Abstract: In order to evaluate the effect of additional application of Azotobacter and Azospirillum inoculants (Biofertilizers) on canola (Brassica napus L.) yield and profitability, a split-plot experimental design with 20 treatments was carried out during 2004-2005 with four replications in the North of Iran. High yielding canola (cv. Hyola 401 hybrid), was grown in rotation after wheat. Two levels of biofertilizers as control and seeds inoculation in main plot and 10 treatments of chemical fertilizers comprising N, P, K and their combinations, NPKS and NPK Zn in sub plots were applied. The treatment T20 resulted in the maximum seed yield (3374 kg ha-1) coinciding with the maximum number of pods per plant (246 pods per plant) followed by the treatments T19, T18 and T15. Out of these 4 treatments, it was discovered that the highest net benefit of adding biofertilizers was observed at T15 (1.07 million rials.ha-1 = 117.7 $.ha-1). The research projects how the efficiency of these biofertilizers was maximum in presence of N and P fertilizers, while in the presence of K and Zn fertilizers at T20 it resulted mainly in the increase of fodder rather than seed. The seed N, protein and the oil percentage remained unaffected by biofertilizers application.
INTRODUCTION
Canola (Brassica napus L.) growing has become attractive to the farmers in Iran, as an indigenous source of vegetable oil and animal meal. A healthy canola plant is considered as nitrogen demanding commercial crop and it has been shown that an addition of 135 kg ha-1 nitrogen would significantly increase the canola yield. Although the application of nitrogen fertilizer along with the others chemical fertilizers have proved to be essential but their higher application may result in environmental disasters like NO-3 pollution of ground water, soil acidification and increased denitrification resulting in higher emission of N2O to the atmosphere, which may impact global warming. These problems have renewed public interest in exploring alternate or supplementary non-polluting sources of N for agriculture. Finding an alternative for such a nutrient has become important. Soil microorganisms like Azotobacter and Azospirillium are free living N2 fixing bacteria which can successfully grown in the rhizospheric zone of crops and fix 10-20 kg N ha-1 cropping season. Besides N2 fixation these bacteria synthesize and secret considerable amounts of biologically active substances like gluconic acid and the ability of direct phosphate solubilization which enhance root growth of plants (Rodriguez et al., 2004). Azotobacter along with other N2 fixing bacteria like rhizobium play important role in yield-attributing characters owing to the production of siderophores which regulates the availability of nutrients to the crop (Boiero et al., 2007).
Rock et al. (1996) examined Rhizobium leguminosarum bv. phaseoli and Pseudomonas strains for their plant growth-promoting potential on lettuce and forage maize. The plants were grown in field conditions in the site having very fertile soil tended to increase the dry matter yield of lettuce shoots (p≤0.10). Lettuce inoculated with rhizobia had a 6% higher P concentration (p≤0.10) than the uninoculated control. In moderately fertile soil the dry matter of maize shoots was significantly increased (p≤0.05) by inoculation with strain 24 plus 17.5 kg ha-1 P-superphosphate, or with strain P31 plus 35 kg ha-1 P-superphosphate. Inoculation with PSM did not affect lettuce P uptake in the less fertile soil but in the moderately fertile soil, maize plants inoculated had 8% higher P concentration than the uninoculated control (p≤0.01). They have concluded that rhizobia function as plant growth promoting rhizobacteria with the nonlegumes lettuce and maize.
Shukla et al. (2002) reported that application of Azotobacter resulted significantly in higher number of seeds/siliquae, TDM, branches/plant and the length of siliquae in Indian mustard. Sharma et al. (1997) reported that the oil content of Indian mustard decreased by successive increasing in N level and application of Azotobacter, suggesting that oil and protein production increased significantly when nitrogen applied either through Azotobacter or urea. Cecilia et al. (2004) reported wheat grains harvested from Azospirillium-inoculated plants contained significantly higher Mg, K and Ca than non-inoculated plants. They also proved that grain yield loss to drought was 26.5 and 14.1% in non-inoculated and Azospirillium-inoculated plants respectively. Khalid et al. (2004) reported that Peat-based seed inoculation with selected PGPR isolates exhibited stimulatory effects on grain yields of tested wheat cv. in pots (up to 14.7% increase over control) and field experiments (up to 27.5% increase over control).
Not much experimental work has been conducted on the use of such N2 fixing bacteria on the growth and yield of canola. The only attempts made on canola refer to the application of inoculation with Penicillium bilaji, Bacillus thuringiensis and Phosphate solubilizing Rhizobacteria for the P-uptake, vegetative growth and grain yield of canola (Freitas et al., 1997). It seems that finding an alternative factor to reducing the harmful effects of nitrogen exceed application in the environment as well as keeping the production level for crops is essential, therefore the study of the effects of Biofertilizers (Azotobacter and Azospirillium) in the growth and productivity of canola have been conducted.
MATERIALS AND METHODS
A split-plot experimental design with 20 treatments and 4 replications was carried out during 2004-2005 cropping season. The canola (cv. Hyola 401 hybrid), a high yielding early maturity canola hybrid, was grown during the months October to May, which is generally a humid season in Northern Iran. The area receives an average of 700-800 mm rain fall and has a relative humidity of 77%. Bacterial strains of Azospirillum and Azotobacter inoculants were applied in the main plots and a combination of chemical fertilizers, both macro and micronutrients were applied in the sub plots. The experiment was carried out at the Baiecola Agricultural Research Station in Mazandaran province (Iran).
The strains of Azotobacter and Azospirillum were isolated from the different samples of the soils of local area. To facilitate the identification of Azotobacter spp., modified mannitol agar medium with 10 g of glucose mannitol per liter as a carbon source was used. For Azospirillum NFb a potato extract media was used. The isolates, thereafter were compared with the reference strains.Combined inoculants of Azotobacter chroococcum, Azospirillum lipoferum and Azospirillum brasilense strains were applied for the biofertilizer treatments.
The canola crop was taken as a second crop in rotation after wheat. It was grown under rain fed conditions. Soil samples were collected and analyzed to know the composition or the nutrients availability and the crop requirements for the nutrients. The chemical fertilizers were chosen and applied accordingly. The experimental soil was texturally silt-clay, with pH 7.6, 1.3% OC, 180 ppm of available K, 7 ppm of available P, 18 ppm Mn, 10 ppm Fe, 1.1 ppm B and 0.96 ppm of Zn. The chemical fertilizers consisting N, P, K, S and Zn were applied prior to cultivation, except the nitrogen fertilizer which was applied in split stages, once basal and twice top-dressed.
The experimental field was divided into 4 blocks. Each block was then divided into 20 plots in all the experiment was done in 80 plots of each of 10 m2 area. For calculating the seed oil, protein and N content number of seed were sampled separately from each treatment, the seed oil and protein content were measured following Nuclear Magnetic Resonance Spectrophotometry (NMR) and Micro Kjeldahl digestion using automated colorimetric analysis, respectively. Data were analyzed following the analysis of variance technique (ANOVA) and then the mean differences were adjudged by Duncan`s Multiple Range Tests (DMRT).
RESULTS AND DISCUSSION
Yield
Application of chemical fertilizers on yield (Treatments T2
to T10): The effect of chemical fertilizers on yield was
statistically significant (p<0.01). The application of N and P fertilizers
at treatments T2 and T3 did enhance the yield by
148 and 133% over the control (Table 1). Favorable reports
exist on canola seed yield by application of N fertilizer (Ozer, 2003;
Hocking et al., 2003) and P being a structural component of nucleic
acid and protein and nucleoprotein its application does favor significantly
the seed yield, LAI and TDM in canola and other brassica species
(Lickfett et al., 1999).
The present experiment indicated that the yield can be augmented (Table 1) substantially, above 3000 kg ha-1 by a mixture of NPKZn (T10) and NPKS (T9) but applying NPK alone (T8) the yield remained below that level. The supplementary addition of Zn and S increased the yield marginally, but these three treatments show the same statistical rank. In certain soils the application of S alone or in combination with NPK has also favored substantially the increase in canola yield (Jayan and Patro, 1999; Hocking and Strapper, 2001; Santonoceto et al., 2002).
Application of biofertilizers (treatments T12 to T20): Application of biofertilizers alone had little effect on the yield (T11) but when applied in conjunction with N (T12) and P (T13) or with NP (T15) it made a tremendous difference in the number of pods per plant that is reflected in the seed yield (Table 1). On the other hand, the application of biofertilizers with K (T14) and with PK (T17) had only little effect on the number of pods per plant as also on the seed yield. The yield obtained was even lower than when N (T12) and P (T13) fertilizers were applied individually with biofertilizers. The number of pods per plant and the yield obtained at T15 far exceeded those obtained at T16 and T17 where biofertilizers were applied with NK and PK fertilizers. Thus the efficiency of biofertilizers in the presence of K fertilizer seems to get subdued, which is also seen at T18. The seed yield obtained at T19 (BF+NPKS) and T20 (BF+NPK Zn) exceeded that at T15, reflected also in the increase in the number of pods per plant.
Positive reports of application of biofertilizers (Azotobacter and Azospirillum and other bacteri) on yield are available on crops like: Indian mustard (Suneja and Lakshminaraya, 2001), cotton (Yue et al., 2007), corn (Albrecht et al., 1981), sorghum (Singh et al., 2005) wheat (Cecilia et al., 2004), tobacco (Li et al., 2007) and barley (Ozturk et al., 2003), which is attributed to the enhancement of factors like N2 fixation nitrate reductase activity, intake of NO3, NH4, H2PO4, K and Fe, plant water status and production of phytohormones such as Indol acetic acid (Wani et al., 1998; Antoun et al., 1998; Arshad and Frankenberger, 1998). It is possible that the enhancement of such factors has been instrumental in the present experiment also, especially at treatments T15, T19 and T20.
Economics: The expenditure incurred per ha for applying different chemical fertilizers varied (Table 2) and becomes costlier every year, but the additional cost of biofertilizers is only marginal. The selling price per kg, as fixed by the Government of Iran, though increases every year, is not sufficient enough to sustain. The yield obtained at most of the treatments exceeds the average canola yield in the world (1750 kg ha-1) and in Iran (1640 kg ha-1), the role played by biofertilizers is visible in all treatments T12 to T20. For achieving the yield comparable to the average level of canola yield in Germany (4133 kg ha-1) and France (3540 kg ha-1), the application of biofertilizers in conjunction with N, P and Zn seems to be the only alternative.
| Table 1: | Effect of chemical and biofertilizers on yield, pods per plant and cost of fertilizers |
| BF = Biofertilizers; alphabets represent statistical
similarities *1 m.Rials = 110 $ |
|
| Table 2: | Monitory benefits (million Rials) at different treatments |
| *1 million Rials = 110 $, **Taking 9.5 million Rials as the cut off value for recognizing profitable treatment by using chemical fertilizers, ***10.5 million Rials as the cut off value for treatments using additional biofertilizers | |
If the benefit is calculated by subtracting the investment cost from the sale value it is maximum at T20 and considering 9.5 million rials.ha-1 as the cut off value one may recognize T5, T8, T9 and T10 as profitable treatments with application of only chemical fertilizers. The cut off value for treatments using additional biofertilizers would be above 10.5 million rials.ha-1 (Table 2). Considering that the treatments T15, T18, T19 and T20 are profitable. If the benefit is viewed in terms of improvement in yield (Table 1), consequent to adding biofertilizers, the results obtained at T15 could be adjudged better than those at T18, T19 and T20.
The efficiency of application of biofertilizers towards nutrient uptake by canola plant is betrayed in the improvement of important morphological and physiological characteristics (Table 3, 4) as well, at treatments T15 and T20.
CONCLUSION
Comparing the results obtained at T15 (Biofertilizers + NP) and T20 (Biofertilizers + NPK and Zn), it is observed that (Table 3, 4) application of biofertilizers was more effective at T15 for seed yield augmentation and oil yield/ha. The seed yield increased from 2622 kg ha-1 at T5 (NP) to 2911 kg ha-1 at T15. At T5 it already showed an increase of 256% over the control (T1), but at T15 the increase was 217% over T11 and 295% over T1. Though the highest seed yield (3374 kg ha-1) was obtained at T20, the improvement over T10 (NPK Zn) was lesser than that at T15 over T5. The treatment T10 already showed an increase of 319% over T1, but the addition of biofertilizers at T20 resulted in an increase of 268% over T11 and 358% over T1.
The increase in the yield at T15 and T20 was consequent to the greater proliferation of the number pods per plant. At T15 the number of pods per plant (196) showed an increase of 9.49% over T5 and 117% over T11. At T5 the number of pods per plant already showed an increase of 138% over control (T1). At treatment T20 the number of pods per plant (249) showed an increase of 8.26% over T10 and 176% over T11. At T10 also an increase of 206 % was observed over control T1. Therefore it is concluded that the increase in the number of pods per plant at T15 over T5 (9.49%) was more than the increase at T20 over T10 (8.26%). Among the four useful treatments T15, T18, T19 and T20, though the minimum number of pods was obtained at T15 (196), the percent increase in the number of pods by using additional biofertilizers was maximum. The achievement of 196 pods per plant was concluded to be essential for obtaining the seed yield beyond 2900 kg ha-1, which is almost doubled the average seed yield of the Mazandaran Province.
The application of biofertilizers decreased substantially the Harvest Index, which is the ratio of economical yield (seed yield) to total plant biological yield including seed yield also at both the T15 (22.46%) and T20 (20.56%) when compared with that at T5 (22.86%) and T10 (22.87%), respectively. But the percentage of lowering the HI at T15 was less than that at T20. The higher HI indicates a corresponding increase in seed yield rather than stover, which seems to have been promoted more at T20. While the increase in HI at T5 and T10 over control (T1) was almost similar the biofertilizers inoculation promoted the seed yield at T15 compared to T20. The TDM at T20 showed a remarkable increase compared to only a marginal increase at T15 (Table 3, 4).
| Table 3: | Plant yield and yield attributing characters at pair T10/T20. |
| Table 4: | Plant yield and yield attributing characters at pair T5/T15. |
The plant height is an important contributing factor towards higher TDM. Among the treatments T15, T18, T19 and T20 the maximum plant height was achieved at T20 (135 cm), which coincided with the maximum TDM (11940 kg ha-1). At T15 the plant height was 118 cm, which is minimum among these four treatments. Obtaining the seed yield beyond 2900 kg ha-1 required to having taller plants (≥118 cm), which can be considered as a bio-indicator for having a healthier plant growth and high seed yield. However plant height alone cannot be a sufficient indicator for expecting a high yield, the application of NP fertilizers together with biofertilizers is essential.
The maximum seed oil content (46.11%) was obtained at T20, which showed an increase of 0.61% over T10. At T15 however, the seed oil percent (44.88%) is lesser than at T20, showing an increase of 0.51% over the respective treatment of T5. When the oil yield is calculated it showed a substantial increase of 11.54% from 1170 kg ha-1 at T5 to 1350 kg ha-1 at T15. At T20 although the maximum oil yield (1556 kg ha-1) was obtained, the percent increase in oil yield over that at T10 was only 9.96%.
Seed protein content is useful for animal feed after oil extraction and is depended on the seed-N contents. The maximum seed-N (4.12%) and protein content (25.75%) was obtained at T15. The percent increase in seed-N at T15 over T5 (7.02%) was more than that at T20 over T10 (5.61%). Similarly the percent increase in seed protein content at T15 over T5 (7.02%) was more than at T20 over T10 (5.16%).
Considering the above discussion it is concluded that using combined inoculants of Azotobacter chroococcum, Azospirillum lipoferum and Azospirillum brasilense as biofertilizers in conjunction with NP fertilizers at T15 was more profitable than using such biofertilizers along with NPK Zn together at T20 in enhancement of canola seed yield.