Evaluation of Indigenous Bradyrhizobium Japonicum Liquid formulated inoculant as a Seed Treatment on Growth

Evaluation of Indigenous Bradyrhizobium Japonicum Liquid formulated inoculant as a Seed Treatment on Growth, Nodulation and Yield of Soybean
ABSTRACT
A liquid formulation of an indigenous, rhizospheric, potentially virulent Bradyrhizobium japonicum strain (APEXBJ2) was developed by amending synthetic production medium with nontoxic polymers like Polyvinyl Pyrrolidone (PVP) and Na-alginate. The strain was isolated from Gazipur, Bangladesh and identified by 16S rDNA sequencing. The liquid bioinoculant formulated with PVP (1.8%) and Na-alginate (0.2%) promoted long term survival of APEXBJ2 with a better shelf life (109cells/ml) compared to control (102cells/ml) at 6 months of storage and supported 107 cells/ml up to 12 months of storage under ambient temperature (28°C±2), whereas PVP (1.8%), Na-alginate (0.2%) recorded the same population up to 8 months and 6 months respectively. Several field performances of the liquid formulation used as seed coating were evaluated in the consecutive years (2013-2015) to evaluate nodulation and yield of soybean in the presence and absence of inoculation, and the efficacy of liquid formulation as an alternative to peat-based inoculants. Nodule number per plant was significantly influenced by APEXBJ2 formulation alone. The yield and the yield components such as number of pods per plant, number of seeds per pod, seed yield, and yield per hectare (P ? 0.05) was affected significantly by inoculation of APEXBJ2 formulation alone. Therefore this liquid formulation of Bradyrhizobium will open a new avenue in the organic cultivation of soybean and associated legumes in the farming arenas of Bangladesh.

INTRODUCTION
Nitrogen gas (N2) is one of the most abundant elements on earth and comprises nearly 80 percent of total atmospheric gases, yet most organisms are unable to use N2 as a source of nitrogen. Legumes, such as soybean, are able to capture atmospheric nitrogen and utilize it through the process of nitrogen fixation. Soybean acts as the host plant and recognizes Bradyrhizobia japonicum when it is placed nearby in the soil. Soybean inoculated with B. japonicum will form structures called nodules on the plant’s roots (Madrzak et al., 1995). These nodules house the bacteria. Atmospheric nitrogen (N2) is converted by B. japonicum to ammonia (NH3) which the soybean plant can use. This symbiotic relationship is of special significance to legume husbandry as seed inoculation with effective strains of B. japonicum can meet the nitrogen requirements of the soybean to achieve increased yields and additionally, does not cause any hazard to the environment. Soybean uses large amounts of nitrogen throughout the growing season with requirements ranging up to 353 kg/ha, that should be guaranteed by biological nitrogen fixation (BNF)( Hungria et al., 2006, Nutrient Management for Agronomic Crops in Nebraska, 2000). Otherwise depletion of the soil N reserves is likely to occur (Peoples et al., 1995). Obviously, such a phenomenon is of world-wide interest because it implies lesser dependence on expensive petroleum based nitrogen fertilizers for soybean.

Soybean extension is important for Bangladesh as it can be a source of much sought-after protein for livestock, fisheries, and humans. Heavy rainfall, overuse of fertilizers and depletion of organic matter has degraded land in most areas of this country. This has resulted in significant soil pH decreases over the years and decline of the microbiome. Wide-scale soybean plantations can improve land conditions as well as produce a profitable and useful crop. Only 9% of oilseed land is planted to soybean in Bangladesh (Bangladesh Oilseeds and Products Annual, 2017). Farmers will only be willing to plant soybean in the Rabi (winter) season instead of other alternative crops like rice and potato if it is profitable to them. Profit will increase with higher yields. It is necessary to inoculate soybean seeds with high-quality rhizobium to get the maximum yields of soybean on a large scale. Seed inoculations promote root nodulation around the primary root near the location where the inoculum was placed on the seed, as B. japonicum is relatively mobile (Ciafardini 1987). Plant improvement generally seeks to increase the proportion of dry matter production that goes to seed production (Lukiwati and Simanungkalit, 2002). It was observed that the symbiotic nitrogen fixation in legume depends upon co-selection of plant and bacterium genotypes (R. Mytton, L ; Skøt, Leif, 1993). Isolation of native symbiotically efficient isolates is proved to be useful in increasing the nitrogen fixation and greater soybean yield (Melchiorre et al., 2011).
Soybean seed inoculation by rhizobial biofertilizers has been practiced in recent years in Bangladesh to improve soybean yields in fields. Yield increases up to 75–200% have been observed in soybean inoculated by B. japonicum compared to non-inoculated fields where soybean has never been grown before (Sattar et al., 1996). Inoculation with highly effective rhizobia, a common practice in agricultural production (Catroux et al., 2001), requires survival and establishment of inoculated rhizobia in the soil (Da and Deng, 2003). Many of the rhizobium inoculants in Bangladesh are based on solid peat formulations. Rhizobium in peat carrier is not very practicable for wide adaptation by farmers as the bulk will be considerable and therefore both transport and storage will be more difficult compared to liquid inoculants. It is also difficult to find peat suitable for inoculant carrier in Bangladesh and they are limited in supply. Different batches of peat may also differ in composition and texture. A break through is needed in the inoculants technology to improve the shelf life and field efficacy of bio-fertilizers in Bangladesh to make them commercially viable and acceptable to farmers. Death of the organisms in the inoculated seed due to desiccation is one of the important factors contributing to the failure of inoculation response under field conditions. Liquid formulations have been produced and widely used for decades in many countries of the world like USA, Canada, Australia, Argentina, Brazil and India. We have tried to adopt the technology for small and marginal farmers of Bangladesh. We found that rhizobium in the liquid formulation has greater shelf life than peat based formulations and will be much easier to transport and be more acceptable to farmers. Therefore this study was conducted to evaluate the effect of indigenous Bradyrhizobium japonicum liquid inoculants on the nodulation of soybean plants and subsequent impact of inoculation on their growth and yield over chemical fertilizers. Soil and climate factors and agricultural practices that affect nodulation, growth and yield of soybean were studied on a widely distributed area of northern Bangladesh over two seasons and several years.
Materials and Methods
Isolation and Identification Bradyrhizobium japonicumIsolation of the bacteria
Soybean plants were planted in soil collected from Boroibari, Gazipur. Nodules formed on these plants were collected after 6 weeks from freshly uprooted plants and washed thoroughly under tap water to remove the soil particles. Nodules were surface sterilized by imbibing in 70% ethanol for 30s and in 50% Clorox for 4 min then washed 6 times with sterile water. The nodules were put in 1.5mL microfuge tubes containing 0.5mL of 0.9% NaCl solution. Then the nodules were crushed with the help of sterile forceps and streaked on CRYEMA plate (Somasegaran and Hoben, 1994). Streaked plate was incubated at 30oC for 5 days. After an inoculation of 5 days at 30oC, single colonies were selected and re-streaked on YEMA for pure culture.
Characterization of the bacteria
The isolates were gram stained and those found to be gram negative were selected. The isolates were tested for growth in glucose peptone agar media and those which failed to grow in this media were selected. The isolates were also tested for acid production by streaking them on YEMA containing bromothymol blue and they did not change the color of the media from blue to yellow. The isolates were grown in liquid media and pH change was monitored. Isolates which did not produce acid were selected.
Definitive identification of the isolate by sequencing 16s rDNA:
Isolate APEXBJ2 was identified as by sequencing the first 500 bases of 16S rDNA. The obtained sequence showed 94% query cover and 98% identity with Bradyrhizobium japonicum USDA 110 strain USDA110 16S ribosomal RNA, complete sequence NCBI Reference Sequence: NR_074322.1 and gene bank accession number KF279677.1
Seedling infectivity test:
Soybean seedlings inoculated with the isolate APEXBJ2 was grown on N-free seedling growth medium (Burton, 1984) and it grew and produced nodules while control seedlings fails to form nodule and subsequent growth.
Formulation of Liquid Inoculant
Production Media and Cell Culture in Fermenter
Synthetic medium containing K2HPO4 (0.5g), NaCl (0.1g), KNO3 (0.9g), (NH4)2HPO4 (0.4g), MnSO4 (0.01g), FeSO4.7H2O (0.05g), Monosodium glutamate (1.0g), Yeast extract (1.0g), Glycerol (12g) was used as a basal production medium for liquid inoculant formulation with selected appropriate concentrations of additives. The compositions of synthetic medium used for cultivating viable B. japonicum (APEXBJ2) to late exponential-early stationary growth phase at pH of 6.8 to 7.0 with 30°C constant temperature. Higher numbers of bacterial cells (CFU 1.0×109 to 1.19×1010) are produced using synthetic medium by 57- 62 hours incubation using 2% pure seed culture in fermenter.

Liquid Inoculant Preparation
Nontoxic polymer like Polyvinyl Pyrrolidone (PVP) (1.8%) and Na-alginate (0.2%) are then mixed with the harvested culture and finally packaged into sterile High-density polyethylene (HDPE) bottles. The survival of strain(s) was enumerated at monthly intervals up to one year by serial dilution technique (Sehrawat et al., 2015). Formulation containing PVP and Na-alginate showed higher survival of bacterial cell till 360 days in HDPE bottles (Table-1). Peat based carrier was sterilized by gamma-irradiation and formulated according to Roughley, 1970.

Field evaluation
Field Trial with Different Carrier Based Formulation
The experiment was conducted at the research field of Apex Biofertilizers and Biopesticides Limited, Gogindaganj, from December 2013 to April 2014 to study the effect of liquid biofertilizer on the yield of BARI soybean-5 over Peat based formulation.
Geographically the experimental site was located at 25º11 N latitude and 89º28 E longitudes which fall in agro-ecological Zone 3 of Bangladesh. The soil of the experimental field was comparatively neutral in reaction having soil pH: 6.85; Soil organic matter: 1.82%; Soil Nitrogen: 0.10%; Soil Phosphorus: 0.43 µg/g; Soil Potassium: 0.07 meq/100g; Sulphur: 14.59µg/g; Magnesium: 0.13meq/100g; Calcium: 0.73meq/100g; Boron: 0.10µg/g and Zinc: 2.09µg/g.

The experiment was laid out in a Randomized Complete Block Design (RCBD) with three replicates. A unit plot size was 3m x 2m. Distance between rows was 30 cm and between plants were 7.62 cm for proper development and growth of plants. On the other hand plot-to-plot and block to block distance were 0.5 m and 1.0 m respectively. Seven treatments were used as experimental variables and assigned randomly in each replication. Applied chemical fertilizers were calculated for soybean using the fertilizer recommendation guide 2010. 100% Application doses of chemical fertilizer for 3m x 2 m area was: Urea (N): 30.26 g; TSP (P): 148.29g; MOP (K): 101.33 g; Gypsum (S): 33.48 g; Boric Acid (B): 0.80 g.

Among the seven treatments, uninoculated fresh seeds were used in two treatments (positive controls) and in the other five treatments seeds were coated with the same stain of B. japonicum (APEXBJ2) biofertilizer in different formulations (liquid based or peat based). Seeds were inoculated in the field using three months old inoculant. The seeds were sown in furrows at an approximate depth of 2 cm. Necessary intercultural operations such as weeding, application of insecticide and fungicide were done as and when necessary.

The crop was harvested at maturity. Ten comparative plants were selected randomly from each unit plot for recording all data of nodulation, morpho-physiological traits and yield attributes. Grain yield of soybean was determined by harvesting an area of 1m x 1m and converting the grain yield value to ton ha-1 at 12% moisture content.

Data were analyzed by analysis of variance (ANOVA) using MSTAT package and the means were compared according to Tukey’s W Test (Table-2).

Field Trial With different Nitrogen Doses
Two field trials were conducted to evaluate the effects of B. japonicum (APEXBJ2) liquid inoculant on growth, nodulation and yield of BARI soybean-5 at Rangpur and Noakhaili in 2014-2015. The experiments were laid out in split plot design with three replications. Treatments comprise two levels of B. japonicum (APEXBJ2) inoculants (I) were three levels of nitrogen viz. without nitrogen (N0), 20 Kg Nitrogen/ha (N20) and 40 kg nitrogen/ha (N40) were used in the study. TSP, MOP, Gypsum, Zinc sulphate and Boron were applied as basal application @ 25, 60,15,2 and 1 kg/ha P,K,S,Zn and B respectively at final land preparation. Data on nodule number and dry weight were recorded at mid flowering stage. Yield contributing parameters and yield were recorded at harvest and data were analyzed statistically (Table-3).

Economic analysis of different Nitrogen Doses and Liquid Inoculant
Economic analysis of inoculation/uninoculation (with and without different N doses fertilizer application) treatments at two sites was done. The estimated variable cost, gross return, net return, marginal return, marginal benefit cost ratio (MBCR) were presented in Table 4.

Results and Discussion
Survival of APEXBJ2 stain in liquid Formulation
Survival of APEXBJ2 stain was also studied in formulated liquid carrier stored at 28°C±2 dark condition. The results showed that formulation containing PVP and Na-alginate supported more than 109 bacteria ml-1 after 180 days of storage (Table: 1). Die-off of APEXBJ2 strain slowly occurred in harvested culture control medium. Thus no viable cells were detected after 240 days in these media. The liquid formulations tested supported a passable survival of APEXBJ2 strain, providing more than 107 bacteria ml-1 at the end of the assay. During the first 120 days formulation containing only PVP had the highest number of viable cells but, at the end of the assay, formulation containing PVP and Na-alginate was the best additives in maintaining high populations of this strain.

Table 1: Survival of B. japonicum (APEXBJ2) in liquid formulation at room temperature
Formulation Colony forming units (CFU)/ml After formulation
Initial CFU
120
days 180
days 240
days 300
days 360
days
Control 5.67×109 4.53×105 3.50×102 – – –
Na-Alginate (0.2%) + PVP (1.8%) 5.30×109 2.53×109 1.93×109 6.10×108 5.10×107 1.53×107
PVP (1.8%) 5.47×109 4.91×109 8.40×108 1.63×107 1.44×106 7.25×105
Na-Alginate (0.2%) 5.38×109 3.70×108 6.80×107 4.60×106 3.90×105 1.96×105
Field Experiments
Evaluation of Carrier Based Formulation
Results show the effects on nodulation, yield contributing characters and yields of BARI soybean-5 of different carrier based formulation of B. japonicum (APEXBJ2) (Table-2). The inoculants had no or diminutive effect on field stand (Plants m-2), Plant height (cm) and seed size (100 Seed weight) but it significantly increased the total number of nodules per plants and nodules on taproot over uninoculated treatments. Seed inoculations promoted root nodulation which subsequently influenced the growth and yield of soybean. The highest number of branches (3.60) were recorded when seeds were coated with liquid Carrier Na-Alginate 0.2% and PVP 1.8% (Table-2). Liquid Carrier (Na-Alginate 0.2% and PVP 1.8%) inoculated treatment gave significantly higher number of Pods plant-1, Seeds Pod-1, Seed weight plant-1 and finally Yield of soybean. However the values were not significantly different from those of Peat Carrier (APEXBJ2 Biofertilizer) inoculated treatment. The lowest yield (1.20 ton ha-1) was observed in the treatment with 25% urea fertilizer and the highest yield (2.45 ton ha-1) was observed in the treatment where the seeds were inoculated with Liquid Carrier (Na-Alginate 0.2% and PVP 1.8%).
Liquid Carrier (B. japonicum APEXBJ2) therefore reduces the use of 75% chemical nitrogen fertilizer and the yield of this treatment was even greater than the yield of 100% chemical fertilizer treatment.

Evaluation of different Nitrogen Doses with Liquid Formulation
Results (Table-3) showed that liquid inoculant increased plant height, nodule number, nodule dry weight, grain yield and stover yield of soybean significantly compared to uninoculated control. Nodule number and dry weight increased by 17.9% and 31 timed higher over uninoculated control treatment. Grain yield showed 30% higher compared to uninoculated whereas stover yield by 19% higher over control at Rangpur, and 27% higher grain yield and 10% higher stover yield at Noakahali. Nitrogen application showed significant effect on plant growth, grain yield and stover yield but no significant effect on nodule number and dry weight rather higher dose of nitrogen showed negative effect on nodule number and dry weight. Grain yield and stover yield increased significantly due to nitrogen application. A dose of 40 Kg N/ha application showed the highest grain and stover yield of soybean. Inoculation without nitrogen recorded similar grain and stover yield with 40 kg N application without inoculation (I0*N40). Significant interaction effect was observed in nodule dry weight, grain yield and stover yield at Rangpur and sotver yield of Noakhali location.

Table 2: Nodulation, yield attributes and yields of BARI soyben-5 using different carrier based formulations of Bradyrhizobium japonicum (APEXBJ2)
Treatment Plants m-2 Plant height (cm) Branch plant-1 Nodule plant-1 Nodule plant-1 on taproot Pods plant-1 Seeds Pod-1 100 Seed wt. (g) Seed weight (g plant-1) Yield
(ton ha-1)
T1 35.33 51.20 3.27 b 2.20 c 0.00 d 39.60 b 1.57 bc9.57 5.64 bc1.72 bcT2 35.00 48.73 2.47 e 3.93 c 0.33 d 28.67 c 1.37 c 8.90 3.98 c 1.20 c
T3 35.00 51.13 2.93 cd 44.47 ab9.67 b 47.53 ab1.67 ab9.77 7.49 ab2.05 abT4 35.33 49.33 3.20 bc41.87 ab8.33 b 39.87 b 1.57 bc9.40 5.52 bc1.65 bcT5 36.00 51.13 2.83 d 39.53 ab5.67 c 39.33 b 1.62 abc9.50 5.77 bc1.69 bcT6 36.00 50.80 3.60 a 46.20 a 12.00 a 50.47 a 1.88 a 9.97 8.65 a 2.45 a
T7 36.00 53.40 3.07 b-d 28.13 b 9.00 b 40.20 ab1.47 bc9.03 5.00 bc1.43 bcCV% 3.80 16.96 9.84 25.18 11.59 10.20 8.59 6.95 15.45 12.70
Values in a column with same letter(s) are not statistically different at 0.05 level of significance by Tukey’s W test.

Note: T1= 100% chemical fertilizer used, T2= 25% urea+100% others chemical used, T3= T2 + Peat Carrier (APEXBJ2), T4= T2 + liquid Carrier Na-Alginate 0.2% (APEXBJ2), T5= T2+ liquid Carrier PVP 1.8% (APEXBJ2), T6= T2+ liquid Carrier Na-Alginate 0.2% + PVP 1.8% (APEXBJ2) and T7= T2+ Cell culture without formulation (APEXBJ2)
14287522606000
3096260306705D
D
47625193675C
B
00C
B

Fig1. Effect of inoculation of liquid formulation of Bradyrhizobium japonicum (APEXBJ2) on root nodulation of soybean 30 days after germination; A= Uninoculated control (100% chemical fertilizers), B= Inoculation of liquid inoculums (Na-Alginate 0.2% and PVP 1.8%), Nodulation after first flowering of BARI-5 soybean; C= Liquid formulation (Na-Alginate 0.2% and PVP 1.8%), D= Conventional peat based solid formulation of B. japonicum.

Economic Analysis of Different Nitrogen Doses with Liquid Formulation
At Ranglpur (Table-4), liquid inoculant (I) exhibited higher gross and net return (64,580/- and 54,995/-, respectively) over uninoculated control (I0). Among N levels N40 treatment showed the highest gross and net return (63,680/- and 53,567/-, respectively) followed by treatment N20 (49,780/- and 40,495, respectively). The highest gross return was found in treatment combination I×N40 (65,140/-) followed by I×N20, I×N0 whereas the highest net return was with treatment I×N20 (54,815/-). Marginal benefit cost ratio (MBCR) was found highest with treatment I×N0 (2.67) followed by treatment combination I×N20 (2.58), I×N40 (2.44) and I0×N40 (2.35). Marginal return was recorded highest in treatment I×N20 (25,472/-) followed by I×N40, I×N0, I0×N40 and I0×N20.

At Noakhali (Table-4), liquid inoculant treatment (I) exhibited the higher gross and net return (84,720/- and 75,135/-, respectively) over uninoculated control (I0) (66,920/- and 57,635/-, respectively). Nitrogen treatment N40 recorded the highest gross and net return (83,180/- and 73,067/-, respectively) followed by treatment N20. From inoculation and nitrogen combination (interaction) the highest gross return (87,320/-) and net return (76,910/-) were observed with treatment combination I×N40 followed by treatment combination I×N20 (84,680/- and 74,795/- as gross and net return, respectively). Similar trend was found in marginal return and marginal benefit cost ration (MBCR). Treatment combination I×N40 showed the highest marginal return (27,687/-) and MBCR (2.66) followed by treatment combination I×N20 (2.59) and I×N0 (2.49).

Conclusion
Considering the findings, it is suggested that, the use of liquid formulation of B. japonicum (APEXBJ2) could augment the N nutrition of soybean leading to increased yield and yield contributing parameters. The liquid formulation produced yield increase over the control with 25% Urea, and even showed significant yield increase over 100% chemical fertilizer treatment. The formulation is thus able to reduce the use of chemical nitrogen fertilizer by 75%. The result also depicts that; comparatively higher yield was also obtained in case of liquid inoculum over peat carrier based treatment.

And from economic analysis, it is revealed that liquid APEXBJ2 inoculant is more beneficial. Farmers can apply this liquid inoculant instead of nitrogenous fertilizer urea in soybean production.

The liquid formulation technology has become more popular than carrier based biofertilizers in recent years, because of extended shelf life and user friendly application procedure. The agricultural microbiologists have utilized their innate expertise to develop Liquid biofertilizer formulations as a promising and updated technology over conventional carrier based production technology.Table 3: Effect of liquid Bradyrhizobium japonicum (APEXBJ2) inoculant on growth , nodulation, grain and straw yield of soybean at Rangpur and NoakhaliPlant height (cm) Nodule Number/ plant Nodule dry weight (mg/plant) Grain yield (t/ha) Straw yield (t/ha)
Treatments RangpurNoakhaliRangpurNoakhaliRangpurNoakhaliRangpurNoakhaliRangpurNoakhaliInoculant Uninoculated (I0) 77.07b 65.60b 1.57b 2.63b 9.84b 6.63b 1.71b 2.40b 2.66b 2.26b
Inoculated (I) 84.24a 74.02a 26.66a 23.96a 333.61a 209.21a 2.24a 3.04a 3.17a 2.84a
Significant level * * ** ** ** ** * ** * *
CV(%) 2.39 2.80 14.11 14.12 13.25 10.71 6.52 7.74 5.28 6.78
Nitrogen N0 78.32b 67.28b 15.43 14.78a 134.35a 114.87 1.75b 2.52a 2.69b 2.27b
N20 80.67ab 69.10b 14.35 13.65ab 129.42a 105.00 1.96b 2.69ab 2.94ab 2.52b
N40 82.98a 73.05a 12.55 11.45b 105.92b 103.90 2.21a 2.96a 3.11a 2.86a
Sig. level ** ** NS * * NS ** * ** *
CV (%) 2.39 2.80 14.11 14.12 13.25 10.71 6.52 7.74 5.28 6.78
Inoculant × Nitrogen I0×N0 73.07 61.67 1.53 2. 30 5.53c 6.63 1.30b 2.09 2.15c 1.82c
I0×N20 77.53 64.80 1.90 2.93 12.17c 6.57 1.67ab 2.33 2.65b 2.22b
I0×N40 80.60 70.33 1.27 2.67 11.83c 6.70 2.15a 2.78 3.16a 2.75a
I × N0 83.57 72.90 29.33 27.27 263.17a 223.10 2.20a 2.94 3.23a 2.73a
I × N20 83.80 73.40 26.80 24.37 146.67b 203.43 2.24a 3.04 3.23a 2.82a
I × N40 85.37 75.77 23.83 20.23 200.00a 201.10 2.27a 3.13 3.06ab 2.97a
Sig. level NS NS NS NS * NS ** NS ** *
CV (%) 2.39 2.80 14.11 14.12 13.25 10.71 6.52 7.74 5.28 6.78
In a colum, having same letter(s) do not differ significantly at 5% level of probability as per DMRT.

**= Significant at 1% level; *= Significant at 5% level

Table 3. Cost benefit ratio of soybean using liquid APEXBJ2 inoculant and urea in winter season 2014-2015
Treatments Rangpur (2014-2015) Noakhali (2014-2015)
Yield (t/ha) Gross Return (TK) Variable Cost
(TK) Net Return
(TK) Marginal Return
(TK) MBCR Yield (t/ha) Gross Return (TK) Variable Cost
(TK) Net Return
(TK) Marginal Return
(TK) MBCR
Grain Straw Grain Straw Liq. Inoculant I0 1.17 2.66 49,780 9,285 40,495 – – 2.40 2.26 66,920 9,285 57,635 – –
I 2.24 3.17 64,580 9,585 54,995 14,500 1.51 3.04 2.84 84,720 9,585 75,135 17,500 1.83
Nitrogen N0 1.75 2.69 50,880 9,057 41,823 – – 2.52 2.27 70,160 9,057 61,103 – –
N20 1.96 2.94 56,840 9,585 47,255 5,432 0.57 2.69 2.52 74,980 9,585 65,395 4,292 0.45
N40 2.21 3.11 63,680 10,113 53,567 11,744 1.16 2.96 3.11 83,180 10,113 73,067 11,964 1.18
Inoculant × Nitrogen I0×N0 1.30 2.15 38,100 8,757 29,343 – – 2.09 1.82 57,980 8,757 49,223 – –
I0×N20 1.67 2.65 48,720 9,285 39,435 10,092 1.09 2.33 2.22 65,020 9,285 55,735 6,512 0.70
I0×N40 2.15 3.16 62,220 9,813 52,407 23,064 2.35 2.78 2.75 77,780 9,813 67,967 18,744 1.91
I × N0 2.20 3.23 63,660 9,357 54,303 24,960 2.67 2.94 2.73 81,900 9,357 72,543 23,320 2.49
I × N20 2.24 3.23 64,700 9,885 54,815 25,472 2.58 3.04 2.82 84,680 9,885 74,795 25,572 2.59
I × N40 2.27 3.06 65,140 10,413 54,727 25,384 2.44 3.13 2.97 87,320 10,413 76,910 27,687 2.66
Price of Soybean grain= 26.00/kg (1US$ = 81 taka); Soybean Stover= 2.00/kg; Urea =12.00/kg; TSP=22.00/kg; MOP= 17.00/kg; Gypsum= 9.00/kg;
Zinc sulphate= 120.00/kg; Boron=140.00/kg; APEXBJ2 liquid inoculant=600.00/liter