Subscribe Now Subscribe Today
Research Article

Effect of Azotobacter chroococcum Application on Quantity and Quality Forage of Rapeseed Cultivars

S. Ebrahimi , H. Iran Nejad , A.H. Shirani Rad , G. Abbas Akbari , R. Amiry and S.A.M. Modarres Sanavy
Facebook Twitter Digg Reddit Linkedin StumbleUpon E-mail

The study was conducted to determine the effect of Azotobacter chroococcum (Azotobacter) application on quality and quantity forage of the rapeseed cultivars (RGS 003, Hyolla 401 and Hyolla 330) and the possibility of cultivation the rapeseed forage in summer. The experimental design was spilt plot laid out in randomized complete block with three replications. Different levels of Azotobacter (zero and one kg ha-1) and cultivars were randomized to main plot and sub plot units, respectively. Results showed that Azotobacter significantly affected on all quantity traits except plant height. There were significant differences among cultivars for dry matter, biomass, sub branch number, plant height and forage glucosinolate concentration. Therefore Azotobacter can be considered as growth promoting for rapeseed in the future studies. Also, RGS003 cultivar was better than other cultivars when Azotobacter was applied.

Related Articles in ASCI
Similar Articles in this Journal
Search in Google Scholar
View Citation
Report Citation

  How to cite this article:

S. Ebrahimi , H. Iran Nejad , A.H. Shirani Rad , G. Abbas Akbari , R. Amiry and S.A.M. Modarres Sanavy , 2007. Effect of Azotobacter chroococcum Application on Quantity and Quality Forage of Rapeseed Cultivars. Pakistan Journal of Biological Sciences, 10: 3126-3130.

DOI: 10.3923/pjbs.2007.3126.3130



Forage Shortage in Iran has imposed the intense pressure on the pastures and followed by the lateral problems such as soil erosion. Increasing cultivation area is not also possible due to water and land limitation. At present the most ready lands are cultivated, so that increasing yield per area and advantages of the second cultivation that fill the time interval between the production of a winter crop (seed) and its further cultivation, is very important.

Most researchers about forage production such as Goihl and Mcelliney (1994), Neilsen et al. (2002) and Amin et al. (2002) have emphasized on compatibility of rapeseed for forage production. They explain the advantages of rapeseed forage production, when it is placed in the cropping pattern and crop rotation of the region.

Clay Pool et al. (1985) also found that 45 day old Holstein calves weight increased 900 kg daily, when they were feed by rapeseed meal during 7 weeks before weaning and 8 weeks after weaning.

Schwab et al. (2001) reported that damage of rodent on potato and carrot fields was reduced by mixed cropping with forage rape.

Application of rapeseed and barley together can increase digestion of forage in rumen of sheep (Jochmann et al., 2002).

Result of research on field without weed showed that forage rape has the capacity of producing up to 12 ton ha-1 (Anonymous, 2004).

Banuelos et al. (2002) also showed that forage rape is appropriate replacement for other forage plants at dry regions.

Therefore, forage production comparison among different rapeseed cultivars as the second cultivation is very important, because the wasting usage of the chemical fertilizer especially potash, nitrogen and phosphorus fertilizers to increase yield in different crops has caused very problems from the economical and environmental perspectives. It seems that the best way is substitution of biological fertilizers instead of chemical fertilizers.

The biological fertilizers attribute not only to the materials from dung and plant remainders, but also including all products that are made from the activity of the microorganisms and its microorganisms. The soil bacteria such as Azotobacter are one of these microorganisms (Kennedy et al., 2004). The different species of Azobacter can be categorized in the group of the plant growth promoting rhizobacteria (PGPR) that in the medium of the root (rhizosphere), has necessary potential to occupy the root system of the plants (Vessey, 2003).

At present, in some of the countries, this bacteria is used as biological fertilizer to produce very agricultural products including cereals, vegetables (Kadder et al. 2002). Ravikumar et al. (2004) studied the effect of Azotobacter on the plant species in the coastal regions and observed that the plant inoculation with Azotobacter increased plant growth and quantity of chlorophyll in the leaf.

Rodelas et al. (1999) reported that Azotobacter increased the yield of the products like sugar beet, carrot and cabbage as much as 10%. These bacteria also help to preserve the health of the plant by controlling the pathogenic agent indirectly as that growth improvement and the crop yield increment is the ultimate aim (Mrkovacki and Milic, 2001).

Kwon et al. (2003) have reported that rape is a useful forage fodder crop with high content of crude protein and low contents of Neutral Detergent Fibre (NDF).

Prasad and Prasad (2004) showed positive effect of azotobacter on the yield and height of Brassica plant. Gupta and Samnotra (2004) conducted several researches in Kashmir, India and concluded that the simultaneous application of azotobacter and manure had a significant effect on plant height and yield of Brassica.

In another study the effect of azotobacter and other biofertilizers were examined on the vegetative growth (shoot length, fresh and dry weights and number of leaves per plant) of mustard. Azotobacter, applied alone, resulted in the highest values for the parameters measured (Abdul et al., 2003).

With respect to above explanation, determining the effects of growth stimulating bacteria on the crop yield in the soils of every region are completely necessary for extension the usage of the biological fertilizers and sustainable agriculture. Therefore the purpose of the present study was to evaluate Azotobacter effects on growth, development and yield of tested varieties.


This research was conducted in Mahdasht of Karaj at a field with 3000 m2 area during growing season 2005. The soil texture was clay loam with pH 7.4. The experimental design was split plot laid out in randomized complete block with three replications. Two levels of the Azotobacter (supplied by Mehr Asia Biotechnology Company, Tehran, Iran, P.O. Box 15875-9498) (0 as control and 1 kg ha-1) and three cultivars (Hyolla 401, RGS 003 and Hyolla 330) were randomized to the main and sub plot units respectively. In this experiment, every sub plot had 6 lines with 3 m long and the interval between the plots was 1 m. At the end of June, land preparation was done with disk to break the clods, leveling the land by leveler and creating the stream and the mound by furrower. Weed was controlled by the terflan herbicide (2-2.5 L ha-1) before cultivation and it was blended with the soil.

Seeds were inoculated with Azotobacter in 2005/June/20. Then inoculation and control seeds were planted directly to soil. The first irrigation was done immediately after seed planting. All treatments were irrigated every 7 days once until harvesting time. The harvesting area was 2.4 m2 per plot. When 25% of the plants had flower they were harvested.

Before final harvesting, 10 plants were selected randomly from each plot and the number of sub branch and plant height was measured. Forage dry and wet weights of all four rows per plot were determined after omitting the effect of the margin (half meter from the beginning and the end of each line).

The raw protein percent (with Kjeldahl method) ash percentage (by the method of burning in the electrical jug with the temperature 500°C for 5 h), organic material percent (the ash-the dry material = the organic material) were determined and glucosinolate concentration (quantity of glucose in the sample by spectrophotometer). The data were subjected to statistical analysis using SAS statistical software (SAS Institute Inc., 1997). When analysis of variance showed significant treatments effects, Duncans multiple range test was used to analyze the mean at p≤0.05.


Analysis of variance showed that the variety and Azotobacter had significant effects on the biomass, so that the inoculated plants with Azotobacter produced more wet forage (39.701 ton ha-1) than control (Table 1). Kennedy (2004), Ravikumar et al. (2004) and Rodelas et al. (1999) reported that the yield in different crops could be increased when crops were inoculated with Azotobacter. Mean comparison among varieties showed that RGS 003 had higher forage (46.957 ton ha-1) than other varieties. This can be attributed to the high genetic potential of RGS 003 cultivar. Some researchers such as McGregor (1987) and Morrison et al. (1997) also attributed difference between varieties for forage to the difference of their genotypes.

The effect of Azotobacter and cultivar on dry weight was significant, but their interaction effect was non significant for dry weight (Table 1).

Table 1: Analysis of variance on traits of inoculated canola cultivars with Azobacter
* and ** significant at 5% and 1% probability levels respectively; ns: Non-significant; V: Variety (V1 = Hyola 401, V2 = Hyola 330, V3 = RGS 003, A: Azotobacter (A1 non application, A2: application)

Table 2: Means comparison for yield and quality in, variety and use of Azotobacter
Mean fallowed by the same letter(s) in each column are not significantly different (Duncan multiple rang test 5%); V: Variety (V1 = Hyola 401, V2 = Hyola 330, V3 = RGS 003); A: Azotobacter; (A1: non application, A2: application)

Inoculated plants with Azobacter had more dry weight (7.365 ton ha-1) than control (Table 2). The researchers like Narula et al. (2000), Ahmad et al. (2005) and Ravikumar et al. (2004) reported that plant inoculation with Azotobacter had positive effect on the dry weight and it was attributed to Azotobacter that stimulated growth hormones production. Dry matter was difference among cultivars. RGS 003 cultivar produced more dry matter than other cultivars.

There were significant differences among cultivars for plant height. Azotobacter did not affect on this trait. The height difference of cultivars is related to their genetic background. Hyolla 401 cultivar was taller than others, but it was ranked with Hyolla 330 in a statistical group (Table 2).

Varieties had different sub branch number (p≤0.01). Inoculated plants with Azotobacter had more sub branch (5.4) number than control, but it was not statistical significant. Barea and Azcon (1975) also reported that sub branch number enhancement could be attributed to production of growth stimulation hormone by Azotobacter. Hyolla 330 cultivar had the highest sub branch number and it was ranked with Hyolla 401 cultivar in two separately statistical groups. RGS 003 had the lowest sub branch number. It was located at third rank. Probably, this difference has the genetic backgrounds (Table 2).

Protein percent was not different among cultivars. This trait was not also affected by Azotobacter (Table 1). Zamber et al. (1984) reported that plant inoculation with Azotobacter increased forage protein percent, but Zaied et al. (2003) observed that seed protein percent in inoculated wheat plants with Azotobacter did not increased.

Cultivars had different glucosinolate. This trait was the lowest in RGS003 cultivar (Table 2). Scientific reports show that herbaceous species of Brassica as naturally and originally have the poison composition that glucosinolate is one of them. The difference in the quantity of glucosinolate rises from genetic difference among the cultivars (Gustine and Jung, 1985). Brassica forage had the lowest glucosinolate concentration (Tookey et al. 1980). The researchers have proved that the most quantity of glucosinolyate was in the non-herbaceous species of Brassica, primary seeds and brassica meal (Vanetten and Daxenbichler, 1977).

In this experiment, the treatments of Azotobacter, cultivar and the interaction effect of the cultivar* Azotobacter did not influence on ash percent. In the standard table (NRC, 2001) researchers has reported that the quantity of ash in the forage of rapeseed is 11.4% of the dry matter, whereas our record showed that ash quantity in rapeseed forage was 17%. Plant population density, urea fertilizer application, type and quantity of the irrigation, sowing season and time of harvest influence effectively on the quality traits (Chao et al., 1998). In the standard table, the harvesting time of product was in the beginning of vegetative growth period, but in this experiment forages were harvested in the beginning of flowering period that perhaps, this case has caused to increase the ash in the forages of varieties.

None of the used treatments did not significantly influence on the organic material percent (Table 1). In the standard table organic material and hay of the rapeseed forage (NRC, 2001; MAFF, 1990) have been reported 88.6 and 91.2, respectively. The little difference in the organic material of our rapeseeds with standard table is due to harvesting time: harvesting time of rapeseed plants in the standard table was in the beginning of the germination, whereas the harvesting time of our rapeseeds was at flowering.


Generally, plant growth promoting rhizobacteria including Azotobacter has advantages in compared to the chemical fertilizers that justify its usage. Environmental pollution reduction, less cost, simple availability, power of reproduction and propagation are the major important factors of growth stimulating bacteria and biological fertilizers in compared to chemical fertilizers.

RGS 003 cultivar had the highest forage yield and the best forage quality. Therefore mentioned cultivar can be used to cultivate in Summer.


We thank to all workers of the lab in the oilseed section of the Seed and Plant Improvement Institute Research of Karaj.

1:  Mir, A.H., S. Hayat and I. Ahmed, 2003. Performance of biofertilizers inoculation to Indian mustard. Brassica, 5: 58-63.

2:  Ahmad, F., I. Ahmad and M.S. Khan, 2005. Indole acetic acid production by the indigenous isolates of Azotobacter and fluorescent Pseudomonas in the presence and absence of tryptophan. Turk. J. Biol., 29: 29-34.
Direct Link  |  

3:  Amin, E.A., A.I. Ghazy and M.K. Tahoun, 2002. The influence of Brassica species and accession on productivity and nutrient quality of forage rape in Egypt.

4:  Anonymous, 2004. Effect of nitrogen on canola plant growth.

5:  Banuelos, G.S., D.R. Bryla and C.G. Cook, 2002. Vegetative production of kenaf and canola under irrigation in central California. Ind. Crops Prod., 15: 137-145.
CrossRef  |  

6:  Barea, J.M. and R. Azcon, 1975. Synthesis of auxins, gibberellins and cytokinins by Azotobacter vinelandii and Azotobacter beijerinckii related to effects produced on tomato plans. Plant Soil, 43: 609-619.

7:  Chao, N.K., W.J. Jin, B.K. Kang and Y.M. Park, 1998. Effect of seeding rate on growth yield and chemical composition of forage rape cultivars. Kor. J. Crop Sci., 43: 54-58.

8:  Clay Pool, D.W., C.H. Hoffman, J.E. Old Field and H.P. Adams, 1985. Canola meal, cotton seed and soybean meals as protein supplements for calves. J. Dairy Sci., 68: 67-70.

9:  Goihl, J.H. and R.R. Mcelliney, 1994. Feed Industry Red Book, Edin Prairie. Comm. Marketing, Inc., MN.

10:  Gupta, A.K. and R.K. Samnotra, 2004. Effect of biofertilizers and nitrogen on growth, quality and yield of cabbage (Brassica n.). Environ. Ecol., 22: 551-553.

11:  Gustine, D.L. and G.A. Jung, 1985. Influence of some management parameters on glucosinolate levels in Brassica forage. Agron. J., 77: 593-597.

12:  Jochmann, K., B.S. Ozyurt, P. Lebzinen and G. Flanchowsky, 2002. Influence of pressure and temperature on in sacco degradation of dry matter and crude protein of barly rape mixtures in the rumen sheep. Landbauforschunq Volkenrode, 50: 50-53.

13:  Kadder, A., M.H. Main and M.S. Hoque, 2002. Effects of azotobacter inoculants on the yield and nitrogen uptake by wheat. Online J. Biol. Sci., 2: 259-261.
Direct Link  |  

14:  Kennedy, I.R., A.T.M.A. Choudhury and M.L. Kecskes, 2004. Non-symbiotic bacterial diazotrophs in crop-farming systems: Can their potential for plant growth promotion be better exploited? Soil Biol. Biochem., 36: 1229-1244.
CrossRef  |  Direct Link  |  

15:  Kwon, B., J. Lim, J. Shin and A. GaeSoo, 2003. Differences in nutrient quality among rape varieties for oil seed and forage. Kor. J. Crop Sci., 48: 372-375.

16:  MAFF, 1990. Table of Nutritive Value and Chemical Composition of Feeding Stuffs for farm Animals. Summary of the Recommendations of a Working Party Sponsored by the Ministry of Agriculture, Fisheries and Food. MAFF Publications, London, pp: 17.

17:  McGregor, D.L., 1987. Effect of plant density on development and yield of rape seed and its significance to recovery from hail injury. Can. J. Plant Sci., 67: 43-51.

18:  Morrison, J.E., R.W. Rickman and K.L. Pfeiffer, 1997. Measurement of wheat residue cover in the Great Plain and Pacific Northwest. Agriculture. Ecosyst. Environ., 39: 187-196.

19:  Mrkovacki, N. and V. Milic, 2001. Use of Azotobacter chrooccocum as potential useful in agricultural allocation. Ann. Microbiol., 51: 145-158.

20:  Narula, N., V. Kumar, R.K. Behl, A. Deubel, A. Gransee and W. Merbach, 2000. Effect of P-solubilizing Azotobacter chroococcum on N, P and K uptake in P-responsive wheat genotypes grown under greenhouse conditions. J. Plant Nutr. Soil Sci., 163: 393-398.
CrossRef  |  Direct Link  |  

21:  Neilsen, J.E., B.A. Rowe and P.A. Lane, 2002. Leaf area development and growth of two irrigated Brassica forage crops.

22:  NRC, 2001. Nutrient Requirement for Dairy Cattle. Academy Press, Washington, DC.

23:  Prasad, M. and R. Prasad, 2004. Effect of mepiquat chloride, Azotobacter and nitrogen on cotton (Gossypium hirsutum) and their residual effect on succeeding transplanted Indian mustard Brassica juncea. Indian J. Agric. Sci., 74: 273-275.
Direct Link  |  

24:  Ravikumar, S., K. Kathiresan, S.T.M. Ignatiammal, M.B. Selvam and S. Shanthy, 2004. Nitrogen-fixation Azotobacters from mangrove habitat and their utility as marine biofertilizers. J. Exp. Mar. Biol. Ecol., 15: 157-160.

25:  Rodelas, B., J. Gonzalez-Lopez, C. Pozo, V. Salmeron and M.V. Martinez-Toledo, 1999. Response of Faba bean (Vicia faba L.) to combined inoculation with Azotobacter and Rhizobium leguminosarum bv. viceae. Applied Soil Ecol., 12: 51-59.
CrossRef  |  Direct Link  |  

26:  SAS Institute, 1996. SAS/STAT User`s Guide. 2nd Edn., SAS Institute Inc., Cary, NC.

27:  Schwab, F.E., F.G. Pittoello and N.P.P. Simon, 2001. Relative palatability of green manure crops and carrots to while tailed deer. Wildlife Soc. Bull., 29: 317-321.

28:  Tookey, H.L., C.H. Vanetten and M.E. Daxenbichler, 1980. Glucosinolate. In: Toxic Constituents of Plant Food Stuffs, Liener, I.E. (Ed.), Academic Press, New York, pp: 103-142.

29:  Vanetten, H.L. and M.E. Daxenbichler, 1977. Glucosinolates and derived products in crucifers vegetables: Total glucosinolates by retention on anion exchange resin and enzymatic hydrolysis to measure released glucose. J. Assoc. Anal. Chem., 60: 946-949.

30:  Vessey, J.K., 2003. Plant growth promoting rhizobacteria as biofertilizers. Plant Soil, 255: 571-586.
CrossRef  |  Direct Link  |  

31:  Zaied, K.A., A.H. Abd El-Hady, H. Aida Afify and M.A. Nassef, 2003. Yield and nitrogen assimilation of winter wheat inoculated with new recombinant inoculants of rhizobacteria. Pak. J. Biol. Sci., 6: 344-358.
CrossRef  |  Direct Link  |  

32:  Zamber, M.A., B.K. Konde and K.R. Sonar, 1984. Effect of Azotobacter chroococcum and Azosprillum brusilense inoculation under graded levels of nitrogen on growth and yield of wheat. Plant Soil, 79: 61-67.

©  2021 Science Alert. All Rights Reserved