Phosphorus (P) is one of the major limiting nutrients to mung bean production. However, the use of imbalance P nutrient and lack of inocula decreased the mungbean yield in Bangladesh. Therefore, this study has been untaken to study the effect of different phosphorus levels and Bradyrhizobium inocula on the yield and protein content of mungbean. Treatment comprised of four levels of phosphorus - P₀, P₃₀, P₆₀, P₉₀ (0, 30, 60 and 90 kg P₂O₅ ha^-1 respectively) and strains of  Bradyrhizobium inocula - I₀, I₁, I₂ and I₃ (not inoculated, inoculation with BINA-MB-THA 301, BINA-MB 441 and BINA-MB 301). The highest seed yield (1066.3 kg ha^-1) was obtained from P₆₀ followed by P₉₀ (957.7 kg ha^-1) and P₃₀ (870.5 kg ha^-1) while the lowest seed yield (578.9 kg ha^-1) was obtained from the control. Protein content was highest (24.5 %) in P₉₀, followed by P₆₀ (24.4%) and P₃₀ (24.4 %), and the lowest (24.1 %) was obtained from control. Inoculation of seed increased the yield and yield components, and harvest index. Better performance of the yield components and protein content of seed was found with the inoculants I₁. The I₁ strain produce the highest seed yield (982.5 kg ha^-1) while the lowest yield (670.5 kg ha^-1) was obtained from uninoculated control plot. The highest protein content of mungbean seed (25.3 %) was estimated when I₁ strain of Bradyrhizobium was used for seed inoculation. Among the treatment combinations, P₆₀I₁ provided better growth and yield performance and increased protein content of mungbean. 

 Keywords: rhizobium strain, inoculation, yield, protein content, orthogonal comparison

Mungbean (Vigna radiata L. Wilczek) is one of the major and short duration pulse crops grown in pre-monsoon season of Bangladesh. It belongs to the family Leguminosae and sub family Papilionaceae. The crop is commonly known as green gram, golden gram, mung dal, mungbean but most commonly as ‘Mung’ in Bangladesh. It is an excellent source of easily digestible protein which complements the stable rich diet in the country. Mungbean seed contains 51 % carbohydrate, 26 % protein, 1 % fat, 4 % minerals, 3 % vitamins and 10 % moisture.¹ Mungbean covered an area of 44006.32 hectares with a production of 37054 tonnes in 2019-20.² The average yield of mungbean is about 0.842 t ha^-1, which is very low compared to the potential yield (1.5-1.6 t ha^-1) of mung bean in Bangladesh.³ The yield difference between potential and actual yield indicates the wide scope for increasing yield of mungbean in Bangladesh with improved management practices and by using proper doses of fertilizer.

Optimum amount phosphorus supply is important to fix nitrogen in the soil by the legumes. As mungbean is a legume crop, it responds well to added phosphorus.⁴ Phosphorus deficiency causes yield reduction by limiting plant growth.⁵ It influences nutrient uptake by promoting root growth and nodulation.⁶ The use of phosphorus nutrient has steadily increased overtime. The P consumption in 1981 was 80000 ton which in 2016 increased to 210000 ton in Bangladesh.⁷ Mungbean responds favourably to phosphorus fertilization.⁸ Phosphorus enhance the uptake of nitrogen, thus increase nitrogen content in the crop which increase protein content of mungbean.⁹

Presently, a number of organisms like Rhizobium/Bradyrhizobium have been identified to use as biological agent for fix atmospheric nitrogen by symbiotic process with legume crops and make it available to the plants. Bangladesh Institute of Nuclear Agricultural (BINA) isolated some Rhizobium/Bradyrhizobium strains especially for mungbean cultivar. To reduce the production cost and to fulfill the demand, more pulse production could be achieved through seed inoculation with Rhizobium/Bradyrhizobium. As per Franco,¹⁰ Rhizobium/Bradyrhizobium strains in association with the host plant were able to fix approximately 20 % of the atmospheric nitrogen through the world annually. Singh et al.¹¹ reported that seed inoculation of mungbean with Bradyrhizobium increased the protein content of mungbean. Hence, there is a large scope of utilizing the biological nitrogen fixing technology for obtaining more protein rich food from mungbean and also to improve the nitrogen status of soils by selecting efficient strains of mungbean inocula. The use of effective strain of mungbean inocula can play a vital role in improving yield and soil health. The strains of mungbean inocula identified by BINA which are suitable for better yield performance of mungbean, but the potentialities of different strain of Bradyrhizobium may vary depending upon the soils and climatic conditions. Therefore it is urgently needed to verify the potentialities of different strains of Bradyrhizobium in mungbean crop. Limited studies are available to find out the effect of phosphorus and inoculum separately, however, to the best of our knowledge, no studies on the combined effect of these two factors on mungbean are available in Bangladesh. Considering the above facts, the present study has been undertaken to find out the optimum level of phosphorus and suitable Bradyrhizobium strain to achieve high yield and protein content of mungbean.

Site of the research

The field experiment was conducted at the Agronomy Field Laboratory, Bangladesh Agricultural University (BAU), Mymensingh, during the period of March to June (2013) in the Kharif-1 season to study the effect of phosphorus level and different strain of Bradyrhizobium inocula on the yield performance and protein content of mungbean (cv. BINA Mung-2). Geographically the experimental field was located at 24.75⁰ north latitude and 90.50⁰ east longitude at an altitude of 18 m above the mean sea level.

Initial Soil properties at 0-15 cm soil depth

The soil is belongs to the Old Brahmaputra Alluvium Soil under Agro-Ecological Zone 9 (AEZ-9, Old Brahmaputra Floodplain) under Sonatala series.¹² The experimental site falls into Non-calcareous Dark Grey Floodplain soils (Aeric Haplaquept). The land topography was medium high, silty loam soil texture.  The soil was sampled from six to seven random spots at 0-15 cm depth of the experimental plot and analyzed for both physical and chemical properties. The initial soil characteristics at 0-15 cm soil depth before laying out the experiment are presented in Table 1. 

Weather condition during study period

The experimental site is under a subtropical monsoon climatic region with a mean monthly maximum and minimum temperature of 29 °C and 19 °C, respectively, with total rainfall of 217 mm during the mungbean growing season (Figure 1). During the mungbean growing seasons (March-May), the highest rainfall of 125 mm was received in April, followed by 76 mm in 2011, while only 5.1 mm and 21.4 mm of rainfall was received in February and March, respectively in 2013. Most of the rainfall was received in April, accounting for nearly 44 % of the total rainfall.

Figure 1 Mean monthly temperature and total rainfall during mungbean growing season of the research area, recorded from the local weather station.

Treatments and experimental design

BINA Mung-2 was used as the test crop. The cultivar is suitable for growing in the summer season.¹³ Four levels of Phosphorus @ 0, 30, 60 and 90 kg P₂O₅ ha^-1 form TSP were applied and four inocula namely not inoculated (I₀), Inoculation with BINA-MB-THA 301 (I₁), Inoculation with BINA-MB 441 (I₂), Inoculation with BINA-MB 301 (I₃) were used in the experiment. Fresh seeds of mungbean were dipped in the culture broth for 18-20 minutes. The experiment was laid out in a Randomized Complete Block Design (RCBD) having three replications. The unit plot size was 4.0 m x 3.0 m.

Crop management

Mungbean seeds were sown in the furrow on 7^th March, 2013. The line to line distance was 30 cm with continuous sowing in the line. Other than phosphorus, the crop was fertilized during final land preparation at the rate of 20 kg N ha^-1 as urea, 20 kg K ha^-1 as muriate of potash (MP), 10.8 kg S ha^-1 as gypsum and 1 kg B ha^-1 as boric acid, as recommended by the Pulses Research Centre, Bangladesh Agricultural Research Institute. All fertilizers were applied by broadcasting and were mixed with soil thoroughly. Weeding and thinning were done at 18^th and 35^th days after sowing (DAS). Plant to plant distance was maintained at 6-7 cm. During the experimental period, there was heavy rainfall for several times. Hence, irrigation was not necessary but it was essential to drainage the excess water from the field. Jute hairy caterpillar and pod borer were successfully controlled by the application of Malathion 57 EC @ 1.5 L ha^-1 at the time of 50 % pod formation stage (55 DAS). At the time when 80 % of the pods turned brown colour, the crop was assessed to attain maturity. The crop was harvested on 21^st May, 2013 from pre-selected 4.0 m² area.

Data and their estimation procedures

Yield components for mungbean, i.e. plant height (cm), branches plant^-1, pods plalnt^-1, sterile pods plalnt^-1, aborted ovule pod^-1, pod length (cm), seeds pod^-1, dry weight plant^-1 (g), 1000-seed weight, seed and stover yield were recorded using 1 m² quadrate from three places in each plot. The pods of each plot were harvested by hand picking as per maturity. Data on yield components were collected from five randomly selected plants plot^-1. Before harvesting, five plants were selected and uprooted randomly from each of the plot for data recording. Harvest index was calculated with the help of following formula: $Harvestindex\left(HI\%\right)= \frac{Seedyield}{Biologicalyield} x100$

Seed yield was recorded at 11 % moisture content. Stover weight was determined after oven-drying at 70 ◦C to a constant weight. Seed and stover yields were taken from the central part of each plot after discarding the border rows. The collected data were compiled and tabulated properly for statistical analysis. Protein content of mungbean seed was estimated by the standard micro Kjeldahl method (total nitrogen estimation method).¹⁴

Statistical analysis

The collected data were compiled and analyzed statistically using the analysis of variance (ANOVA) technique with the help of MSTAT-C programme and the mean separation were adjudged by Duncan’s Multiple Range Test.¹⁵

Effect of phosphorus on growth and yield of mungbean

Different phosphorus levels had significant effect on plant characters, yield and yield attributes of mungbean (Table 2). Plant treated with P₆₀ gave the tallest plant (70.0 cm) and it was statistically identical with P₃₀ (67.8 cm) and P₉₀ (67.1 cm). Plant grown without phosphorus (control) produced the shortest plant (64.4 cm) though it was identical to the plant when received P₉₀ (67.1 cm). These findings are collaborate with the results reported by Patel and Patel¹⁶ and Imran et al.¹⁷ where they observed that plant height of mungbean showed superiority at 60 kg P₂0₅ ha^-1 followed by 40 kg P₂0₅ ha^-1. The highest number of branches plant^-1 (2.6) was counted from the treatment P₆₀ which was statistically similar to P₉₀ (2.5) and P₃₀ (2.4). The lowest number of branches plant^-1 (1.8) was counted from control.

The highest 1000-seed weight (25.2 g) was recorded from P₆₀ treatment which was followed by P₉₀ (23.6 g) (Figure 2). The crop was fertilized with P₉₀, P₃₀ and control gave statistically identical results.  These findings are partly similar with the findings of Reddy et al.²⁰ where they observed that application of 50 kg P₂O₅ ha^-1 resulted highest 1000-seed weight of mungbean. The seed yield was significantly influenced by different phosphorus levels (Figure 2). It was found that P₆₀ produced the highest seed yield (1066 kg ha^-1) while the lowest seed yield (578.9 kg ha^-1) was obtained from the control. Seed yield increased due to the application of P₆₀ fertilizer, it might have fulfilled the requirements of the crop which helped better uptake of other plant nutrient at balance proportion to result in better plant growth and subsequently improve the yield contribution attributes and finally increased the seed yield. Significantly the highest stover yield m^-2 (476.7 g) was produced by the crop when received P_60. The lowest amount of stover yield (3576 kg) obtained from control plot (Figure 2). The highest plant height, number of branches plant^-1, number of pods plant^-1, number of leaves plant^-1 might be contributed to increase stover yield of mung bean. Similar finding was found by Sharma et al.²¹ reported that stover yield of mungbean increased with increase of phosphors up to or equivalent of 60 kg P₂O₅ ha^-1. Different phosphorus levels exerted a significant influence on the harvest index of mungbean (Figure 2). Harvest index was recorded maximum (22.7 %) with P_60. The harvest index was statistically similar under P₉₀ and P₃₀ treatments. The lowest harvest index (15.8 %) was obtained from the control plot. Response curve in orthogonal comparison in regression indicates that the optimum phosphorus level for mungbean cultivation was 63 kg P₂O₅ ha^-1 and its corresponding seed yield should be 1307.0 kg ha^-1 and seed yield would be reduced by 0.98 kg ha^-1 for reducing 1 kg of phosphorus fertilizer (Figure 3). These findings are similar with the findings of Raundal et al.²² where they reported that application of 60 kg P₂O₅ ha^-1 significantly increased the seed yield of mungbean.

Figure 2 Effects of phosphorus level on thousand seed weight – TSW (a), seed yield (b), stover yield (c) and harvest index (%) (d) of mungbean.
p₀ = No phosphorus, P₃₀ – 30 kg P₂O₅ ha^-1_, P₆₀ – 60 kg P₂O₅ ha^-1_, P₉₀ – 90 kg P₂O₅ ha^-1

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Figure 3 Response curve showing the relationship between level of phosphorus and seed yield of mungbean; P₀ = No phosphorus, P₃₀ = 30 kg P₂O₅ ha^-1_, P₆₀ = 60 kg P₂O₅ ha^-1 and P₉₀ = 90kg P₂O₅ ha^-1

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 The estimated protein content of Mungbean seed varied significantly due to different phosphorus levels (Figure 4).  The highest protein content of mungbean seed was recorded from P₉₀ treatment. The lowest protein content was estimated from the control plot. The figure indicates that the protein content of mungbean seed increased with the increased phosphorus levels.

Figure 4 Protein content of mungbean as influenced by phosphorus levels; P₀ = No phosphorus, P₃₀ = 30 kg P₂O₅ ha^-1_, P₆₀ = 60 kg P₂O₅ ha^-1 and P₉₀ = 90 kg P₂O₅ ha^-1

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Effect of Bradyrhizobium strain

Different strain of  Bradyrhizobium had significant effect on plant height, number of branches plant^-1_, number of sterile pods plalnt^-1_, number of aborted ovule pod^-1_, pods number plant^-1, Pod length, number of  seeds pod^-1 _, dry weight plant^-1_, stover yield, seed yield kg ha^-1 _, thousand seed weight and harvest index (Table 2). The tallest plant (70 cm) was obtained when crop was treated with I₁ strain which was statistically identical to I₂ (68 cm) and I₃ (67 cm). The shortest plant height (64 cm) was obtained from the crop when the seed were uninoculated. These might be due to continuous nitrogen supply by Bradyrhizobium inoculum to the plant after a stage of growth and ultimately enhanced plant height. These results are close related with the findings of Kavathiya and Pandey²³ stated that inoculated seed gave significantly increased plant height compared with uninoculated control. Significantly the maximum number of branches plant^-1 (2.7) was found when the seeds were inoculated with I₁ strain which was statistically similar with I₂ (2.5). The lowest branches number plant^-1 was noticed from the control plot (2.0), though it was statistically identical to the crop treated with I₃ (2.2).The plot treated with I₁ produced the maximum number of pods plant^-1 (15.5). The inoculum strain I₂ and I₃ produced statistically identical number of pods plant^-1 12.8 and 11.9 respectively, where I₃ and I₀ gave statistically similar result and the lowest pod number plant^-1(11.5) was observed from control (I₀) strain.

The highest number of sterile pods plant^-1 (2.4) was found from the control plot (I₀) followed by inoculation of seed with I₃ and I₂ strain. The lowest number of sterile pods plant^-1 (1.46) was obtained when the crop was treated with I₁ strain of Bradyrhizobium. The highest number of aborted ovules pod^-1 (2.0) was counted from the control plot (I₀) and the lowest number of aborted ovules pod^-1 (0.9) was counted from the plot treated with I₁ strain of Bradyrhizobium. This might be due to the highest potential of I₁ for more nitrogen fixation which enhanced more uptakes of other nutrients and thus reduced the sterility of the crop and produced lower number of aborted ovules. Different strain of Bradyrhizobium showed a significant effect on the pod length of mungbean. The highest pod length (5.7 cm) was measured when the seed was treated with I₁ strain of the inoculum. The crop treated with I₂ and I₃ strain produced statistically identical pod length. The lowest pod length (5.0 cm) was measured from the control (I₀) treatment. The crop treated with I₁ strain produced the highest number of seeds pod^-1 (10.0) which was statistically similar with I₂ (10.0) and I₃ (9.4). The lowest number of seeds pod^-1 (8.4) was counted from control treatment. The result indicated that inoculated plant produced higher number of seeds pod^-1 and increased the pod length than that of uninoculated plant. The lowest dry weight plant^-1 (10.2 g) was obtained from the control plot.

Inoculated and uninoculated plants showed significant variation on the 1000-seed weight of mungbean (Figure 5). The highest 1000-seed weight (25.94 g) was measured when the seed was treated with I₁ strain of Bradyrhizobim. The lowest 1000-seed weight (22.0 g) was observed from uninoculated treatment (control). Seed yield of mungbean was significantly the highest (982.50 kg ha^-1) when I₁ strain of Bradyrhizobium was used for seed inoculation and the lowest (670.50 kg ha^-1) from the control plot (Figure 5). The crop treated with I₁ produced highest amount of stover yield m^-2 (4451 kg ha^-1) followed by the treatment I₂ and I₃ (Figure 5)_. But the Bradyrhizobium strain I₂ and I₃ produced statistically identical stover yield. These findings are in close conformity with the findings of Chowdhury et al.²⁴ reported that mungbean seed inoculation with Bradyrhizobium strain significantly increased the stover yield by about 50%. Harvest index was also significantly affected by different strain of Bradyrhizobium (Figure 5). The highest harvest index (20.58%) was recorded in the crop treated with I₁ strain of Bradyrhizobium which was statistically identical with I₂. The lowest harvest index (18.0 %) was calculated in control plot.

Figure 5 Effects of Bradyrhizobium strain on thousand seed weight – TSW (a), seed yield (b), stover yield (c) and harvest index (%) (d) of mungbean
I₀ – Not inoculated; I₁ – Inoculation with BINA-MB-THA 301; I₂ – Inoculation with BINA-MB 441 and I₃ – Inoculation with BINA-MB 301

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Bradyrhizobium strain played a significant role on protein contain of mungbean (Figure 6). It was observed that the maximum protein content (25.28%) was obtained when the seeds were inoculated with I₁ strain of Bradyrhizobium, which was followed by I₂ and I₃ strains of Bradyrhizobium. The minimum protein content (22.84%) was estimated from the seeds of control plot. This might be due to the absence or minimum number of Bradyrhizobium bacteria present in the soil, that could not supply sufficient nitrogen as to the requirements of the crop by the process of biological nitrogen fixation and ultimate low nitrogen content of the seed, thus finally reduced the protein content of mungbean seed.

Figure 6 Effects of Bradyrhizobium strain on the protein content of mungbean seed
I₀ – Not inoculated; I₁ – Inoculation with BINA-MB-THA 301; I₂ – Inoculation with BINA-MB 441 and I₃ – Inoculation with BINA-MB 301

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Interaction effect of different phosphorus level and Bradyrhizobium strain

Different phosphorus levels and Bradyrhizobium strain interacted to  influence sterile pods number plalnt^-1 , aborted ovules number pod^-1 _, seed yield, stover yield and protein content (%) of mungbean but did not significant to influence the plant height, number of branches plant^-1 _, pods number plant^-1_,pod length, number of  seeds pod^-1 _, dry weight plant^-1_, thousand seed weight and harvest index (%) (Table 4 and Figure 7).

Figure 7 Interaction effects of phosphorus level and different Bradyrhizobium strain on yield (a), stover yield (b), harvest index (%) (c) and protein content (%) (d) of mungbean
P₀ = No phosphorus, P30 =30 kg P₂O₅ ha^-1_{, P60} =60 kg P₂O₅ ha^-1 _and    P₉₀ =90 kg P₂O₅ ha^-1

I₀ – Not inoculated; I₁ – Inoculation with BINA-MB-THA 301; I₂ – Inoculation with BINA-MB 441 and I₃ – Inoculation with BINA-MB 301

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The tallest plant (73.2 cm) was found from the plant treated with P₆₀I₁. The shortest plant (58.7 cm) was obtained with P₀I₀. This might be due to the lack of phosphorus and nitrogen element of the plant which reduced the cell division, carbohydrate and protein synthesis and also lowers the normal activity of the cambium tissue which results shorter plant height. The maximum number of branches plant^-1 (2.9) was counted when P₆₀ interacts with I₁ and the lowest number of branches plant^-1 (1.6) was recorded from P₀I₀. The highest number of pods plant^-1 (18.5) was obtained from P₆₀I₁ and the lowest number of pods plant^-1 was observed with P₀I₀ (8.3). Significantly the highest number of sterile pods plant^-1 (2.9) was counted in P₀I₀. This might be due to the insufficiency of phosphorus and nitrogen nutrients which lowered the plant growth and development, flowering and fruit setting and finally increased the pod sterility of mungbean. The lowest sterile pods number plant^-1 (1.0) was counted from the crop treated with P₆₀I₁ which was statistically identical with P₆₀I₂. The highest number of aborted ovules pod^-1 (3.2) was significantly observed from the control plot (P₀I₀) and the lowest number of aborted ovules pod^-1 (0.8) was counted when P₆₀ treated with I₁ and it was statistically identical with P₆₀I₂ (0.9) and P₃₀I₁ (0.8). The highest pod length (6.3 cm) was obtained by P₆₀ when coupled with I₁ strain of Bradyrhizobium and the shortest pod length (4.4 cm) was recorded from the control (P₀I₀) plot. However the highest number of seeds pod^-1 (10.1) was counted when P₆₀ used coupled with I₁ strain of Bradyrhizobium. The lowest number of seeds pod^-1 (7.1) was counted from the control plot where neither phosphorus nor inocula were given. The maximum dry weight plant^-1 (13.8 g) was obtained when P₆₀ interacts with I₁ strain of Bradyrhizobium and the lowest dry weight plant^-1 (8.88 g) was recorded in control plot (P₀I₀). Significantly the highest seed yield (1226 kg ha^-1) was obtained when P₆₀ was used with I₁ strain of Bradyrhizobim and the lowest amount of seed yield (432.3 kg ha^-1) was found from the control plot (P₀I₀) (Figure 7). The highest seed yield was obtained from P₆₀I₁ that might be due to the sufficient amount of available phosphorus from P₆₀ and adequate level of nitrogen supplied by the high potential I₁ strain of Bradyrhizobium bacteria by fixing atmospheric nitrogen through the process of biological nitrogen fixation. Significantly the maximum stover yield m^-2 (5129 kg ha^-1) was obtained when the crop was treated with P₆₀I₁ and the lowest straw yield m^-2 (3311 kg ha^-1) was recorded in control (P₀I₀) plot (Figure 7).

In case of 1000-seed weight was not significantly affected by phosphorus fertilizer and combined use of Bradyrhizobium strain inoculation (Table 4). However, the highest 1000 seed weight (27.7g) was found when P₆₀ coupled with I₁ strain of Bradyrhizobuim.  The lowest 1000-seed weight (21.7 g) was recorded from control (P₀I₀)_. Numerically the maximum harvest index (23.7 %) was recorded when P₆₀ interacts with I₂ strain of Bradyrhizobium. The lowest harvest index (14.2 %) was calculated from the absolute control plot (P₀I₀) (Figure 7).

Interaction effect of phosphorus and Bradyrhizobium strains found significant effect in case of protein content of mungbean (Figure 7). When P₆₀ used with I₁ strain of Bradyrhizobium produced the highest protein content (25.9 %) of mungbean seed which was statistically identical with P₉₀ at the same strain of Bradyrhizobium. Significantly the lowest protein content (22.5 %) was estimated from the seed of absolute control plot.

The major objective of this study was to evaluate to study the effect of different phosphorus levels and strains of Bradyrhizobium inocula on the growth, yield and protein content of mungbean compared with control (not inoculated and no phosphorus levels). In accordance with our hypothesis, the highest seed yield was obtained from 60 kg P₂O₅ ha^-1 while the lowest seed yield was obtained from the control. However, the orthogonal comparison in regression shows that 63 kg P₂O₅ ha^-1 is the optimum phosphorus level for the maximization of yield. With increasing phosphorus levels up to 90 kg ha^-1, the protein content of mungbean seed increased. Inoculation of seed significantly increased the yield components, seed and stover yield, and harvest index of mungbean. The inoculants I₁ (BINA-MB-THA 301) of Bradyrhizobium increased seed yield of mungbean and protein content of mungbean seed. In a nutshell, among the treatment combinations, 60 kg P₂O₅ ha^-1 with the inoculants I₁ (BINA-MB-THA 301) of Bradyrhizobium improved crop growth and yield, and protein content of mungbean in Bangladesh. Nevertheless, the result of the present study could be verified further experimentation in different agro-ecological zones of Bangladesh.

The authors acknowledged the Bangladesh Agriculture University, Mymensingh, Bangladesh for supporting to conduct the experiment. We are also thankful to Professor Selim for his valuable suggestions throughout the study period.

The authors declare that they have no conflicts of interest that could have appeared to influence the work reported in this paper.

The authors received no direct funding for this research.

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