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0Annals of Agrarian Science xxx (2017) 1—6
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Evaluation of indigenous rhizobacterial strains with reduced dose of chemical fertilizer towards growth and yield of mustard (Brassica campestris) under old alluvial soil zone of West Bengal, India
Shampa Dutta a' *, Jayanta Kumar Datta a, Narayan C. Mandal b
a Department of Environmental Science, The University ofBurdwan, Burdwan, 713104, West Bengal, India b Department of Botany, Visva-Bharati, Santiniketan, 731 235, West Bengal, India
ARTICLE INFO ABSTRACT
A field experiment had been carried out in the Crop Research and Seed Multiplication Farm of The University of Burdwan, West Bengal, India during the two consecutive winter seasons of 2011-2012 and 2012-2013 to study the effect of indigenous rhizospheric bacterial strains on growth, physiology and yield of mustard variety. Pseudomonas putida, Burkholderia cepacia, Burkholderia sp. and their mixture were used as seed inoculants for mustard cultivation. The experiment was laid down in a randomized complete block design (RCBD) with three replications. Results revealed that indigenous inoculation (with reduced dose of chemical fertilizer) significantly increased (p < 0.05) the yield of mustard as compared to uninoculated control (full recommended dose of NPK fertilizers). A combination treatment of bio-fertilizer and chemical fertilizer also increased plant height, plant biomass and other yield components compared to control. The comprehensive approach of plant growth promoting rhizobacteria (PGPR) in agriculturally important crops should be carried out to explore the hidden potential of PGPR and to promote the quality and yield of crop under field conditions.
© 2017 Production and hosting by Elsevier B.V. on behalf of Agricultural University of Georgia. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Article history: Received 13 October 2016 Received in revised form 23 February 2017 Accepted 27 February 2017 Available online xxx
Keywords:
Indigenous rhizobacteria
Mustard
1. Introduction
Economic growth of India is mostly dependent on agriculture. About 114 million hectares, out of the 329 million hectares of India's geographical area, are under cultivation [1]. Three types of fertilizers are basically used in India-they are either chemical or organic or biofertilizers. Among them Indian farmers mostly used chemical fertilizers which cause not only soil degradation, pollution but also posed severe health and environmental hazards. Organic farming methods (or use of biofertilizers) would solve these issues and make the ecosystem healthier. Biofertilizers mainly plant growth promoting rhizobacteria (PGPR) play an important role in improving soil fertility by fixing atmospheric nitrogen, solubilize insoluble phosphorus and produce plant growth promoting substances.
In India, mustard is the second most important edible oilseed
* Corresponding author. E-mail addresses: ssduttashampa@gmail.com (S. Dutta), jkd.envs@gmail.com (J.K. Datta), mandalnc@rediffmail.com (N.C. Mandal).
Peer review under responsibility of Journal Annals of Agrarian Science.
after groundnut. Brassica campestris and B. juncea have higher number of siliquae per plant and higher number of seeds per sili-quae, that's why higher yield, were found to be important for the breeders. Industrialization and green revolution have brought about an increase in productivity but they have also resulted in massive abuse of environment. Hence, there is a need to search for alternative strategies to improve soil health without causing damage to environment. 'Sustainable agriculture' is a method of farming system that are productive and profitable, conserve the natural resources as well as protect the environment for a long period [2].
To achieve the highest priority of agricultural sustainability, use of biofertilizer is dominated over chemical fertilizer. Biofertilizers are the preparations containing live and efficient strains of rhizo-bacteria with plant growth promoting activities. These are used mainly in seed, soil or other organic manure for increasing their number; as a result accelerate their metabolic processes and the metabolites ultimately assimilated by plants [3]. Biofertilizers not only provides nitrogen but also provides plant growth promoting substances such as indole acetic acid, gibberellins, vitamin B etc. [4]. Biofertilizers can enhanced the yield by 20—30% and activate the soil biologically [5]. Moreover, biofertilizer applications not only
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S. Dutta et al. / Annals of Agrarian Science xxx (2017) 1—6
increase root development of plants but also increase the level of mineral and water uptake [6]. However, absolute use of biofertilizer may not enhance the productivity of the food grain. They cannot replace the chemical fertilizers for getting maximum crop yields. The improved level of productivity was achieved by combined application of biofertilizer and chemical fertilizer [7].
There is very limited information that rhizobacterial strains enhanced growth and productivity of agricultural crops under old alluvial soil. Gupta [8] reported that biofertilizers can fix atmospheric nitrogen in cereal crop without any symbiosis. Some other workers [9] also demonstrated that biofertilizers provide balanced nitrogen and phosphorus to wheat crops. But very limited work was reported on oilseed crops, especially, mustard. With this backdrop, present work dedicated to study the impact of combined doses of chemical fertilizer and biofertilizer on mustard crop. Three potent rhizobacteria (Pseudomonas putida, Burkholderia cepacia and Burkholderia sp.) were isolated from the crop field rhizosphere and their plant growth promoting activities were studied in vitro [10]. These indigenous species are potent phosphate solubilizers, nitrogen fixers, indole acetic acid producers and also they have some antifungal activities [10].
The present work was done in relation to combined doses of chemical fertilizer (reduced dose of nitrogen fertilizer) and bio-fertlizers (Pseudomonas putida, Burkholderia cepacia and Burkholderia sp.) and their applications on mustard crop. With this idea an experiment was laid down in the Crop Research and Seed Multiplication Farm of The University of Burdwan to evaluate the impact of these indigenous microflora with reduced chemical nitrogen fertilizer and the productivity of mustard crop under this old alluvial soil zone of Burdwan district, West Bengal, India.
2. Material and methods
2.1. Experimental field
Field experiments were conducted during the two winter seasons of 2011—2012 and 2012—2013 with mustard (Brassica cam-pestris L. cv. B9) crop under the old alluvial soil zone of the Crop Research and Seed Multiplication Farm, The University of Burdwan, 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 mean sea level.
2.2. Climatic condition
Meteorological data were recorded and some data were collected from the College of Agriculture, Bidhan Chandra Krishi Viswavidyalaya, West Bengal. Maximum and minimum temperature, rainfall, relative humidity was represented graphically in Fig. 1. Climatic factors of both the growing seasons were more or less similar and were suitable for mustard cultivation.
23. Physico-chemical parametes of soil
The experimental soil is mainly old alluvial type. The percentage of the different components of soil was: sand (0.02—0.2 mm), slit (0.002—0.02 mm) and clay (<0.002 mm) ranges between 22.34%, 38.35% and 26.24%, respectively [11]. pH of the soil was slightly acidic. The level of NPK and organic carbon were measured and tabulated in Table S3 and Table S4.
2.4. Production of inoculants
These strains (Pseudomonas putida, Burkholderia cepacia and Burkholderia sp.) were isolated from rice rhizosphere, purified and
plant growth promoting potential traits were studied [10]. These strains were grown to high population in liquid broth culture (nutrient broth medium) in conical flasks and were subsequently transferred to larger fermentation vessels at laboratory level. Like all pure bacterial culture, selected strains must be grown under sterile condition and do require minimum aeration provided by shaking (120 rpm) at 28 °C. After an incubation period of 2—3 days the broth culture became ready to be mixed into a suitable carrier material.
2.5. Seed inoculation
Crop was Brassica campestris, variety Bg and the seeds were collected from the Crop Research and Seed Multiplication Farm, The University Burdwan, West Bengal, India. At first 30 g mustard seeds were surface sterilized with 0.1% mercuric chloride (HgCl2) solution (seed application rate was 2 g/12 m2). Then rinse with distilled water thoroughly. The seeds were dipped for 5 h in bacterial suspension, both as individual (for T1-T3) and in combined (for T4) culture. 2 g seeds were dipped in distilled water and served as control (T5). The seeds were coated with actively growing bacterial culture (108 cfu/ml) by the help of a chelating agent (a sticker, gum acacia), so that a high number of superior bacteria remained present when the crop root emerges. Seeds were then air dried slightly and used for field experiment. These inoculants, PGPR, can then quickly colonize the root and start the process of seedling growth.
2.6. Experimental treatment details
Five different combinations of bacterial inoculants and chemical fertilizers were applied in the field as separate treatments including:
T1 - Pseudomonas putida + 25% reduced dose of chemical nitrogen fertilizer (75 kg ha-1 N: 50 kg ha-1 P: 50 kg ha-1 K); T2 - Burkholderia cepacia + 25% reduced dose of chemical nitrogen fertilizer (75 kg ha-1 N: 50 kg ha-1 P: 50 kg ha-1 K); T3 - Burkholderia sp. + 25% reduced dose of chemical nitrogen fertilizer (75 kg ha-1 N: 50 kg ha-1 P: 50 kg ha-1 K); T4 - (Pseudomonas putida + Burkholderia cepacia + Burkholderia sp.) + 25% reduced dose of chemical nitrogen fertilizer (75 kg ha-1 N: 50 kg ha-1 P: 50 kg ha-1 K) and T5 - Control - Full recommended dose of chemical fertilizer (100:50:50, i.e., 100 kg ha-1 N: 50 kg ha-1 P: 50 kg ha-1 K) for both years.
Chemical fertilizers were applied according to the Directorate of Agriculture, Government of West Bengal at the recommended dose (100:50:50) for mustard [12]. Chemical fertilizers were used in the form of urea, single super phosphate and muriate of potash as the source of N, P and K, respectively. With bacterial inoculant application, 25% reduced dose of nitrogen fertilizer (urea) was used. As per the guidelines of the Department of Agriculture, chemical fertilizers were used in two splits such as lA N + Full P + Full K as basal and lA N as top dressing. Two hand-weeding at 15 DAS (days after sowing) and 40 DAS were carried out in each season.
2.7. Experimental design
The experiment was conducted in the randomized complete block design (RCBD) technique with three replications; individual plot sizes was 4 m x 3 m, row to row spacing was 25—30 cm and plant to plant spacing was 10—15 cm. The land was prepared by mechanically ploughed and harrowed. Irrigation channels were 0.5 m wide in between the replications for the easy and
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S. Dutta et al. / Annals of Agrarian Science xxx (2017) 1—6
—■— Rainfall (mm) -■- RH(%) I I Temp(Deg C)
Dec'11 Jan'12
Experimental months
|-85 £
Dec'12 Jan'13
Experimental months
Fig. 1. Meteriological data of the two growing seasons of mustard. (A) Winter season of 2011—2012 and (B) Winter season of 2012—2013.
Nov'11
Feb'12
Feb'13
uninterrupted irrigation of the individual plot independently until crop grown up to the maturity stage.
2.8. Experimental output
Plant samples were collected at intervals of 15 DAS (days after sowing) from 30 DAS to 60 DAS of crop growth and again at the time of harvest. The observations on agronomic traits including growth attributes, yield attributes and yields were recorded at these stages of crop growth. Ultrastructure of plant anatomy (stem) was also studied. The crops of each plot were harvested separately when 90% of the plant with siliquae became golden yellow in colour.
Ten plant samples were uprooted randomly from each plot under different treatments to determine the growth attributes (root and shoot length, fresh weight and dry weight of leaf, root and shoot), yield attributes (length of siliqua, diameter of siliqua, number of siliquae per plant, number of seeds per siliqua, test weight of seed) and yield (weight of seed). The length (cm) of root and shoot were recorded using a centimeter scale. For determination of fresh weight of the leaf (g), root and shoot (g), the samples were washed thoroughly with tap water and after slight blot drying fresh weight was taken. For dry weight determination, collected samples were transferred to hot air oven at 80 °C for 72 h. The total number of filled siliquae per plant was recorded at harvest, while the total number of seeds per siliqua was recorded by random selection of 10 siliquae from each plant per plot at harvest. Length (cm) and diameter (cm) of the siliquae were measured using centimeter scale from 10 randomly selected plots. 1000 seed weight (test weight) was recorded in grams (g). The seed grain yield of each plot was recorded by harvesting plants followed by sun-drying, threshing and cleaning on the threshing floor. The seed yield was recorded in gm~2.
2.9. Statistical analysis
All the experimental data were analyzed separately with two-way ANOVA analysis and values were expressed as the mean of three replicates. All the experimental data were subjected to statistical analysis using MINITAB-16 software. The statistical significance of differences between the different treatments was compared using DMRT (Duncan's Multiple Range Test) at 5%
confidence interval [13,14].
2.10. Scanning electron microscopic (SEM) study
Ultrastructural anatomy of transverse section of stems of both treated and control mustard plants (on 30 DAS) were studied by scanning electron micrograph (HITACHI S-530).
3. Results and discussion
Three potent biofertilizer strains viz., Pseudomonas putida, Burkholderia cepacia and Burkholderia sp. were used in this study to evaluate their potential towards growth and yield of mustard crop. In vitro potentials of these strains have already been tested [10]. They able to grow in nitrogen free Burk's medium which is the preliminary indication of their nitrogen fixing phenotype. The strains could grow in presence of commercially used pesticides such as 2,4-D, bavistin, mancozeb, blytox and lindane indicating their applicability in pesticide added crop field. All the three strains were found to be good phosphate solubilizers and could solubilize tricalcium phosphate (TCP) and rock phosphates like Jordan and Mussori rock phosphates. All of them produced more than 400 mg/ mL soluble phosphates from TCP. They also produced good amount of indole acetic acid (IAA) when the medium was supplemented with L-Tryptophan. Moreover, all the strains have broad spectrum antifungal potentials and they could kill a wide range of phyto-pathogenic fungi as well as spoilage fungi efficiently. Interestingly, the strains were unable to kill one biopesticide fungus Trichoderma sp. and two phosphate solubilizing bacterial species (Bacillus sub-tilis and Burkholderia tropica). There was no antagonistic behavior observed too when they were tested against each other (Table S1 and Table S2).
Crop growth attributes data were presented in Tables 1—3. From Table 1, it is clear that 19.6% and 40.27% higher root length was recorded in treatment T5 at 30 DAS in two consecutive experimental years, respectively. However, root length at T5 treatment on 45 and 60 DAS showed much lower in comparison to other treatments. On the other hand, combined biofertilizer treated plots (T1-T4) showed better root and shoot growth in comparison to sole chemical fertilizer treated (T5) plot in both the growing seasons of mustard (Tables 1—3).
The betterment of growth attribute towards application of
4 S. Dutta et al. / Annals of Agrarian Science xxx (2017) 1—6
Table 1
Crop growth attributes of B9 mustard variety under the influence of combined doses of biofertilizer and chemical fertilizer during the winter seasons of 2011—2012 and 2012-2013.
Treatments Root Length Shoot Length
2011—2012 2012—2013 2011—2012 2012—2013
30DAS 45DAS 60DAS 30DAS 45DAS 60DAS 30DAS 45DAS 60DAS 30DAS 45DAS 60DAS
T1 6.03ab 7.00ab 18.29a 5.96a 7.86a 20.47a 6.04a 19.23a 88.82a 7.11a 20.29a 93.12Ь
T2 4.70a 7.43ab 16.34a 5.84a 7.73a 17.16a 8.14a 17.76a 86.65ab 9.72a 19.65a 89.02ab
T3 4.87a 6.91ab 16.07a 5.73a 7.84a 16.43a 7.49a 19.57a 80.04b 10.27a 19.65a 81.42a
T4 5.10a 8.31b 18.69a 7.71ab 8.56a 20.71a 9.44a 18.31a 89.71a 11.21a 21.08a 93.85Ь
T5 7.21b 6.28a 14.64a 8.3бЬ 7.14a 16.02a 8.31a 17.25a 79.75Ь 10.34a 18.69a 82.79a
SEm (±) 0.47 0.53 1.99 0.б2 0.59 2.2б 1.35 1.89 2.42 1.57 1.81 2.35
CV (%) 14.б 12.7 20.5 1б.0 12.9 21.б 29.8 17.7 4.9 27.9 15.8 4.б
LSD(0.05%) 1.54 1.71 б.48 2.02 1.91 7.39 4.42 б.15 7.89 5.11 5.91 7.бб
Notes: Means followed by the same letter between treatments are not significantly different at the 5% level using Duncan's multiple range test (DMRT). Means of three replicates are taken. SEm (±), Standard errors of means; CV, Coefficient of variance; LSD, Least significant differences of means at 5% level.
Table 2
Crop growth attributes of B9 mustard variety under the influence of combined doses of biofertilizer and chemical fertilizer during the winter seasons of 2011—2012 and 2012—2013.
Treatments Fresh weight (g) of leaf Fresh weight (g) of root & shoot
2011—2012 2012—2013 2011—2012 2012—2013
30DAS 45DAS 60DAS 30DAS 45DAS 60DAS 30DAS 45DAS 60DAS 30DAS 45DAS 60DAS
T1 2.90a 5.3бЬ 5.52a 3.90a б.18Ь 6.34a 1.22a 6.99a 45.79a 2.04a 8.20a 49.87a
T2 2.22a 4.78b 7.19a 3.59a 5.57ab 7.06a 2.02ab 5.61a 29.70a 3.06ab 7.36a 31.94a
T3 3.98a 4.43ab 5.76a 5.23a 4.61ab 7.41a 2.69ь 6.75a 35.99a 3.82b 7.83a 38.71a
T4 4.08a 4.37ab 6.18a 4.87a 5.85ab 8.47a 2.09ab 5.77a 47.82a 2.85ab 6.90a 51.62a
T5 3.09a 2.70a 4.41a 5.09a 3.54a 5.79a 1.54a 4.36a 29.21a 2.43a 5.74a 31.21a
SEm (±) 0.83 0.59 1.13 0.89 0.б7 1.24 0.32 1.52 8.40 0.35 1.б1 9.23
CV (%) 44.4 23.7 33.7 34.0 22.4 30.5 29.3 44.б 38.б 21.5 38.7 39.3
LSD(0.05%) 2.72 1.93 3.б9 2.91 2.17 4.03 1.05 4.96 27.40 1.15 5.2б 30.10
Note: For abbreviations, see footnote Table 1.
Table 3
Crop growth attributes of B9 mustard variety under the influence of combined doses of biofertilizer and chemical fertilizer during the winter seasons of 2011—2012 and 2012—2013.
Treatments Dry weight (g) of leaf Dry weight (g) of root & shoot
2011—2012 2012—2013 2011—2012 2012—2013
30DAS 45DAS 60DAS 30DAS 45DAS 60DAS 30DAS 45DAS 60DAS 30DAS 45DAS 60DAS
T1 0.65a 0.39a 1.05a 0.83a 0.68a 1.44a 0.40a 0.46a 10.10a 0.52a 0.56a 12.67a
T2 0.73a 0.67a 1.09a 0.85a 0.85a 1.62a 0.44a 0.57a 11.26a 0.60a 0.67a 14.81a
T3 0.53a 0.60a 1.20a 0.71a 0.84a 1.55a 0.20a 0.59a 10.43a 0.32a 0.73a 10.95a
T4 0.76a 0.72a 1.35a 0.90a 0.96a 1.67a 0.62a 0.62a 18.29a 0.70a 0.78a 24.03a
T5 0.60a 0.34a 0.91a 0.70a 0.40a 1.21a 0.40a 0.38a 8.24a 0.52a 0.44a 11.94a
SEm (±) 0.28 0.14 0.21 0.33 0.17 0.28 0.27 0.16 3.34 0.31 0.20 4.19
CV (%) 75.1 45.6 31.9 72.1 38.2 32.3 113.4 54.0 49.6 100.5 55.2 48.8
LSD(0.05%) 0. 93 0.47 0.67 1.08 0.54 0.91 0.88 0.53 10.90 1.01 0.66 13.68
Note: For abbreviations, see footnote Table 1.
biofertilizer is well documented [15,1б]. Researchers suggested that biofertilizers, especially plant growth promoting rhizobacteria (PGPR), are the most reliable replacements for the chemical ones in sustainable agroecosystems nutrient management. However, enhancement of plant growth promoting traits do not occur independently, the multiple mechanisms, such as phosphate solubili-zation, dinitrogen fixation, ACC deaminase and antifungal activity, IAA and siderophore biosynthesis etc. are responsible for the plant growth promotion and increased yield [17].
Throughout the growing seasons, higher level of shoot lengths were recorded by applying biofertilizers in combination with reduced dose of chemical fertilizers in both the experimental years (2011—12 and 2012—13). Combined applications of biofertilizers and chemical fertilizers increased the nutrient use efficiency of the
plants because this fertilizer acted as an excellent source of macro and micro nutrients, and, therefore, shoot and root lengths were increased [18].
Results revealed that fresh weight accumulations were higher in combined biofertilizer treated plots (T-1-T4) in comparison to sole chemical fertilizer treated plot (T5) from the mid phase of growth of mustard in both the consecutive years of experiment (Table 2). Fresh weights of leaves were ranges between 4.43 and 5.36 g in 2011-12 and 4.61-6.18 g in 2012-13 on 45 DAS with treatment T1-T4. Similarly, on 60 DAS, enhanced leaf fresh weight accumulations were recorded with treatment T1 -T4 in both the experimental years. On the other hand, T5 showed the minimum value of leaf fresh weight accumulations i.e., 2.70 g on 45 DAS, 4.41 g on 60 DAS (2011-2012) and 3.54 g on 45 DAS and 5.79 g on 60 DAS
S. Dutta et al. / Annals of Agrarian Science xxx (2017) 1—6 5
Table 4
Yield and yield components of B9 mustard variety under the influence of combined doses of biofertilizer and chemical fertilizer during the winter seasons of 2011—2012 and 2012—2013.
Treatments Number of siliqua Siliqua length (cm) Siliqua diameter Number of seeds per Test weight of seeds Seed yield (g/m2) Straw yield (g/m2) per plant (cm) siliqua (g)
2011-12 2012-13 2011-12 2012-13 2011-12 2012-13 2011-12 2012-13 2011-12 2012-13 2011-12 2012-13 2011-12 2012-13
T1 59.79a 62.95a 3.95a 4.23a 1.05a 1.33a 13.49a 15.39a 2.69a 2.82a 113.3a 136.7ab 230.0b 240.0b
T2 62.72a 64.23a 4.13a 4.36a 1.07a 1.21a 13.80a 15.66a 2.55a 2.63a 118.3a 138.3b 220.0ab 233.3ab
T3 63.28a 66.89a 4.11a 4.37a 0.98a 1.32a 14.44a 15.32a 2.62a 2.78a 118.3a 135.0ab 208.3ab 218.3ab
T4 69.57a 70.99a 4.03a 4.63a 1.07a 1.41a 14.73a 16.19a 2.88a 2.95a 130.0a 150.0b 250.0b 263.3b
T5 56.43a 58.37a 3.97a 4.07a 0.93a 1.18a 12.96a 15.31a 2.46a 2.57a 105.0a 121.7a 173.3a 183.3a
SEm (±) 4.20 4.09 0.30 0.27 1.00 0.11 1.00 1.54 0.16 0.16 7.66 4.55 15.38 15.83
CV(%) 11.7 10.9 12.8 10.8 17.6 14.9 12.5 17.1 10.2 10.2 11.3 5.8 12.3 12.0
LSD (5%) 13.68 13.33 0.97 0.88 0.34 0.36 3.26 5.02 0.51 0.53 25.0 14.84 50.17 51.62
Note: For abbreviations, see footnote Table 1.
(2012—2013). Fresh weight of root and shoot also followed the similar results with leaf fresh weight accumulations. On 45 DAS, fresh weight of root and shoot was 5.61—6.99 g (2011—2012) and 6.90—8.20 g (2012—2013) with (T1-T4). Similarly, on 60 DAS, 29.70—47.82 g (2011—2012) and 31.94—51.62 g (2012—2013) fresh weight of root and shoot was observed with treatments T1-T4. However, control crop (T5) always remains the least values on 45 and 60 DAS in both the years of experiments (Table 2).
Dry weight accumulations of plant followed almost similar results of fresh weight accumulations of mustard crop. Here maximum dry weight accumulation occurs with treatment T4. The leaf biomass was recorded as 0.76 g, 0.72 g and 1.35 g on 30, 45 and 60 DAS, respectively in 2011—12; and, 0.90 g, 0.96 g and 1.67 g on 30, 45 and 60 DAS, respectively in 2012—2013. In case of root and shoot dry weight, 18.29 g (2011 —2012) and 24.03 g (2012—2013) on 60 DAS showed the maximum value with T4 treatment. Whereas, least biomass of leaf, root and shoot were observed with T5 treatment from the midpoint of plant growth (Table 3) in both the experimental years.
Experimental results revealed that the chemical fertilizer treated crop plants gain more or less same weight with combined biofertilizer treated crop plants on 30 DAS, but as the day proceed, sole chemical fertilizer treated crop can no longer maintain the same trend of weight gain. This finding indicates that as the day proceeds, dry weight accumulation enhanced with the combined
use of biofertilizer and chemical fertilizer [19]. On the other hand, fresh biomass for leaf, root and shoot showed remarkable significant (p < 0.05) changes between different treatments at 45 DAS. But fresh weight of leaf does not exhibit any significant changes between treatment to treatment at initial stage of observation (30 DAS). Results also highlighted that different treatment combinations has no significant change (p > 0.05) towards dry biomass of leaf, root and shoot. From the result it was also clear that plant growth promoting rhizobacteria play a vital role in fresh and dry weight accumulations of plants with reduced dose of chemical fertilizer. Increased dry matter may be due to the ability of bio-fertilizers to transport major nutrients like N and P as well as secreting plant growth promoting substances. A study by Zarei et al. [20] had also shown that nitrogen fixing bacterial inoculation increased both fresh and dry weights of soybean.
The highest siliquae per plant was recorded for the treatment T4 in both first (69.57) and second (70.99) year of experiment. However, sole chemical fertilizer treated plot (T5) exhibited much lower number of siliquae (56.43 and 58.37) in both the experimental years. The length and diameter of siliquae were recorded maximum for treatment T2 as 4.13 cm and 1.07 cm, respectively. But in second year both length (4.63 cm) and diameter (1.41 cm) of siliquae were highest for the treatment T4. As siliquae per plant, both length and diameter also showed much lower in chemical fertilizer treated plot (T5). On the other hand, almost similar upliftment of the yield
S. Dutta et al. / Annals of Agrarian Science xxx (2017) 1—6
attributes such as number of seeds per siliqua, test weight (g) of seed, seed yield (g/m2) and straw yields (g/m2) were recorded for the combined applications of biofertilizer and reduced dose of chemical fertilizer over only chemical fertilizer treatment (T5) (Table 4). Bacteria used in this study may increase seed yield by providing essential micro and macro nutrients for plant growth, producing growth stimulating material, root system development and anti-pathogenic effect of crop plant [21].
Scanning electron microscopic (SEM) study also revealed that, biofertilizer treated plants (T4) had prominent vascular system (Fig. 2A) as compared to only chemical fertilizer treated plants (T5). Moreover, in chemically treated plants, few wrinkles in the vascular tissues were observed (Fig. 2B) and major impairment was recorded in the vascular tissues (xylem and phloem) of the plants. This is perhaps due to excessive accumulations of chemical fertilizer into the cellular body. Almost similar cellular deformation was reported by Mondal et al. [22] under varying cadmium stresses on chickpea (Cicer arietinum L.).
Therefore, our result highlighted that dual application of bio-fertilizer and reduced dose of chemical fertilizer has some additive effect over chemical fertilizer. This is perhaps due to the perfect balancing of nutrient uptake in the root environment which equally support for beneficial bacterial growth and plant growth [20]. Previous research [20] also support that proper supply of mineral, especially, phosphorus can increase flowering rate. Moreover, both phosphorus and nitrogen can improve the reproductive growth and fruit production in the plant.
4. Conclusions
Our present investigation revealed that, based on the attributes of growth and in terms of yield, the best treatment was T4 [Pseudomonas putida + Burkholderia cepacia + Burkholderia sp. + 25% reduced dose of chemical nitrogen fertilizer (75 kg ha-1 N: 50 kg ha-1 P: 50 kg ha-1 K)] out of the five applied combination treatments under this old alluvial soil agro-climatic zone. The mustard cultivar Bg can be cultivated for a better yield of mustard with this treatment combination. The mixed biofertilizer was more effective than other three indivisual biofertilizers. This may due to the synergistic interactions exists between the applied microorganisms. Throughout the study it was found that, individual bacterial inoculum with 25% reduced dose of chemical N-fertilizer significantly enhanced overall plant growth. This means 25% nitrogen fertilizer dose can easily be supplemented by natural resource to make mustard cultivation more productive over a long period toward achieving the ultimate goal of sustainability in mustard cultivation. It was also found that plant growth attributes were enhanced in the second experimental year in all the studied parameters. The reason may probably that biofertilizers restore the soil natural nutrients status and balance the nutrient level of soil as well as soil health. The wide scale application of PGPR may decrease the global dependence on agricultural chemicals.
Therefore, it can be suggested that judicious use of chemical fertilizers can lead toward increase in yield under this agro-climatic condition of the old alluvial soil of Burdwan, West Bengal, India. This type of practice can, therefore, be applied in the field for commercial scale of crop production with less environmental pollution with good economic returns.
Acknowledgements
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http:/ dx.doi.org/10.1016/j.aasci.2017.02.015.
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