Abstract: For investigating the effect of chemical fertilizer as well as biofertilizers on seed yield and quality i.e. oil, protein and nutrients concentration of rapeseed (Brassica napus L.), a split-plot fertilizers application experimental design in 4 replications was carried out during the 2005-2006 growing season, at the Gharakheil Agricultural Research Station in the Mazandaran province of Iran. Rapeseed was grown as a second crop in rotation after rice. Biofertilizers treatments were two different levels: control (no seed inoculation) and seeds inoculation with a combination of Azotobacter chroococcum and Azosprillum brasilense and Azosprillum lipoferum, as main plot and chemical fertilizers comprised N, P, K and their combinations, NPKS and NPK Zn as sub plots. The maximum value of seed yield obtained at (BF+NPK Zn) 3421.2 kg h-1 corresponding to 244.5 pods per plant and maximum concentration of Zn in leaves as well as seeds. The highest weight of 1000 seeds (4.45 g) happened to obtain at (BF+NPK S) which coinciding with the maximum K levels in leaves. The highest number of branches was obtained at (BF+NPK Zn) with 4.43 branches per plant i.e., 46.2% increase over the control. The maximum value of rapeseed oil content 47.73% obtained at T16 (BF+NK) but maximum protein concentration of seed obtained at T12 (BF+N). Overall the results indicated that inoculation resulted in increase in seeds yield (21.17%), number of pods per plant (16.05%), number of branches (11.78%), weight of 1000 grain (2.92%), oil content of seeds (1.73%) and protein (3.91%) but decrease (-0.24%) in number of seeds per pods comparing to non-Biofertilizers treatments. Irrespective to the treatments, results showed that application of Biofertilizers coincided with 3.86, 0.82, 2.25, 0.75 and 0.91% increase in concentrations of N, P, K, S and Zn in the seeds over the non-Biofertilizers treatments.
INTRODUCTION
Amongst the factors that influence rapeseed growth and yield, the choice of fertilizers and provision of conditions for optimum nutrient availability is of paramount importance since rapeseed plant is very efficient at taking up nutrients. This efficiency is reflected in the high nutrient contents in rapeseed leaf and seed relative to other crops. While on one hand it means that the vegetative matter and the seed are healthy for animal feed, taking a rapeseed crop as a rotation crop depletes the soil nutrients. Problems arising out of such depletion are overcome to a great extent by returning dry matter to the soil. Rapeseed is also viewed as a cleaning crop as it provides a break in the cycle of the pests and diseases hosted by other crops and for managing grass weeds. Using a judicious combination of chemical fertilizers as well as biofertilizers to obtain the best results can be strategic step.
Positive reports of application of biofertilizers (Azotobacter and Azosprillum and other bacteria) 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), mustard (Sharma et al., 1997), wheat and mustard (Gupta Pramila and Gupta Vishal, 2006), tobacco (Li et al., 2007) and barley (Ozturk and Sahin, 2003), which are mainly 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 (Antoun et al., 1998; Arshad and Frankenberger, 1998; Wani et al., 1998).
Since attention has seldom been given to the nutrient requirement of the soil, rapeseed crop has suffered in most cases, either from lack or excess of these nutrients. Hence, the necessity to evaluate the best nutrient uptake for improving rapeseed yield, quality and fortification in Mazandaran crops, based on soil test results.
MATERIALS AND METHODS
In order to evaluate the rapeseed growth and nutrient uptake a split-plot experimental design with 20 treatments and 4 replications was carried out in 2005-2006, where Azosprillum and Azotobacter inoculants were as main plot and combination of chemical fertilizers including macro and micronutrients were as sub plots in Gharakheil Agricultural Research Station in Mazandaran province (Iran). Azotobacter and Azosprillum strains were isolated from the different samples of the soils of local area. To facilitate the recognition of Azotobacter sp., modified Mannitol agar medium with 10 g of glucose, Mannitol per liter as a carbon source (Thompson and Skerman, 1979) and for recognition of Azosprillum NFb and potato extract media were used. The isolates were compared with reference strains. Combined inoculants of Azotobacter chroococcum, Azosprillum lipoferum and Azosprillum brasilense strains were used as Biofertilizers treatments.
Rapeseed (cv. PF 7045.91 or Sarigol), was grown under rain fed condition. The experimental soil was texturally silt-clay, with pH 7.2, 1.5% OC, 193 ppm of available K, 8.7 ppm of available P, 19 ppm Mn, 11 ppm Fe, 1 ppm B and 1 ppm of Zn. The chemical fertilizers consisting N, P, K, S and Zn were incorporated with soil prior to cultivation, except nitrogen fertilizer which was applied in split into basal and top-dressed in two times. Treatments were consist of: T1 = control, T2 = N, T3 = P, T4 = K, T5 = N.P, T6 = N.K, T7 = P.K, T8 = N.P.K, T9 = N.P.K.S, T10 = N.P.K.Zn, T11 = Biofertilizers, T12 = Biofertilizers+N, T13 = Biofertilizers+P, T14 = Biofertilizers +K, T15 = Biofertilizers+NP, T16 = Biofertilizers +NK, T17 = Biofertilizers+PK, T18 = Biofertilizers+NPK, T19 = Biofertilizers +NPKS and T20 = Biofertilizers+NPKZn.
RESULTS
Yield and yield components: The yield and yield components were all influenced significantly by the treatments (Table 1). The effect on rapeseed yield was statistically significant at p<0.01, the best results obtained at (BF +NPK Zn) touching 3421.2 kg h-1 i.e., almost five times increase over the control.
The effect on the number of pods per plant was also statistically significant p<0.01 (Table 1), showing a definite improvement. The best result obtained at (BF+NPK Zn) with 244.5 pods per plant, applying Biofertilizers along with NPK Zn, showing a 138% increase over the control. Result also showed that BF+NPK Zn coinciding with 51% increase comparing to non-Biofertilizers treatment of NPK Zn.
The increase in the number of seeds per pod (of main stem) was also significant. The number of seeds per pod did not change much from the control with the different treatments (LSD = 1.671) except at NPK, BF + NPKS and BF+NPK Zn. With the application of Zn along with NPK, the best result with 25.88 seeds per pod showed a distinct increase of 11.21% over the control. However seeds inoculation with the Biofertilizers did resulted in more increase in the number of seeds per pod compared to the chemical fertilizer treatments. The results indicated that an increase in the yield achieved was consequent mostly to the proliferation in the number of pods per plant. A similar observation was made by Hocking et al. (2003) in experiments using N fertilizer in canola growing.
The number of branches appears to be statistically significant at p<0.05
levels (Table 1), the best result was obtained at BF+NPKZn
i.e., 46.20% increase over the control.
| Table 1: | Effect of Biofertilizers and chemical fertilizers on rapeseed yield, yield component and seed oil and protein contents |
| N: Nitrogen fertilizer (Urea at 250 kg h-1), P: Phosphorus fertilizer (Triple super phosphate at 150 kg h-1), K: Potassium fertilizer (Potassium sulfate at 150 kg h-1), S: Sulphur fertilizer at 100 kg h-1), Zn: Zinc sulfate at 40 kg h-1), BF: Biofertilizers (combination of Azotobacter chroococcum and Azosprillum brasilense and Azosprillum lipoferum), LSD: Least significant differences at 0.05 level, Values with different letter(s) indicate significant difference from each other | |
The effects on 1000 grain weight thought positive but did not show much difference between different treatments. The best result was at the BF+NPKS i.e., 19.30% increase over the control, but was not statistically different from the other treatments except the control. Despite the differences in the number of pods and the yield with every treatment, the 1000 grain weight remained between 4 and 4.45 g. This appears to be the range only up to which the improvement could be achieved by the treatments. Considering this narrow range of variation, it is obvious that the change in the 1000 grain weight was not determining the improvement in the yield at high yielding treatments (BF+NPK, BF+NPKS and BF+NPK Zn).
Seed quality
Seeds oil and protein contents: Oil and protein content of
the rapeseed seeds also significantly were influenced by the treatments
(Table 1). The effects of oil content of seeds were
statistically significant at p<0.01 level, the maximum seed oil content
was obtained at BF+NK showing a 46.73% i.e., 11.68% increase over the
control. The treatments BF+NPKS and BF+NPK Zn also resulted in the oil
contents of more than 46%.
The results showed that the maximum protein contents of the seed resulted at BF+N (25.22%) i.e., 18.96% increase over the control, which was followed by BF+NPK, BF+NPKS and BF+NPK Zn. The results indicated the role of nitrogen in increasing the protein content of the seed which is supported by those observed by Ahmad et al. (2007).
Nutrient contents of leaves and seeds
Nitrogen: The application of N fertilizer, either as chemical fertilizer
or Biofertilizers resulted in a substantial increase in N content of the leaves
and seeds. The leaves maximum N concentration at BF+NP (4.24%) was
about 45.20% more over the control (Table 2). The maximum
N concentration reported in leaves at flowering stage of rapeseed plant is 3-4.5%
(Holmes, 1998). The result showed thereby the capacity
of the rapeseed plant to extract N from the soil, which was more efficient at
the presence of Azotobacter and Azosprillum showing their ability
to make available more N for the rapeseed. There were 14, 29 and 34% more N
concentration in leaves in non-Biofertilizers treatments of control, N and NP
comparing to BF, BF+N and BF+NP respectively. However in the presence of other
nutrient components the effectiveness of the bacteria for nutrient availability
was less.
| Table 2: | Effect of Biofertilizers and chemical fertilizers on nutrients contents of leaves at flowering stage |
| N: Nitrogen fertilizer (Urea at 250 kg h-1), P: Phosphorus fertilizer (Triple super phosphate at 150 kg h-1), K: Potassium fertilizer (Potassium sulfate at 150 kg h-1), S: Sulphur fertilizer at 100 kg h-1), Zn: Zinc sulfate at 40 kg h-1), BF: Biofertilizers (combination of Azotobacter chroococcum and Azosprillum brasilense and Azosprillum lipoferum), LSD: Least significant differences at 0.05 level. Values with different letter(s) indicate significant difference from each other | |
Application of N fertilizer resulted also in an increase in N concentration in seeds at all the treatments (Table 3), the higher levels found at BF+N (4.04%) showing 19.17% increase over control which statistically was similar to the rest of the treatments except control. Nitrogen content of seed in the present study was more than the range of N concentrations (2.7 to 3.84%) recorded by Hocking and Strapper (2001). Kakati and Kalita (1996) reported 3.69% N in the grains of Indian mustard (Brassica juncea) by applying 100 kg ha-1 N.
Phosphorus: The application of P fertilizer individually and in combination with N and K resulted in increase in P concentration in leaves at treatments: P, NPKS, BF+K, BF+NP and BF+NPK. However maximum P concentration in the leaves (Table 2) obtained at T15 when the treatment included Biofertilizers along with NP (0.287%) showing 82.80% increase over the control.
The results showed that P concentration on seeds was also affected by the treatments. The maximum P concentration was at PK and BF+P (0.69%) having 18.96% increase over the control. Ahmad and Abdin (2000) reported that rapeseed seed P concentration was ranged between 0.2 and 0.6%, although Holmes (1998) suggested that seed P concentration was within 0.68-0.81% of the TDM (Total dry Matter). In the present study (Table 3), however, the seed P level ranged between 0.51 to 0.69%. Together with nitrogen fertilizer, phosphorus plays an important role in rapeseed yield and seed oil content. Its concentration in the seed composition is an obvious value addition to the animal feed.
Thakur and Chanel (1998) reported that application of 120 kg N and 40 kg P ha-1 significantly increased the seed yield along with the uptake of 56% N and P 12% respectively in gobhi sarson (Brassica napus).
There results showed that there was 21% more P concentration in leaves in non-Biofertilizers treatments of control comparing to Biofertilizers. However in the presence of P there was no increase in P concentration in BF +P. It was concluded that in the presence of the specific nutrient in soil solution the activity and ability of the bacteria in making available nutrient decreased.
Potassium: The application of K fertilizer individually or in combination
with N, P, S and Zn in the presence or absence of Biofertilizers showed an overall
increase in the concentration K in leaves (Table 2), in treatments
of chemical fertilizers the most significant being at treatments NPKS (1.853%)
showing 49.79% increase over the control but it was not statistically different
from the rest of the treatments except with control, N, P and K. application
of Biofertilizers resulted in higher increase relative to the corresponding
treatments without Biofertilizers. The maximum concentration K in leaves obtained
at BF = NPKS (2.043%) showing 65.15% increase over the control. Increase in
K concentration of leaf have already been observed (Kopsell
et al., 2004) in Leafy Brassica oleracea cultivars, the leaf
K levels ranged between 2.1 and 3.5% while in the present study, leaf K levels
ranged from 1.2 to 2.04%. Holmes (1998) reported the level
of K in the plant between 1-2.2% of TDM in leaves at flowering. Asare
and Scarisbrick (1995) also reported that N and S concentration of oilseed
rape (Brassica napus L.) improved considerately in treated plots as compared
to control plots.
| Table 3: | Effect of Biofertilizers and chemical fertilizers on nutrients contents of seeds at maturity |
| N: Nitrogen fertilizer (Urea at 250 kg h-1), P: Phosphorus fertilizer (Triple super phosphate at 150 kg h-1), K: Potassium fertilizer (Potassium sulfate at 150 kg h-1), S: Sulphur fertilizer at 100 kg h-1), Zn: Zinc sulfate at 40 kg h-1), BF: Biofertilizers (combination of Azotobacter chroococcum and Azosprillum brasilense and Azosprillum lipoferum), LSD: Least significant differences at 0.05 level. Values with different letter(s) indicate significant difference from each other | |
K concentration of seeds was also affected by the treatments. The maximum value obtained at BF+K resulting in 0.69% (23.21%) increase over the control. It was observed that application of K fertilizer along with Biofertilizers resulted in 7% more increase as compare with the application of K alone.
Sulfur: Application of S fertilizer in non Biofertilizers plots showed an increase in the concentration of S in leaves only at treatments NPKS and NK, while in the rest of the treatments it was lower and statistically similar. The results indicated that N and K have been supportive for mobilization of S to the leaves as at P the concentration was less than the control. The application of Biofertilizers resulted in higher increase relative to the corresponding treatments of non-Biofertilizers plots. The maximum concentration of S in leaves obtained at BF+NPKS (127.17 ppm) showing 37% increase over the control.
S concentration of seeds was also affected by the treatments. The maximum value obtained at (NPKS) resulting in 0.370 ppm (15.62%) increase over the control which was similar with those of NPK Zn and BF+ NPK S.
Zinc: Application of Zn fertilizer in non-Biofertilizers plots showed an increase in the concentration of Zn in leaves only at treatments NPK Zn 27.17 ppm showing 26.37% increase over the control. In Biofertilizers treatments at T20 the maximum concentration of Zn in leaves (29.44 ppm) was resulted which showed that application of Biofertilizers resulted in 3% more increase in Zn concentration of leave as compare to the corresponding treatment with no Biofertilizers treatment.
The Zn concentration in the seed was highest at NPK Zn showing >90% increase over the control. However in treatment at presence of Biofertilizers i.e. at BF + NPK Zn (56.88 ppm) resulted in 96.81% increase over the control. The result obtained in the present study was comparable with the Holmes (1998) suggesting 70 ppm of Zn concentration in seeds at the maturity. Malewar et al., (2001) reported an increase of 15% in yield of Indian mustard by application of Zn as ZnSO4 (30 kg ha-1), which also resulted in high uptake of Zn in stover and seed.
DISCUSSION
Seeds inoculation with Azotobacter and Azosprillum resulted in 21.17% increase in seed yield over the control (chemical fertilizers), which was mainly due to increase in number of pod/plant (16.05%). Increase in number of branches (11.78%) and weight of 1000 grain (2.92%), oil content of seeds (1.73%) and protein content of seeds (3.91%) was also observed with application of biofertilizers (Table 4). However, a decrease in the number of seeds per pod in inoculated seeds was observed. The results of the present experiment was fairly comparable with those of the earlier workers, Shukla et al. (2002) report that application of Azotobacter resulted significantly in higher number of seeds per siliquae, TDM, branches/plant and the length of siliquae in Indian mustard. Sharma et al. (1997) suggested that oil and protein production increased significantly when nitrogen applied either through Azotobacter or urea.
Azotobacter and Azosprillum are free living N2 fixing bacteria which in the rhizospheric zone have the ability to synthesize and secret some biologically active substances like B vitamins, nicotinic acid, pentothenic acid, biotin, heteroauxins, gibberellins etc. which enhances the root growth (Kader, 2002; Khalid et al., 2004). The Azotobacter and Azosprillum association helps the crop improvement also by excretion of ammonia in the presence of root exudates that enhances and regulates the nutrient uptake by plants (Chandra et al., 2007). The higher dry-matter production by the inoculated plant might be because of the augmented uptake of nitrogen, phosphorous and potassium, which in turn was consequent to the root proliferation.
The results showed that although application of chemical fertilizers
have definite increase especially in the corresponding element but seed
inoculation with Azotobacter and Azosprillum resulted
even more in accumulation of the element either in leaves during flowering
period or in seeds at maturity. Irrespective to the treatments, results
showed that application of Biofertilizers coincided with 3.86, 0.82, 2.25,
0.75 and 0.91% increase in concentrations of N, P, K, S and Zn in the
seeds over the non-Biofertilizers treatments.
| Table 4: | Gain of Biofertilizers on yield, yield contributing characters and oil content of seeds comparing to control |
Cheema et al. (2001) suggested that the high yield of rapeseed depended on an optimum concentration of all elements in plant. Enhancing the yield is the prime objective for agronomical research while an enriched animal feed, available after seed oil extraction, also indirectly benefits the human health. The human need of quality food and proteins is fulfilled to a great extent by the animal production industry which has a synergistic relationship with oilseed production and processing. The economic sustainability of both depends on this mutually beneficial relationship and is critical to any evaluation of the role of nutrients in producing enriched oilseed and animal meal.
In rapeseed cultivation equally important are the TDM and protein rich seed, the two useful by-products, which depend upon the mobility of nutrients to the leaves and seeds. These two treatments promoted a healthy plant growth and resulted also in concentrating the nutrients in leaf as well as in seed. This enrichment and fortification of seed will ultimately result in improving the rapeseed yield and quality in successive seasons. Care must be taken however to ensure that the levels of S in seed after oil extraction does not exceed a limit beyond which it may be harmful (Glucosinolate) as direct animal meal. The dry matter by-product also makes the fertilizer application cost-effective in returning the nutrients back to the soil for other crops like soybean, rice and wheat.