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0Journal of the Saudi Society of Agricultural Sciences (2015) xxx, xxx-xxx
King Saud University Journal of the Saudi Society of Agricultural Sciences
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SAUDI SOCIETY FO« AGRICULTURAL SCIENCES
FULL LENGTH ARTICLE
Chemical fertilizer in conjunction with biofertilizer and vermicompost induced changes in morpho-physiological and bio-chemical traits of mustard crop
Tanushree Mondal *, Jayanta Kumar Datta , Naba Kumar Mondal
The University of Burdwan, Department of Environmental Science, Golapbag More, Post - Rajbati, Burdwan, West Bengal 713104, India
Received 27 May 2014; revised 15 April 2015; accepted 2 May 2015
KEYWORDS
Biofertilizer; Vermicompost; Morpho-physiology; Pigment content; Secondary metabolites
Abstract To study the impact of reduced dose of chemical fertilizer and its combination with biofertilizer and vermicompost on morpho-physiological and biochemical traits of mustard (Brassica campestris cv. B9), field experiments were conducted during winter seasons of November to February 2011-2012 and 2012-2013 respectively in an old alluvial soil zone of Crop Research and Seed Multiplication Farm, Burdwan University, Burdwan, West Bengal, India. Mustard was cultivated using a full recommended dose of chemical fertilizer (N:P:K-100:50:50) and along with six different reduced doses of chemical fertilizer combined with biofertil-izers and vermicompost. The performance of the crop was adjudged in terms of various parameters viz. leaf area index (LAI), leaf area duration (LAD), leaf area ratio (LAR), crop growth rate (CGR), net assimilation rate (NAR), photosynthetic rate (PR), harvest index (HI) and biochemical attributes such as total chlorophyll, sugar and proline content of physiologically active leaves of mustard. Differential significant (p < 0.05) treatment response was reflected for the studied traits during crop maturity. The data revealed that vermicompost application significantly stimulated most of the studied attributes. It was concluded that 25% reduced dose of chemical fertilizer and
* Corresponding author. Tel.: +91 8972219960. E-mail addresses: tanus53@yahoo.com (T. Mondal), profjkdatta@ yahoo.co.in (J.K. Datta), nkmenvbu@gmail.com (N.K. Mondal).
1 Tel.: +91 94746348300.
2 Tel.: +91 9434545694.
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its combination with vermicompost (T4) was optimum for most of the parameters studied as compared to the control at both crop stages.
© 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
India is the third largest producer of oil seeds in the world. It accounts for 19% of world's area and 9% of the global production (Sinha, 2003). Mustard (Brassica campestris) is an important oil seed crop, next to sunflower. Application of chemical fertilizers has contributed significantly to the huge increase in the world food production. But the adverse impacts of excessive inputs of chemical fertilizers in conventional agricultural practices are being well documented (Banerjee et al., 2011; Garai et al., 2014). Chemical fertilizers also have contributed significantly toward the pollution of water, air and soil. In agro-ecosystems, the use of synthetic toxic chemical pesticides affects the soil fertility and growth of cultivated crops (Ignacimuthu and Vendan, 2007). At present we are using chemical fertilizers in great quantities to compensate the deficiency of nutrients in soil. For minimizing the accumulation of pollutants in agro-ecosystems we should avoid the use of toxic chemicals especially synthetic chemical pesticides and fertilizers in agricultural process. Organic products are eco-friendly natural sources, which can be considered as an alternative to sustainable agriculture development. In India, as a step toward the expansion of native sources, the application of organic waste materials will be useful for achieving higher production. The impact of organic agriculture on natural resources favors interactions within the agro-ecosystem that is vital for both agricultural production and nature conservation. The current trend is to explore the possibility of supplementing chemical fertilizers with organic ones that are eco-friendly and cost-effective.
In the above context, eco-friendly and environmentally safe biofertilizer, vermicompost, etc. became handy to minimize chemical fertilizer use as well as a carbon sink in such crop fields. In recent years, vermicomposting has turned out to be promising way out for safe disposal of organic waste. It is a technique of biodegradation or stabilization of organic waste (natural/anthropogenic) by using earthworms and microbes (Garg et al., 2006; Suthar, 2007; Mainoo et al., 2009). Earthworm plays a major role in plant material degradation and this concept is used in vermicomposting technology with the supplement of cow dung source to enhance plant growth. Cow dung is an organic and nitrogen rich material, and it can be easily degraded in the soil. Cow dung also exhibits plant growth promoter properties (Nattudurai et al., 2014).
The use of biological nitrogen fixation by living nitrogen fixers will help to minimize use of chemical nitrogen fertilizer and to improve plant growth to decrease the production cost and environmental risk (El-Hawary et al., 2002). Bacterial fertilization of non-legume crops by nitrogen fixing bacteria has assumed great importance in recent years. In our present investigation, phosphate solubilizing bacteria (PSB) and Azotobacter are used as biofertilizer. PSB secrete some organic acids which can solubilize P from insoluble and fixed forms to plant available forms, whereas Azotobacter can convert
atmospheric N2 into plant available form of N in the soil. In the recent years, among the various sources of organic manure, efficacy of vermicompost was reported manifold. Earthworms consume large quantities of organic matter and excrete it as cast and this cast contains several enzymes and is rich in plant nutrients, which are beneficial for bacteria and mycorrhizae (Reddy and Reddi, 2002). They also noted that vermicompost is an excellent base for the establishment of beneficial non-symbiotic and symbiotic microbes. Different levels of nitrogen significantly influenced LAI, total dry matter accumulation, CGR, but plant height was not significantly influenced by the different levels of nitrogen application. On the other hand, Stamp (2003) proposed that increased crop growth and development rates and greater biomass accumulation in well-fertilized crops would also correlate with decreased allocation of resources toward the production of starch, cellulose, and non-nitrogen-containing secondary metabolites, important group of bioactive compounds.
This study, therefore, was conducted to evaluate the effects of integrated nutrient management (chemical fertilizers, biofer-tilizer and vermicompost) on the morph-physiology and macromolecular changes in leaves of mustard (B. campestris cv. B9) and to screen out the best fertilizer combination under this field condition.
2. Materials and methods
2.1. Experimental site
Field experiments were conducted at the Crop Research and Seed Multiplication Farm, Burdwan University, Burdwan, West Bengal, India. The latitude is 87°50'37.35"E and longitude is 23°15'7.29''N with an average altitude of 30 m above the mean sea level during the winter seasons of 2011-2012 and 2012-2013 with rapeseed (B. campestris L. cv. B9).
2.2. Climatic condition
Weekly minimum and maximum temperature, total rainfall, sunshine hours, wind speed, and relative humidity (RH) were recorded. Some climate factors were collected and analyzed. Climate factors in both growing periods were similar but not the same. Both the growth periods started with moderate temperature where the maximum temperature was around 29 °C and the minimum temperature was around 16 °C, and then they cooled and ended again with similar maximum and minimum temperatures. The mean temperature and high relative humidity (ranging between 75% and 90%) were similar for both growing cycles. The mean wind speed (1.3-8.1 km h_1) and mean sunshine (4.23-7.15 h day-1) were almost the same in both growing seasons. No rainfall was recorded in the first growing season (2011-2012), but in the second season 0-5 mm rainfall was recorded on average.
2.3. Treatment combinations and design of experimental field The treatments used were as follows:
T1 - Full recommended dose of chemical fertilizers (100:50:50, that is 100 kg ha"1 N:50kgha" P:50kgha" K).
T2 - 50% of the recommended dose of chemical fertilizers (50 kg ha"1 N:25kgha" P:25kgha" K) + vermicompost (2.5 t ha"1).
T3 - 50% of the recommended dose of chemical fertilizers (50 kg ha"1 N:25 kg ha"1 P:25kgha"1 K) + dried cow dung (2.5 t ha"1).
T4 - NPK (75% of full dose) + vermicompost (2.5 t ha"1). T5 - NPK (75% of full dose) + dried cow dung (2.5 t ha"1).
T6 - NPK (75% of full dose) + vermicompost (2.5 t ha"1) + Azotobacter and PSB (7.5 kg ha"1 each). T7 - NPK (75% of full dose) + dried cow dung (2.5 t ha"1) + Azotobacter and PSB (7.5 kg ha"1 each).
The treatment combinations were replicated thrice and arranged in a randomized block design (RBD). Individual plot sizes were 4 m x 2.5 m. Row-to-row and plant-to-plant spacing was 30 cm and 15 cm, respectively. Irrigation channels measuring 0.5 m wide were between the replications to ensure easy and uninterrupted flow of irrigation for each individual plot. The same treatments were followed for two consecutive years. Chemical fertilizers were applied at the recommended NPK dose (100:50:50) for mustard as per the Directorate of Agriculture, Government of West Bengal (Bhattacharyya, 1998). Treatment T1 was the control. A biofertilizer containing Azotobacter chrococcum and phosphate solubilizing microorganism (Bacillus polymyxa) was applied at the rate of 7.5 kg ha"1, each through seed inoculation. The biofertilizer was collected from Nitrofix Laboratories, 25, Bansdoroni Avenue, Kolkata-70. The colony-forming unit (cfu) of Azotobacter and PSB was 2.3 x 109 and 2.1 x 108 cells per gram of carrier material, respectively.
2.4. Pre-harvesting soil condition
Physical, chemical and biological characteristics of the initial soil (0-15 cm depth) were mechanical fractions such as sand (0.02-0.2 mm) (%) - 22.34, silt (0.002-0.02 mm) (%) - 38.35, clay (<0.002 mm) (%) - 26.24, pH (1:2.5, soil: water) - 5.89, CEC (cmol kg"1) - 10.21, Organic C (%) - 0.98, Available N (mg kg"1) - 12.02, Available K (mgkg"1) - 10.19, Available P (mg kg"1) - 12.53, Total bacteria (cfu g"1 of soil) - 8.6 x 106, and Total Fungi (cfu g"1 of soil) - 1.2 x 103. All the detailed pre and post-harvesting soil characteristics of this experiment were previously discussed in our published article (Mondal et al., 2015).
2.5. Crop establishment
The seeds were soaked in distilled water for 24 h. The biofertil-izer (0.1 kg) was mixed with seeds (1.0 kg) and after that the seeds were sown separately in (4 x 2.5 m2) plots. Sowing dates were 6 December 2011 and 11 December 2012. The plant density was approximately 28-32 plants per m2 land area. Dates of
harvesting were 11 March 2012 and 20 March 2013. Chemical fertilizers were used in the form of urea, single super phosphate, and muriate of potash and it was added after layout preparation. Seed sowing was done after 10 days of application of chemical fertilizers. Vermicomposts and organic fertilizers were used after 5 days of seed sowing at a basal dose (2.5 t ha"1), and a gap of 15 days was maintained between the application of organic fertilizers and chemical fertilizers. As per the guidelines of the Department of Agriculture, chemical fertilizers were used in two splits such as 1/2 N + full P + full K as basal and 1/2 N as top dressing. Two hand-weedings at 15-18 DAS (days after sowing) and 38-40 DAS were carried out. The crops of each plot were harvested separately when 90% of the plant with silique became golden yellow in color.
2.6. Vermicompost preparation and application
For the preparation of vermicomposts, a pit of 1.5 m x 2 m and 1.5 m deep was prepared. Then the pit was filled with cow dung collected from the surrounding villages and Eichhornia from local wetlands. Here, cow dung and dried chopped Eichhornia are used as composting materials. Cow dung and chopped dried Eichhornia are mixed in the proportion of 3:1 and are kept for partial decomposition for 15-20 days. No replication was done. Special types of earthworms such as Eisenia fetida (1500-2000 numbers per pit) were used for the production of vermicomposts. A final layer of soil was applied over the compost pit and allowed to remain for 3 months for bacterial decomposition to take place. The vermi-composting unit should be kept in a cool, moist, and shady site. Therefore, 18-25 0C temperature should be maintained for proper decomposition. The optimum moisture level (30-40%) was maintained. By regular sprinkling of water, the optimum moisture level was maintained. After 3 months, the compost was taken out from the pit and applied to the experimental field. The nutrient availability in vermicomposts was 1.71% N, 1.18% P, 0.98% K, 0.0088% Zn, 0.094% Fe, 0.024% Mn, and 0.012% Cu and that in cow dung was 0.98% N, 1.01% P, 0.54% K, 0.0056% Zn, 0.077% Fe, 0.016% Mn, and 0.009% Cu (Mondal et al., 2014). The first irrigation was applied after seed sowing, and afterward the crop was irrigated at regular intervals ranging from 15 days up to 55 days. The crop exhibited no sign of insect/pest attack and disease incidence. The experimental site was kept free of weeds by intercultural and hand hoeing. All the morphological and yield attributes and changes in soil physical and physico-chemical properties of this experiment were previously discussed in our two previously published articles (Mondal et al., 2014, 2015) and here only pre- and post-harvesting soil characteristics are investigated and described.
2.7. Plant materials
Dry matter and Leaf area were sampled at an interval of 15 days from 30, 45 and 60 days of sowing to quantify effects of environmental influences on effect of different combined doses of chemical and bio-fertilizer with vermicompost and compost. Rate of dry matter accumulation varies across to the life cycle of a crop and physiologically active leaves. During these two consecutive years, influence of seven
different combined doses of chemical, biofertilizers and vermicompost on morpho-physiological traits and biochemical attributes viz., LAI, LAD, LAR, CGR, NAR, PR, HI, total chlorophyll, sugar and proline of physiologically active leaves of mustard was studied and the field data were analyzed statistically.
2.8. Parameters studied
The observations on morpho-physiological traits, LAI, LAD, LAR, CGR, NAR, PR, HI were recorded at different stages, i.e., 30 days after sowing (DAS), 45 days after sowing (DAS), and 60 days after sowing (DAS) of crop growth. Leaf area was measured with an electronic leaf area meter (LAM 101, Disha Online Pvt. Ltd., Raipur, Chattisgarh, India).
The leaf index was calculated using the formula (1) given by Watson (1947)
Leaf Area Land Area
Leaf area ratio is the ratio of leaf area and total biomass. It was calculated with the following formula (2):
LAR = — m2 g-1 W 5
where LA = leaf area in m2 and W = dry weight of plant.
Leaf area duration (LAD) was calculated using the following formula (3):
(LAIi + LAI2)x(t2 - ti)
where LAI1 and LAI2 are leaf area indices recorded at times t1 and t2, respectively.
The dry weight per plant was calculated and used to estimate crop growth rate (CGR) as proposed by Hunt (1978) (4):
CGR = W2 - W1 gm-2day-1 (4)
t2 — t1
where Wi and W2 are dry weights (g m-2) at first and second harvests taken at times t1 and t2 respectively.
Net assimilation rate (NAR) was calculated by using the formula (5) given by Watson (1952):
L2 - L
XT»™ W2 - Wl NAR = —-1 x
t2 - ti log L2 - log Li
g m 2day
where W2 and W1 are the final and initial dry weights of aerial plant parts per unit area at the times t1 and t2, respectively, and L2 and L1 are the final and initial leaf area indices at respective times.
Photosynthetic rate (PR) was calculated by using LICOR machine (LI-6400XT) which is a portable photosynthesis system on physiologically active leaves of mustard.
Harvest index percentage was calculated by using the formula (6) given by Donald and Hamblin (1976):
HI (%) = EconomicYJelf x 100 (6)
BiologicalYield
This study also pertained to assess the biochemical changes in leaves of mustard under different treatments of biofertilizer, chemical fertilizer and vermicompost and was evaluated in terms of total chlorophyll, sugar and proline content to enhance crop growth, metabolism and optimum protection
against pathogens and pest. Physiologically active leaf (2nd and 3rd from the top) samples from ten different plants of each plot were plucked randomly for the determination of different biochemical parameters. Composite samples of leaves were crushed separately in a mortar with 10 ml of distilled water, poured in test tube and centrifuged at 10,000 rpm for 10 min. The supernatant was used for soluble sugar estimation by using anthrone reagent as described by Mc Cready et al. (1950). Chlorophyll content (mgg-1 fresh weight of leaves) was determined through organic solvent (80% acetone) extraction method as described by Arnon (1949). Proline content in leaves was determined by following the method of Bates et al. (1973).
2.9. Statistical analysis
Statistical analysis was done using the MINITAB software (version 8.0, Inc., Des Moines, IA, USA). Means of three replicates were taken. Means followed by the same letter within a treatment were not significantly different at the 5% level using Duncan's multiple range test (DMRT).
3. Results and discussion
In the present study, 25% reduction of NPK and combination with vermicompost (T4) proved significantly effective on various morpho-physiological and bio-chemical attributes studied at different growth stages.
3.1. Crop morpho-physiology
3.1.1. Leaf area index and photosynthetic rate
The data presented in Table 1 revealed greater LAI value shown by treatment T4 in all phases of both the experimental years. LAI values increased in the initial stage of development of crop i.e., from 30 to 45 DAS but gradually declined in the later stage of development from 45 to 60 DAS under all treatments. With maturity of crop growth, LAI increased due to newly emerged leaves and then it decreased gradually due to leaf senescence toward the maturity of the crop. A similar finding was reported by Banerjee et al. (2012a,b). Correspondingly, an enhancement in shoot and root fresh and dry weight was recorded at similar sampling stages (Mondal et al., 2014). Stimulating effects of biofertilizer application also increased greater amount of light interception by the crop plants which have contributed toward the vegetative growth of crop plants under all treatments (Aduloju et al., 2009). Greater light interception by the T4 treated plants leads toward higher rate of photosynthesis or photosynthetic rate (Table 6) which contributed significantly toward the vegetative growth of crop plants of B9 variety leading to higher LAI value. These findings are in line with some earlier findings in case of soybean crop (Aduloju et al., 2009) and sunflower plants (Gorttappeh et al., 2000).
3.1.2. Leaf area ratio
The effect of various treatment performances on leaf area ratio is shown in Table 2. LAR indicates how a system is efficient in growth and in broad sense it reflects the ratio of photosynthe-sizing to respiring material within the plant. The revealed
Table 1 Leaf area index (LAI) values of mustard (Brassica campestris cv. B9) depending on vermicompost, biofertilizer and chemical fertilizer treatments during the winter cropping seasons of 2011-2012 and 2012-2013. Different letters within the same row indicate mean values statistically different at p 6 0.05 as determined by LSD test. The data shown are means of three replicates.
Leaf area index
2011-2012 2012-2013
30 DAS 45 DAS 60 DAS 30 DAS 45 DAS 60 DAS
T1 0.37bc 1.59d 1.49ab 0.13b 6.68d 5.54bc
T2 0.81ab 2.14bcd 1.24ab 0.15b 8.30abc 6.23bc
T3 0.29c 1.77cd 1.01ab 0.19ab 7.94bcd 5.82bc
T4 1.08a 4.32a 1.65a 0.22ab 9.48a 8.66a
T5 0.71abc 2.59bcd 1.19ab 0.21ab 7.21cd 4.68c
T6 0.98a 3.34ab 0.88ab 0.23ab 8.84ab 7.44ab
T7 0.76abc 2.95bc 0.82b 0.26a 7.17cd 6.57bc
SEM± 0.152 0.37 0.237 0.03 0.39 0.643
CD AT 5% 1.63 2.54 2.04 0.723 2.62 3.358
CV% 36.9 22.73 34.8 26.0 8.6 17.4
LSD AT 5% 0.467 1.14 0.732 0.092 1.21 1.981
Notes: Means followed by the same letter between treatments are not significantly different at 5% level using Duncan's multiple range test (DMRT). Means of three replicates are taken, where T1 - Full recommended dose of chemical fertilizer, T2 - 50% of recommended dose of chemical fertilizer + vermicompost (2.5 t ha"1), T3 - 50% of recommended dose of chemical fertilizer + dried cow dung (2. 5 t ha"1), T4 -NPK (75% of full dose) + vermicompost (2.5 t ha"1), T5 - NPK (75% of full dose) + dried cow dung (2.5 t ha"1), T6 - NPK (75% of full dose) + vermicompost (2.5tha"1) + Azotobacter and PSB (7.5 kg ha"1 each), T7 - NPK (75% of full dose) + dried cow dung (2.5 t ha"1) + Azotobacter and PSB (7.5 kg ha"1 each). SEM, standard error of the means; CV, coefficient of variation; CD, critical difference; LSD, least significant difference; DAS, days after sowing.
Table 2 Leaf area ratio (LAR) values of mustard (Brassica campestris cv. B9) depending on vermicompost, biofertilizer and chemical fertilizer treatments during the winter cropping seasons of 2011-2012 and 2012-2013. Different letters within the same row indicate mean values statistically different at p 6 0.05 as determined by LSD test. The data shown are means of three replicates.
Leaf area ratio (m2 g -1)
2011-2012 2012-2013
30 DAS 45 DAS 60 DAS 30 DAS 45 DAS 60 DAS
T1 0.17b 2.37c 4.08d 2.51a 5.59cd 2.63d
T2 0.08bcd 3.36c 5.09cd 2.05abc 4.01e 3.29d
T3 0.06cd 2.70c 4.40d 1.34c 4.93de 2.93d
T4 0.28a 7.03a 7.68a 2.01abc 10.11a 7.63a
T5 0.01d 3.37c 5.58bcd 1.47bc 5.56cd 4.59c
T6 0.15bc 6.37ab 6.29abc 2.21ab 8.11b 6.59b
T7 0.08bcd 5.37b 6.94ab 1.78abc 6.49c 5.57c
SEM± 0.03 0.36 0.47 0.24 0.45 0.33
CD AT 5% 0.76 2.53 2.88 2.04 2.8 2.41
CV% 17.5 14.5 14.3 21.6 12.1 12.1
LSD AT 5% 0.1 1.12 1.45 0.73 1.38 1.02
Note: For abbreviations, see footnote Table 1.
values indicated that in the later stage of development i.e., 45 and 60 DAS, T4 and T1 showed highest and lowest values respectively in both the experimental years. Maximum LAR values mean maximum biomass was deposited in the shoot and branches of the studied B9 mustard variety of this investigation.
3.1.3. Crop growth rate
CGR is a simple and important index of agricultural productivity on rate of dry matter production. Again treatment T4 showed the highest CGR values in two consecutive
experimental years (Table 3). When LAI is large to intercept 95% of sunlight then plant gets optimum crop growth rate and also greater light interception stimulates CGR which in turn increases total dry matter and LAI. Greater LAI causes higher light interception which further enhances CGR and thus results in higher yield (Datta et al., 2012).
3.1.4. Leaf area duration
LAD can be defined as a measure of the ability of plant to produce and maintain leaf area and its whole opportunity for assimilation (Watson, 1947). Highest value of LAD in later
Table 3 Crop growth rate (CGR) values of mustard (Brassica campestris cv. B9) depending on vermicompost, biofertilizer and chemical fertilizer treatments during the winter cropping seasons of 2011-2012 and 2012-2013. Different letters within the same row indicate mean values statistically different at p 6 0.05 as determined by LSD test. The data shown are means of three replicates.
Crop growth rate (g m-2 day-1)
2011-2012 2012-2013
30-45 45-60 30-45 45-60
DAS DAS DAS DAS
T1 2.38abc 4.23c 2.85c 5.06e
T2 3.50ab 5.03bc 3.58bc 6.72d
T3 3.42abc 3.88c 2.53c 5.13e
T4 3.74a 10.54a 5.01a 10.49a
T5 2.02bc 6.75abc 3.11bc 8.11c
T6 2.50abc 9.09ab 4.19ab 9.21b
T7 1.81c 7.69abc 2.94c 7.99c
SEM± 0.49 1.24 0.34 0.29
CD AT 5% 2.94 4.66 2.45 2.24
CV% 15.39 16.51 17.2 6.6
LSD AT 5% 1.52 3.82 1.06 0.88
Note: For abbreviations, see footnote Table 1.
Table 5 Net assimilation rate (NAR) values of mustard (Brassica campestris cv. B9) depending on vermicompost, biofertilizer and chemical fertilizer treatments during the winter cropping seasons of 2011-2012 and 2012-2013. Different letters within the same row indicate mean values statistically different at p 6 0.05 as determined by LSD test. The data shown are means of three replicates.
Net assimilation rate (gm2 day 1)
2011-2012 2012-2013
30-45 45-60 30-45 45-60
DAS DAS DAS DAS
T1 0.41a 0.32c 0.1a 0.17ab
T2 0.55a 0.89c 0.12a 0.13bc
T3 0.45a 0.67c 0.11a 0.09c
T4 0.67a 2.41a 0.1a 0.21a
T5 0.42a 0.97bc 0.09a 0.14bc
T6 0.42a 1.79a 0.09a 0.16ab
T7 0.41a 1.69ab 0.08a 0.15abc
SEM± 0.08 0.24 0.01 0.02
CD AT 5% 1.2 2.06 0.48 0.59
CV% 19.7 13.6 23.8 22.5
LSD AT 5% 0.25 0.75 0.04 0.06
Note: For abbreviations, see footnote Table 1.
Table 4 Leaf area duration (LAD) values of mustard (Brassica campestris cv. B9) depending on vermicompost, biofertilizer and chemical fertilizer treatments during the winter cropping seasons of 2011-2012 and 2012-2013. Different letters within the same row indicate mean values statistically different at p 6 0.05 as determined by LSD test. The data shown are means of three replicates.
Leaf area duration
2011-2012 2012-2013
30-45 45-60 30-45 45-60
DAS DAS DAS DAS
T1 4.74a 6.79b 4.71a 7.59bc
T2 3.42a 7.97b 4.89a 6.59bc
T3 5.4a 7.12b 5.98a 6.24c
T4 6.35a 11.46a 4.92a 10.91a
T5 3.91a 8.33b 5.64a 5.99c
T6 5.62a 9.16ab 5.10a 8.39b
T7 6.27a 8.76ab 5.52a 6.95bc
SEM± 1.01 0.9 0.57 0.62
CD AT 5% 4.21 3.97 3.15 3.31
CV% 19.7 18.3 18.7 14.7
LSD AT 5% 3.11 2.77 1.74 1.97
Note: For abbreviations, see footnote Table 1.
Table 6 Photosynthetic rate of physiologically active leaves of mustard (Brassica campestris cv. B9) depending on vermicom-post, biofertilizer and chemical fertilizer treatments during the winter cropping seasons of 2011-2012 and 2012-2013. Different letters within the same row indicate mean values statistically different at p 6 0.05 as determined by LSD test. The data shown are means of three replicates.
Photosynthetic rate (imol m-2 s-1) on 45 DAS
2011-2012 2012-2013
T1 19.15f 22.58c
T2 22.18d 23.43abc
T3 20.05e 22.74bc
T4 24.03a 25.38a
T5 23.12c 24.12abc
T6 23.65ab 24.67ab
T7 23.52bc 24.50abc
SEM± 0.13 0.59
CD AT 5% 1.54 3.22
CV% 1.1 4.3
LSD AT 5% 0.42 1.82
stage of the crops maturity means the treatment performance of combined and balanced dose of fertilizers (Table 4). The enhanced rate of LAD on 30-45 DAS to 45-60 DAS may be attributed toward higher rate of dry matter accumulation by the crop plants under various treatments.
3.1.5. Net assimilation rate
NAR is related to photosynthetic activities of leaf, i.e., rate of increase in dry weight of whole plant per unit leaf area. There is a considerable amount of variation in the NAR values of the seven applied treatments in the later stages of crop growth which can be due to the formation of siliquae that led to differential rate of supply of photo-assimilate toward the vegetative growth of crop plants (Table 5). During the siliquae formation stage the rate of photosynthesis in leaves decreases and siliquae
increases (Miri, 2007). Steady increase in NAR value at the later stages of crop development can be explained on the basis of the fact that higher nutrient availability to crop plants in the presence of vermicompost, biofertilizer and chemical fertilizer caused more cell elongation in shoot and root development which lead to the progressive development of the CGR and NAR. Our findings corroborate the finding of Shukla and Warsi (2000).
3.1.6. Harvest index
In the current investigation there was considerable variation in the percent ratio of economic and biological yield of the crop i.e., HI value among the seven applied treatment combinations (Table 10). This is due to variable rate of translocation of assimilate toward grain development, leading to variability in
yield and HI value among the seven applied treatments (Banerjee et al. (2012a,b)).
3.2. Bio-chemical
This study pertained to assess the biochemical changes in physiologically active leaves of mustard under different treatments of biofertilizer, chemical fertilizer and vermicompost and was evaluated in terms of chlorophyll and proline content to enhance crop growth, metabolism and optimum protection against pathogens and pest.
3.2.1. Total chlorophyll content
Regarding the effect of combined dose of chemical, vermicom-post and bio-fertilizer, it was noted that all the combined doses
Table 7 Total chlorophyll contents of physiologically active leaves of mustard (Brassica campestris cv. B9) depending on vermicompost, biofertilizer and chemical fertilizer treatments during the winter cropping seasons of 2011-2012 and 2012-2013. Different letters within the same row indicate mean values statistically different at p 6 0.05 as determined by LSD test. The data shown are means of three replicates.
Total chlorophyll (mg g"1 fresh weight of leaves)
2011-2012 2012-2013
30 DAS 45 DAS 60 DAS 30 DAS 45 DAS 60 DAS
T1 1.45b 2.53b 1.71ab 1.61abc 1.42b 1.40ab
T2 0.31c 1.26c 1.18b 1.31c 1.41b 1.3ab
T3 1.35b 2.36b 1.87a 1.70ab 1.4b 1.43ab
T4 2.21a 3.26a 2.03a 1.93a 1.92a 1.56a
T5 1.58b 2.44b 6 b 1.37bc 1.39b 1.07ab
T6 1.45b 2.48b 1.37ab 1.41bc 1.69ab 1.27ab
T7 1.23b 1.96b 1.38ab 1.26c 1.55b 1.00b
SEM± 0.16 0.19 0.16 0.11 0.09 0.14
CD AT 5% 1.66 1.85 1.66 1.36 1.29 1.6
CV% 19.8 12.8 13.7 12.2 10.8 19.5
LSD AT 5% 0.48 0.53 0.39 0.33 0.29 0.45
Note: For abbreviations, see footnote Table 1.
Table 8 Total soluble sugar contents of physiologically active leaves of mustard (Brassica campestris cv. B9) depending on vermicompost, biofertilizer and chemical fertilizer treatments during the winter cropping seasons of 2011-2012 and 2012-2013. Different letters within the same row indicate mean values statistically different at p 6 0.05 as determined by LSD test. The data shown are means of three replicates.
Total soluble sugar (mg g"1 fresh weight of leaves)
2011-2012 2012-2013
30 DAS 45 DAS 60 DAS 30 DAS 45 DAS 60 DAS
T1 5.85b 6.52b 11.00ab 3.63c 4.93c 10.29bc
T2 8.02ab 9.21ab 8.78b 4.79bc 7.26bc 8.15c
T3 6.52b 7.97ab 8.45b 4.74bc 6.79bc 8.89c
T4 12.34a 13.71a 15.95a 8.51a 16.32a 17.20a
T5 8.30ab 9.48ab 11.01ab 3.71c 8.96bc 13.36abc
T6 10.53ab 12.74ab 14.01ab 7.22ab 11.48ab 15.84ab
T7 9.65ab 11.97ab 12.08ab 6.68ab 8.30bc 11.91abc
SEM± 1.55 1.87 1.91 0.77 1.65 1.89
CD AT 5% 5.21 5.72 5.79 3.68 5.39 5.75
CV% 14.22 13.37 11.9 15.67 14.06 11.23
LSD AT 5% 4.76 5.76 5.89 2.38 5.10 5.81
Note: For abbreviations, see footnote Table 1.
of treatments increased the total chlorophyll content gradually over the control (T1). Application of vermicompost proved optimum (T4) at all stages of growth (Table 7). The productivity of any crops depends on the process of photosynthesis, which in turn depends on the chlorophyll content of leaves in plants (Banerjee et al. (2012a,b)). The significant variation in the level of total chlorophyll content in physiologically active leaves of mustard with respect to T1 can be due to variable rate of biosynthesis of chlorophyll and photosynthesis depending upon the genetic potential of the mustard against different treatment combinations and increased uptake of magnesium from the soil in the form of Mg + 2 under the influence of vermicompost application. Beneficial effects of bacterial inoculation on increased chlorophyll content are due to higher availability of nitrogen to the growing tissue and organs supplied by nitrogen fixing Azotobacter species (Chandrasekhar et al., 2005), which also produced growth promoting substances resulting in more efficient absorption of nutrients through photosynthetic pigments and consequently the chlorophyll content increased (Hassan, 2009). The positive effect of N supply on the formation of chloroplasts during leaf growth also enhances chlorophyll content of leaves (Singh et al., 2014). In turn, the chloroplast formation leads to an increase in the lipid content of leaves and chloroplast constituents such as chlorophyll and carotene.
3.2.2. Total soluble sugar
Significant level of variation in total soluble sugar content in leaves among the different treatments was attributed toward the variable rate of photosynthesis leading to production of variable amount of photosynthate in the leaves along with adequate supply of nutrients (Table 8). Our findings were similar with the earlier findings on Sideritis montana (El-Sherbeny et al., 2005) and on Iris (Ali, 2005). Higher biosynthesis of chlorophyll and photosynthesis in flag leaf of plants under the vermicompost treated plots resulted toward higher level of total soluble sugar content of leaves. The results also reveal that biofertilizers have pronounced influence on biosynthesis
of carbohydrates in leaves (Rao et al., 2007). These improved levels of sugar changes are of particular importance because of their direct relationship with such physiological processes such as photosynthesis, translocation and respiration. The sugar content in leaves increased up to 25% of reduced dose of nitrogenous chemical fertilizer and then decreased further with gradual increment of reduction of nitrogen fertilizer dose which was attributed toward higher rate of translocation of sugar transported from leaves to flowering parts and its subsequent utilization for the development of seeds under reduced supply of nitrogen to crop plants. This therefore indicates the role of nitrogen toward biosynthesis and accumulation of
Table 10 Harvest Index of mustard (Brassica campestris cv. B9) depending on vermicompost, biofertilizer and chemical fertilizer treatments during the winter cropping seasons of 2011-2012 and 2012-2013. Different letters within the same row indicate mean values statistically different at p 6 0.05 as determined by LSD test. The data shown are means of three replicates.
Harvest index
2011-2012 2012-2013
T1 54.82ab 63.35c
T2 59.42ab 57.92c
T3 58.07ab 86.99b
T4 71.27a 113.27a
T5 54.99ab 69.41bc
T6 64.98ab 86.31b
T7 54.25b 73.97bc
SEM± 4.85 5.69
CD AT 5% 9.22 9.99
CV% 14.1 12.5
LSD AT 5% 14.93 17.54
Note: For abbreviations, see footnote Table 1.
Table 9 Proline contents of physiologically active leaves of mustard (Brassica campestris cv. B9) depending on vermicompost, biofertilizer and chemical fertilizer treatments during the winter cropping seasons of 2011-2012 and 2012-2013. Different letters within the same row indicate mean values statistically different at p 6 0.05 as determined by LSD test. The data shown are means of three replicates.
Proline (mg g 1 fresh weight of leaves)
2011-2012 2012-2013
30 DAS 45 DAS 60 DAS 30 DAS 45 DAS 60 DAS
T1 7.24ab 6.36b 6.44b 4.39a 3.51b 3.83b
T2 4.85b 8.45ab 7.73b 2.15a 9.42ab 10.47ab
T3 4.03b 7.11ab 7.56b 3.38a 6.18b 3.29b
T4 13.18a 14.10a 14.7a 1.11a 14.81a 14.30a
T5 7.70ab 6.5b 6.71b 2.40a 7.46b 7.85ab
T6 10.54ab 11.43ab 11.13ab 1.70a 13.89a 12.64ab
T7 7.9ab 9 39ab 9.67ab 1.10a 8.93ab 10.2ab
SEM± 2.14 2.19 1.59 1.09 1.9 2.80
CD AT 5% 6.12 6.2 5.27 4.38 5.77 7.01
CV% 18.46 16.35 13.79 15.08 15.02 17.86
LSD AT 5% 6.59 6.75 4.89 3.37 5.84 8.77
Note: For abbreviations, see footnote Table 1.
sugar in crop plants. Similar findings were reported by Setua et al. (2005) in mulberry leaves.
3.2.3. Proline content
The perusal of data revealed that proline content in leaves of the different studied treatments of mustard reflects the differential response of the seven treatments toward environmental stress and different levels of osmotic adjustment (Ozturk and Demir, 2002). For regulating the osmotic balance of crop plants synthesis and accumulation of secondary metabolites such as proline in leaves of mustard were recorded under different treatments of compost (Table 9). Our findings support the earlier findings of Sheteawi and Tawfik (2007).
4. Conclusions
Present study concludes that combined application of chemical fertilizer, biofertilizers and vermicompost reflects the variable morpho-physiological and biochemical response of mustard plants toward synthesis and accumulation of simple secondary metabolites and pigment content such as sugar, proline and chlorophyll respectively in leaves which therefore played a role in regulation of plant osmosis and crop plant metabolism leading to better crop growth and yield. Combined application of different fertilizers has enhanced LAI, CGR, NAR, PR, LAD and HI of mustard crop plants. Efficient utilization of applied inputs in a particular set of environments is reflected by crop growth rate and net assimilation rate which are in fact the gain in weight of community of plants per unit of land and time. Combined application of organic and inorganic fertilizers stimulated the accumulation of certain metabolites for optimum plant growth along with enhanced plant defense system against insect and disease attack as well as through antioxidant defense mechanism led to the growth and yield attributes of crop plants. The present study will be useful to enhance crop production and it may combat global demand. We further infer from the study that at least 25% of NPK fertilizer amount can easily be substituted by vermicompost (T4) under this agroclimatic condition of old alluvial soil, Burdwan, India, which can help in increasing economic conditions of farmers and at the same time government has to spend less money to import chemical fertilizers from other countries.
Conflict of interest
There is no conflict of interest. Acknowledgments
This work was supported by University Grants Commission (UGC) under major research project having basic research grant from Government of India (Ref No. R.T.I/1249/55) dt. 01.07.2011 to Prof. J.K. Datta, the Principal Investigator of this project.
References
Aduloju, M.O., Mahmood, J., Abayomi, Y.A., 2009. Evaluation of soybean [Glycine max (L.) Merrill] genotypes for adaptability to a southern Guinnea Savanna environment with and without P
fertilizer application in north Central Nigeria. Afr. J. Agric. Res. 4, 556-563.
Ali, A.F., 2005. The role of organic manure and some growth regulators on growth, flowering and bulb production and chemical composition of Iris plants. In: The 6th Arabian Conference of Horticulture, Islamia, Egypt, Orman No. 90.
Arnon, D.I., 1949. Copper enzymes in isolated chloroplast. Polyphenol oxidase in Beta vulgaris. Plant Physiol. 24, 1-15.
Banerjee, A., Datta, J.K., Mondal, N.K., Chanda, T., 2011. Influence of integrated nutrient management on soil properties of old alluvial soil under mustard cropping system. Commun. Soil Sci. Plant Anal. 42, 2473-2492.
Banerjee, A., Datta, J.K., Mondal, N.K., 2012a. Biochemical changes in leaves of mustard under the influence of different fertilizers and cycocel. J. Agric. Technol. 8 (4), 1397-1411.
Banerjee, A., Datta, J.K., Mondal, N.K., 2012b. Changes in morpho-physiological traits of mustard under the influence of different fertilizers and plant growth regulator cycocel. J. Saudi Soc. Agric. Sci. 11, 89-97.
Bates, L.S., Walden, R.P., Teare, I.D., 1973. Rapid determination of free proline for water stress studies. Plant Soil 39, 205.
Bhattacharyya, B.K., 1998. Soil Tests based Fertilizer Recommendations for Principal Crops and Cropping Sequences in West Bengal. Pub. No. Bulletin No. 2. Department of Agriculture, Government of West Bengal, Calcutta.
Chandrasekhar, B.R., Ambrose, G., Jayabalan, N., 2005. Influence of biofertilizer and nitrogen source level on the growth and yield of Echinochloa frumentacea (Roxb.) Link. J. Agric. Technol. 1, 223234.
Datta, J.K., Ghosh, A., Banerjee, A., Mondal, N.K., 2012. Biochemical response of selected plant species under air pollution stress. Ecol. Environ. Conserv. 18, 957-962.
Donald, C.M., Hamblin, J., 1976. The biological yield and harvest index of cereals as agronomic and plant breeding criteria. Adv. Agron. 28, 361-405.
El-Hawary, M.I., Talman, I.E.I., El-Ghamary, A.M., Naggar, E.El., 2002. Effect of application of biofertilizer on the yield and NPK uptake of some wheat genotypes as affected by the biological properties of soil. Pak. J. Biol. Sci. 5, 1181-1185.
El-Sherbeny, S.E., Khalil, M.Y., Naguib, N.Y., 2005. Influence of compost levels and suitable spacing on the productivity of Sideritis montana L. plants recently cultivated under Egyptian conditions. Bull. Fac. Cairo Univ. 56, 373-392.
Garai, T.K., Datta, J.K., Mondal, N.K., 2014. Evaluation of integrated nutrient management on Boro rice in alluvial soil and its impacts upon growth, yield attributes yield and soil nutrient status. Arch. Agron. Soil Sci. 60, 1-14.
Garg, P., Gupta, A., Satya, S., 2006. Vermicomposting of different types of waste using Eisenia foetida: a comparative study. Bioresour. Technol. 97, 391-395.
Gorttappeh, A.H., Ghalavand, A., Ahmady, M.R., Mirnia, S.K., 2000. Effect of organic inorganic and integrated fertilizers on qualitative and quantitative traits of different cultivars of sunflower (Helianthus annuus L.) in western Azerbaijan. Iran J. Agric. Sci. 6, 85-104.
Hassan, F., 2009. Response of Hibiscus sabdariffa L. plant to some biofertilizer treatments. Ann. Agric. Sci. Ain Shans Univ. Cairo 54, 437-446.
Hunt, R., 1978. In: Plant Growth Analysis Studies in Biology, vol. 96. Edward Arnold, London, pp. 26-38.
Ignacimuthu, S., Vendan, S.E., 2007. Botanical pesticides in insect pest management. Uttar Pradesh J. Zool. 4, 141-154.
Mainoo, N.O.K., Barrington, S., Whalen, J.K., Sampedro, L., 2009. Pilot-scale vermicomposting of pineapple wastes with earthworms native to Accra, Ghana. Bioresour. Technol. 100, 5872-5875.
Mc Cready, R.M., Guggolz, J., Silviera, V., Owens, H.S., 1950. Determination of starch and amylase in vegetables. Anal. Chem. 22, 1156.
Miri, H.R., 2007. Morphophysiological variation basis of variation in rapeseed (Brassica napus L.) yield. Int. J. Agric. Biol. 9 (6), 845850.
Mondal, T., Datta, J.K., Mondal, N.K., 2014. An alternative eco-friendly approach for sustainable crop production with the use of indigenous inputs under old alluvial soil zone of Burdwan, West Bengal, India. Arch. Agron. Soil Sci. http://dx.doi.org/10.1080/ 03650340.2014.921807.
Mondal, T., Datta, J.K., Mondal, N.K., 2015. Influence of indigenous inputs on the properties of old alluvial soil in a mustard cropping system. Arch. Agron. Soil Sci. http://dx.doi.org/10.1080/ 03650340.2014.1000877.
Nattudurai, G., Vendan, S.E., Ramachandran, P.V., Lingathurai, S., 2014. Vermicomposting of coirpith with cowdung by Eudrilus eugeniae Kinberg and its efficacy on the growth of Cyamopsis tetragonaloba (L.) Taub. J. Saudi Soc. Agric. Sci. 13, 23-27.
Ozturk, L., Demir, Y., 2002. In vivo and in vitro protective role of proline. Plant Growth Regul. 38, 259-264.
Rao, D.M.R., Kodandara, J.M., Reddy, R.S., Katiyar, V.K., 2007. Effect of VAM fungi and bacterial biofertilizers on mulberry leaf quality and silkworm cocoon characteristics under semiarid condition. Caspian J. Environ. Sci. 5, 111-117.
Reddy, T.Y., Reddi, G.H.S., 2002. Mineral nutrition, manures and fertilizers, third ed.. Principles of Agronomy Kalyani, Ludhiana, India, pp. 204-256.
Setua, G.C., Das, N.K., Banerjee, N.D., Sengupta, T., Sudhakar, P., Sen, S., Saratchandra, B., 2005. Effect of integrated nutrient
management on quality leaf production in mulberry (Morus alba) under rainfed, alluvial soil conditions. Ind. J. Agric. Sci. 75, 474478.
Sheteawi, S.A., Tawfik, K.M., 2007. Interaction effect of some biofertilizers and irrigation water regime on Mung bean (Vigna radiata) growth and yield. J. Appl. Sci. Res. 3 (3), 251-262.
Shukla, S.K., Warsi, A.S., 2000. Effect of sulfur and micronutrients on growth, nutrient content and yield of wheat. Ind. J. Agric. Res. 34, 203-205.
Singh, M., Khan, M.M.A., Naeem, M., 2014. Effect of nitrogen on growth, nutrient assimilation, essential oil content, yield and quality attributes in Zingiber officinale Rosc. J. Saudi Soc. Agric. Sci. http://dx.doi.org/10.1016/j.jssas.2014.11.002.
Sinha, S., 2003. Effect of different levels of nitrogen on the growth of rapeseed. Environ. Ecol. 21, 741-774.
Stamp, N., 2003. Out of the quagmire of plant defense hypotheses. Quart. Rev. Biol. 78, 23-55.
Suthar, S., 2007. Vermicomposting potential of Perionyx sansibaricus (Perrier) in different waste materials. Bioresour. Technol. 98, 12311237.
Watson, D.J., 1947. Comparative physiological studies on growth of field crops. 1. Variation in net assimilation rate and leaf area between species and varieties and within and between years. Ann. Bot. 11, 41-76.
Watson, D.J., 1952. The physiological basis of variation in yield. Adv. Agron. 4, 101-144. Academic Press Inc., New York.
