chroococcum DSM2286. Treatment with PGPR(s) significantly increased plant height, shoot and seed dry weight, ear dry weight and length and number of seeds per row. Plants nutrient uptake of N, P, K, Fe, Zn, Mn and Cu were also significantly influenced by application of PGPR(s). These results indicate some PGPR inoculants have the potential to increase maize growth yield and nutrients uptake.
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Increasing demand for food and livestock feed caused maize to be introduced as an important crop in temperate and semi-arid regions. Intensive farming practices that aim to produce higher yield, require extensive use of agrochemicals which are costly and create environmental pollutions (Kozdro et al., 2004; Tate, 1995). Inappropriate application of chemical fertilizers and water irrigation systems in maize production in semi-arid regions of Iran has resulted in pollution and salinization of agricultural land and water resources. Colonization of plant roots by bacteria has been observed for a long time, but only lately its importance for plant growth and development become clear (Glick, 1995). Plant growth-promoting rhizobacteria (PGPR) are beneficial soil bacteria that colonize plant roots and increased plant growth (Glick, 1995). Many rhizobacteria have the capacity to fix atmospheric nitrogen (Dobbelaere et al., 2003), although it has been reported that in the most cases this amount of nitrogen is negligible for plant demand and they can promote plant growth through the production of plant growth regulators (Dobbelaere et al., 2003; Verma et al., 2001). Roots of Azospirillum-inoculated maize seedlings were found to have higher amounts of both free and bound IAA as compared to control. The amount of free IAA significantly increased in the inoculated roots 2 weeks after sowing (Falik and Okon, 1989). In other cases these bacteria increased water and nutrient uptake (Jacoud et al., 1999; Okon and Labandera-Gonzalez, 1984). PGPR can also enhance the plant competitiveness and responses to external stress factors as well as inhibiting soil-borne plant pathogens through antifungal activity (Sharma and Chahal, 1987) and also siderophore production (Neiland, 1981). Different plant growth-promoting rhizobacteria, including free-living and associative bacteria such as Azospirillum, Azotobacter, Bacillus and Pseudomonas have been used in agricultural systems as biofertilizer for their beneficial effects on plant growth (Tilak et al., 1982). Inoculation of maize and wheat with Azotobacter and Azospirillum increased plant growth, nutrient uptake and yield (Abbass and Okon, 1993; Dobbelaere et al., 2001; Okon and Labandera- Gonzalez, 1994; Tilak et al., 1982). It was found that Triticum aesriuum cv. Miriam inoculated with Azospirillum accumulated 20% more N at the booting stage than did the uninoculated control (Kapulnik et al., 1985). Also, co-inoculation with Giomus mosseae and G. fasciculatum in the presence of Azospirillum brasilense produced significantly higher dry matter production and grain yield of barley than their corresponding controls (Suba Rao et al., 1985).
The aim of the present study was to evaluate the effects of seed inoculation with PGPR on growth and nutrient uptake of corn grown in a semi-arid environment of Iran.
MATERIALS AND METHODS
Microorganisms: The bacterial strains studied were Azospirillum lipoferum s-21 and Azotobacter chroococcum s-5 with known positive effects on wheat (Mahour, 2005) and canola (Khalilian, 2006). Azospirillum lipoferum DSM 1691, Azospirillum brasilense DSM 1690 and Azotobacter chroococcum DSM 2286 were used as reference strains.
Experimental conditions and plant material: Field experiment was carried out at research farm of Shahrood University of Technology in 2006. The area is located at latitude of 36° 25`N and longitude of 54° 57` E at an elevation of 1345 m. The annual mean precipitation, temperature and relative humidity are 156.6 mm, 14.4°C and 48%, respectively. The soil was clay loam characterized by a pH, 7.8; EC, 3.9 dS m-1 and organic carbon, 0.75% (Table 1). The experiment was conducted using randomized complete block design as factorial with two factors (Azospirillum and Azotobacter strains) and three replications. Four-row plots were prepared with row width and intra-row space of 70 and 20 cm, respectively. There was a space of 70 cm between plots and 3 m between replications. Seeds of maize (Zea mays, hybrid 647) were washed with distilled water and then moistened with 20% solution of sugar before inoculation with bacteria at concentration of 108 cfu mL-1. Before sowing, the soil was irrigated and 300 kg of urea, 150 kg of single super phosphate and 50 kg of potassium sulphate per hectare were applied according to the results of soil analysis. Nitrogen application was at the rate of 50:50 at sowing time and the starting of reproductive stage. For planting, two seeds were placed at 5 cm depth and at three-leaf stage plants were thinned to one plant per hill and the final plants were considered as population. Plants were harvested at November and the following data were collected: shoot dry weight, ear, grain and 100-seeds weight, number of rows per ear, number of seeds per row, ear length and plant height. Dry weights of the separate organs were measured after oven dried at 75°C for 72 h.
Seed nutrient analysis: For nutrient analysis, the following procedure was applied: 1 g seed was digested in an acid mixture of HNO3 and HClO4 (9:4). The resulting ash was analyzed for nutrient content. The concentration of K was determined by flame photometer. The concentration of organic C was determined by the Walkley and Black method (Walkley and Black, 1934). Total nitrogen was determined by Kjeldhal`s method (Nadeem et al., 2006). Phosphorus content was determined with the molybdenum-ascorbic acid colorimetric method (Hanson, 1950). Zn, Fe, Mn and Cu were determined by atomic absorption.
Statistical analysis: Analysis of variance (ANOVA) was performed on all experimental data and means were compared using the Duncan`s multiple range test with SAS software (SAS Institute, 1998). The significance level was p>0.05 unless otherwise stated.
Growth and yield: Responses of field-grown maize to PGPR inoculation depend on strain and plant growth parameter. In all cases, apart from the strain, inoculation significantly increased plant growth parameters except for the number of rows per ear (Table 2). The inoculation of maize increased shoot dry weight from 63 to 115%. The most effective treatment was A1Z1, A0Z1 followed by A1Z0 with a small difference. Ear dry weight was significantly affected by bacterial inoculants (p<0.01), A0Z1 increased ear dry weight by 141% as compared to the control plants. This increase in ear dry weight resulted in significantly higher ear length and grain dry weight. Inoculation promoted number of seed per row from 32 to 64%. Co-inoculation of maize with A1Z1 enhanced 100-seed weight. The results of the study showed, when plants inoculated with A. lipoferum s-21 alone or in combination with Azotobacter sp. strain 5, growth parameters increased compared to all other treatments. The exception was strain A. chroococcum DSM 2286 which produced highest number of seed per row.
The maize yield and growth enhancement due to bacterial inoculation used in this study could be explained with N2-fixing and phosphate solubilizing capacity of bacteria. Another researches also showed the positive effects of PGPR on the yield and growth of plants such as apricot, tomatoes, sugar beet and barley were explained by N2 fixation ability, phosphate solubilizing capacity and production of antimicrobial substances by PGPR (Abbasi et al., 2002; Cakmakci et al., 2001; Esitken et al., 2003).
Plant Nutrient Element (PNE) contents of seeds: The PNE content in corn seeds treated by PGPR strains may provide important information about the effect of bacterial inoculation in crop growth. The results showed a clear and significant increase in total nutrient content of N, P, K, Fe, Mn, Zn and Cu in seeds due to bacterial inoculation (Table 3). Nearly in all treatments, plants in A0Z1 plot showed the best performance and increased seed content of N (130%), P (113%), K (100%), Fe (153%), Mn (147%), Zn (107%) and Cu (127%) which was significantly different from non-inoculated control plants.
|Table 1:||Soil chemical properties of the top soil layer (0-30 cm)|
|Table 2:||The effects of inoculation with Azospirillum and Azotobacter on yield and yield component of corn|
|A0: Control, A1: Azotobacter sp. Strain 5, A2: Azotobacter chroococcum DSM2286, Z0: Control, Z1: Azospirillum sp. Strain 21, Z2: Azospirillum lipoferum DSM 1691, Z3: Azospirillum brasilense DSM1690, The means with same letter(s) in column have no differences at the 0.05 probability level|
|Table 3:||The effects of biofertilization with co-inoculated Azospirillum and Azotobacter on plant nutrient element (PNE) in seed of corn|
|A0: Control, A1: Azotobacter sp. Strain 5, A2: Azotobacter chroococcum DSM2286, Z0: Control, Z1: Azospirillum sp. Strain21, Z2: Azospirillum lipoferum DSM 1691, Z3: Azospirillum brasilense DSM1690, The means with same letter(s) in column have no differences at the 0.05 probability level|
Plant growth promoting rhizobacteria (Azospirillum and Azotobacter) used in this study had positive effects on yield and growth parameters of corn grown under field conditions. All bacterial inoculations caused significant increase on growth parameters, such as shoot dry weight, ear and seed dry weight with respect to the control, although differences between various bacterial strains almost were insignificant. The results showed the Azospirillum lipoferum s-21 had the most effect on ear and seed dry weight. Similar findings reported by Dobbelaere et al. (2002), who assessed the inoculation effect of 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 root and shoot.
The potential use of Azotobacter spp. as biofertilizer has been reviewed by Brown (1982), who concluded that inoculation with these microorganisms occasionally promoted yields, probably by mechanisms other than biological N fixation. Also similar results have been reported in Azospirillum spp. (Okon, 1984; Zimmer et al., 1988).
Woodard and Bly (2000) reported that the corn inoculated with A. brasilense increased shoot dry weight and grain yield. In other study, Nandakumar et al. (2001) reported that application of PGPR strains increased the yield of rice. The seed treatment with PGPR resulted in increased plant growth and yield of potato under field conditions (Kloepper et al., 1980). The possible reason might be associated with the initial increase in root growth by the application of PGPR strains, which could promote better absorption of essential nutrients. PGPR synthesize phytohormones that promote plant growth at various stages (Kloepper et al., 1986).
The present experiment revealed that seed co-inoculation with Azospirillum s-21 and Azotobacter sp.-5 resulted in an increased 100-seed weight compared to control. This finding in the present study was supported by Sharaan and El-Samie (1999) finding. They showed that co-inoculated Azospirillum and Azotobacter and some of other PGPR, increased 1000-seed weight, number and weight of seeds per spike in wheat under field condition.
Bacterial inoculation did not influence number of row per ear. Similarly, Zaied et al. (2007) reported that biofertilization of corn with Azospirillum strains did not any significant effect on number of row per ear.
Results have also showed that the number of seed per row increased by inoculation. Naidu et al. (2003) reported that Azospirillum sp. increased the number of tillers, dry matter, number of panicles, number of filled grains and 1000-grain weight in rice under field experiment. Results have also showed that, all of PGPR strains affected the ear length and plant height significantly (Table 2). Azospirillum s-21 was the most effective strain on increase the ear length. Although, Co-inoculation with Azospirillum s-21 and Azotobacter sp-5 had highest plant height than control and also the other strains tested.
Azospirillum inoculation increased the rice yield significantly by 1.6-10.5 g plant-1 (32-81% increase) in greenhouse conditions (Malik et al., 2002). However, in field conditions, the estimated yield increase was around 1.8 t ha-1 (22% increase) as reported by Balandreau (2002). This species can also increase the height and tiller number of rice plants (Nayak et al., 1986). They mentioned that the reason may be due to the increase in photosynthetic ability of the plants with high chlorophyll content due to PGPR treatment. Similarly, the efficacy of PGPR formulations on yield attributes of various crops was reported by Weller and Cook (1983). The other possible reason might be associated with the initial increase in root growth by the application of PGPR strains, which could help in promoting better absorption of essential nutrients that are responsible for high rate of photosynthesis. Better root growth may be also responsible for better synthesis of hormones like auxin and cytokinin which could help in better partitioning efficiency which resulted in increased economic yield. (Dobbelaere et al., 2003; Glick, 1995; Kloepper et al., 1986). A. brasilense alter pH of the rhizosphere (Carrillo et al., 2002) and inoculation with Azospirillum may change root physiology and patterns of root exudation (Heulin et al., 1987).
The present study indicates that, Inoculation with the Azotobacter and Azospirillum significantly increased content of N and P in seed of corn. The higher total N and P uptake of corn indicated that Azotobacter and Azospirillum were able to fix N and solubilize P and consequently promotion of plant growth (Dobbelaere et al., 2001; Lynch, 1990; Marschner, 1995).
Plants inoculated with the PGPR generally have higher N content than the uninoculated plants (Puente et al., 2004). However Azospirillum brasilense and Azospirillum irakense strains stimulated overall plant growth, including root development and grain yield of spring wheat and maize, but both rhizobacteria did not change the N concentration in plants or grains (Dobbelaere et al., 2002).
Azospirillum inoculation can increase PO43_ and NH4+ uptake by rice plants (Murty and Ladha, 1988).
Results showed that all bacterial inoculations caused significant differences on the K, Fe, Mn and Zn contents in seed corn, although differences between various bacterial strains were insignificant. In general, PGPR may affect the initiation and development of lateral roots (Rolfe et al., 1997), increase root weight and nutrient-uptake (Canbolat et al., 2006). Also phosphate and potassium K-solubilizing bacteria may enhance mineral uptake by plants through solubilizing insoluble P and releasing K from silicate in soil (Goldstein and Liu, 1987). This evidence confirms that the percentage of P, K, Ca, Mg, Fe, Mn and Zn and Cu was significantly and/or relatively increased in the bacteria-treated plants. Amending soil with beneficial bacteria able to compensate for nutrient deficiency and maintain at least partly a normal plant development. This biofertilizer therefore may have a potential to decrease the input cost of agricultural production and be applied to the revegetation of low commercial value sites, such as metal tailings ponds (Carlot et al., 2002).
We thank Shahrood university of technology and the Soil Microbiology Department, Soil and Water Research, Tehran for helping to carry out this research.
- Abbass, Z. and Y. Okon, 1993. Plant growth promotion by Azotobacter paspali in the rhizosphere. Soil Biol. Biochem., 25: 1075-1083.
- Falik, F., Y. Okon, E. Epstein, A. Goldman and M. Fischer, 1989. Identification and quantification of IAA and IBA in Azospirillum brasilense inoculated maize roots. Soil Biol. Biochem., 21: 147-153.
- Heulin, T., A. Guckert and J. Balandreau, 1987. Stimulation of root exudation of rice seedlings by Azospirillum strains: Carbon budget under gnotobiotic conditions. Biol. Fertil. Soils, 4: 9-14.
- Jacoud, C., D. Job, P. Wadoux and R. Bally, 1999. Initiation of root growth stimulation by Azospirillum lipoferum CRT1 during maize seed germination. Can. J. Microb., 45: 339-342.
- Kapulnik, Y., M. Feldman and Y. Okon, 1985. Contribution of nitrogen fixed by Azospirillum to the N nutrition of spring wheat in Israel. Soil Biol. Biochem., 17: 509-515.
- Kozdro, J., J.T. Trevors and J.D. Van Elsas, 2004. Influence of introduced potential biocontrol agents on maize seedling growth and bacterial community structure in the rhizosphere. Soil Biol. Biochem., 36: 1775-1784.
- Malik, K.A., M.S. Mirza, U. Hassan, S. Mehnaz, G. Rasul, J. Haurat, R. Bally and P. Normand, 2002. The Role of Plant-associated Beneficial Bacteria in Rice-wheat Cropping System. In: Biofertilisers in Action, Kennedy, I.R. and A.T.M.A. Choudhury (Eds.). Rural Industries Research and Development Corporation Canberra, pp: 73-83.
- Marschner, H., 1995. Mineral Nutrition of Higher Plants. 2nd Edn., Academic Press Ltd., London, New York, ISBN-13: 978-0124735439, Pages: 889.
- Naidu, V.S.G.R., J.D.S. Panwar and K. Annapurna, 2003. Yield response in rice to auxin application and inoculation with Azospirillum brasilense. Indian J. Plant Physiol., 8: 96-98.
- Puente, M.E., C.Y. Li and Y. Bashan, 2004. Microbial populations and activities in the rhizoplane of rock-weathering desert plants. II. Growth promotion of cactus seedlings. Plant Biol., 6: 643-650.
- Suba Rao, N.S., K.V.B.R. Tilak and C.S. Singh, 1985. Synergistic effects of vesicular arbuscular mycorrhizas and Azospirillum brasilense on the growth of barley in pots. Soil Biol. Biochem., 17: 119-121.
- Verma, S., V. Kumar, N. Narula and W. Merbach, 2001. Studies on in vitro production of antimicrobial substances by Azotobacter chroococcum isolates/mutants. J. Plant Dis. Protect., 108: 152-165.
- Zaied, K.A., A.H. Abd El-Hady, A.E. Sharief, E.H. Ashour and M.A. Nassef, 2007. Effect of horizontal DNA transfer in Azospirillum and Azotobacter strains on biological and biochemical traits of non-legume plants. J. Applied Sci. Res., 3: 73-86.