Morphophysiological responses of black soybean to nitrogen-fixing bacterial inoculants from diverse agroecosystems
Main Article Content
Abstract
Abstract. Hibatullah FH, Yaseen U, Ahmad F, Khumairah FH, Karuniawan A, Sofyan ET, Nurbaity A, Simarmata T. 2026. Morphophysiological responses of black soybean to nitrogen-fixing bacterial inoculants from diverse agroecosystems. Biodiversitas 27 (1): d270133. https://doi.org/10.13057/biodiv/d270133. Black soybean (Glycine max) is a legume crop of high nutritional and economic value, especially in sustainable agricultural systems. Its productivity, however, is often limited by nutrient-deficient soils. We hypothesized that inoculation with Symbiotic Nitrogen-Fixing Bacteria (SNFB) sourced from diverse agroecosystems could enhance morphophysiological performance under controlled conditions. This study aimed to evaluate the morphophysiological responses of black soybean to inoculation with SNFB isolated from diverse agroecosystems. The study comprised (i) isolation and phenotypic characterization of SNFB strains and (ii) greenhouse evaluation using a randomized complete block design with six bacterial treatments (BJ-H1, BJ-H2, BJ-H3, BJ-H4, BJ-H5, and BJ-H6) and an uninoculated control, each with four replicates. Parameters measured included plant height, chlorophyll content, fresh and dry biomass, and nodulation. Analysis of Variance (ANOVA) followed by post-hoc comparisons (p<0.05) revealed that BJ-H5 significantly increased plant height (2.41%), chlorophyll content (14.7%), fresh shoot biomass (59.33%), and dry shoot biomass (21.11%) relative to the control, BJ-H2 and BJ-H3 induced the highest nodulation, indicative of enhanced nitrogen fixation. Correlation and principal component analyses demonstrated robust positive associations between inoculation treatments and growth traits. These findings provide statistically substantiated evidence that targeted SNFB inoculation, particularly with strain BJ-H5, represents a viable biofertilizer strategy for improving black soybean productivity on marginal or degraded soils, thereby reducing dependence on synthetic nitrogen inputs and supporting sustainable crop intensification.
Article Details
Issue
Section

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.
References
Abiala M, Sadhukhan A, Sahoo L. 2023. Short Communication: Isolation and characterization of stress-tolerant Priestia species from cowpea rhizosphere under drought and nutrient deficit conditions. Curr Microbiol 80: 140. https://doi.org/10.1007/s00284-023-03246-8.
Bhartiya A, Aditya JP, Pal RS, Chandra N, Kant L, Pattanayak A. 2020. Bhat (Black soybean): A traditional legume with high nutritional and nutraceutical properties from nw himalayan region of india. Indian J Trad Knowledge 19: 307-319. https://doi.org/10.56042/ijtk.v19i2.35346.
Central Bureau Statistics Agency. 2021. Soybean Production (tons) and Soybean Consumption (tons). Central Statistics Agency, Jakarta. [Indonesian]
Cho K M, Ha T J, Lee Y B, Seo W D, Kim J Y, Ryu H W, Jeong S H, Kang, Y M, Lee J. H. 2013. Soluble phenolics and antioxidant properties of soybean (Glycine max L.) cultivars with varying seed coat colours. J Funct Foods 5 (3): 1065-1076. https://doi.org/10.1016/j.jff.2013.03.002.
de Castilho CL, Volpiano CG, Ambrosini A, Zulpo L, Passaglia L, Beneduzi A, de Sá ELS. 2021. Growth-promoting effects of Bradyrhizobium soybean symbionts in black oats, white oats, and ryegrass. Braz J Microbiol 52: 1451-1460. https://doi.org/10.1007/s42770-021-00523-1.
Deng Y-Q, Cao X-Y, Tan C-Y, Sun L-J, Peng X, Bai J, Huang S-P. 2020. Strengthening the effect of Bacillus megaterium on remediation of Cd-contaminated farmland soil by Sedum plumbizincicola. J Appl Ecol 31 (9): 3111-3118. https://doi.org/10.13287/j.1001-9332.202009.036. [Chinese]
Dunne KS, Holden NM, Daly K. 2021. Predicting phosphorus sorption isotherm parameters in soil using data of routine laboratory tests. Pedosphere 31 (5): 694-704. https://doi.org/10.1016/S1002-0160(21)60012-7.
Egamberdieva D, Jabborova D, Wirth SJ, Alam P, Alyemeni MN, Ahmad P. 2018. Interactive effects of nutrients and Bradyrhizobium japonicum on the growth and root architecture of soybean (Glycine max L.). Front Microbiol 9: 1000. https://doi.org/10.3389/fmicb.2018.01000.
Esringü A, Turan M, Güneş A, Karaman MR. 2014. Roles of Bacillus megaterium in remediation of boron, lead, and cadmium from contaminated soil. Commun Soil Sci Plant Anal 45 (13): 1741-1759. https://doi.org/10.1080/00103624.2013.875194.
Etesami H, Glick BR. 2024. Bacterial indole-3-acetic acid: A key regulator for plant growth, plant-microbe interactions, and agricultural adaptive resilience. Microbiol Res 281: 127602. https://doi.org/10.1016/j.micres.2024.127602.
Fahde S, Boughribil S, Sijilmassi B, Amri A. 2023. Rhizobia: A promising source of plant growth-promoting molecules and their non-legume interactions: Examining applications and mechanisms. Agriculture 13 (7): 1279. https://doi.org/10.3390/agriculture13071279.
Fatimah, Fadilah RLA, Millah AI, Nurhariyati T, Irawan B, Ni’matuzahroh, Affandi M, Zuhri ARNI, Widhiya EW, Salsabila S, Ramly ZA. 2022. Ability test of IAA (Indole-3-Acetic Acid) hormone-producing endophytic bacteria from Lamongan mangrove. Jurnal Riset Biologi dan Aplikasinya 4 (1): 42-50. https://doi.org/10.26740/jrba.v4n1.p42-50.
Gitonga NM, Njeru EM, Cheruiyot R, Maingi JM. 2021. Bradyrhizobium inoculation has a greater effect on soybean growth, production and yield quality in organic than conventional farming systems. Cogent Food Agric 7: 1935529. https://doi.org/10.1080/23311932.2021.1935529.
Hall TA. 1999. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41: 95-98.
Hamidah MN, Rianingsih L, Romadhon R. 2019. Aktivitas antibakteri isolat bakteri asam laktat dari peda dengan jenis ikan berbeda terhadap E. coli dan S. aureus. Jurnal Ilmu dan Teknologi Perikanan 1 (2): 11-21. https://doi.org/10.14710/jitpi.2019.6742. [Indonesian]
Handoko I, Kodarsih T, Ariyani A. 2010. Koefisien pemadaman tajuk dan efisiensi penggunaan radiasi surya pada tanaman kentang (Solanum tuberosum L.) varietas granola di Galudra, Cianjur, Jawa Barat. Agromet 24 (2): 27-32. https://doi.org/10.29244/j.agromet.24.2.27-32. [Indonesian]
Hartono A, Nadalia D, Sulaeman D. 2021. Development of quick test method for soil ph, nitrate, phosphorus, and potassium combining chemicals and phone cellular application. Agrivita 43 (2): 367-377. https://doi.org/10.17503/agrivita.v43i2.2760.
Hiltenbrand R, Thomas J, McCarthy H, Dykema KJ, Spurr A, Newhart H, Winn ME, Mukherjee A. 2016. A developmental and molecular view of formation of auxin-induced nodule-like structures in land plants. Front Plant Sci 7: 1692. https://doi.org/10.3389/fpls.2016.01692.
Ilangumaran G, Subramanian S, Smith DL. 2024. Complete genome sequences of Rhizobium sp. strain SL42 and Hydrogenophaga sp. strain SL48, microsymbionts of Amphicarpaea bracteata. Front Microbiomes 3: 1309947. https://doi.org/10.3389/frmbi.2024.1309947.
Jain A, Taylor RW. 2023. Determination of cation exchange capacity of calcareous soils: Comparison of summation method and direct replacement method. Commun Soil Sci Plant Analys 54 (6): 743-748. https://doi.org/10.1080/00103624.2022.211876.
Janda JM, Abbott SL. 2007. 16S rRNA Gene sequencing for bacterial identification in the diagnostic laboratory: Pluses, perils, and pitfalls. J Clin Microbiol 45 (9): 2761-2764.
Jarecki W, Borza IM, Rosan CA, Vicas SI, Domuța CG. 2024. Soybean response to seed inoculation with Bradyrhizobium japonicum and/or nitrogen fertilization. Agriculture 14 (7): 1025. https://doi.org/10.3390/agriculture14071025
Jo Y, Sliti A, Shin J-H. 2023. Complete genome sequence of Priestia megaterium S1, isolated from the soybean soil. Microbiol Biotechnol Lett 51 (3): 300-302. https://doi.org/10.48022/mbl.2307.07011.
Kabała C, Musztyfaga E, Gałka B, Łabuńska D, Mańczyńska P. 2016. Conversion of soil pH 1:2.5 KCl and 1:2.5 H2O to 1:5 H2O: conclusions for soil management, environmental monitoring, and international soil databases. Polish J Environ Stud 25 (2): 647-653. https://doi.org/10.15244/pjoes/61549.
Kan L, Nie S, Hu J, Wang S, Bai Z, Wang J, Zhou Y, Jiang J, Zeng Q, Song K. 2018. Comparative study on the chemical composition, anthocyanins, tocopherols and carotenoids of selected legumes. Food Chem 260: 317-326. https://doi.org/10.1016/j.foodchem.2018.03.148.
Khosravi A, Razavi SH. 2021. Therapeutic effects of polyphenols in fermented soybean and black soybean products. J Funct Foods 81: 104467. https://doi.org/10.1016/j.jff.2021.104467.
Kiprotich K, Muema E, Wekesa C, Ndombi T, Muoma J, Omayio D, Ochieno D, Motsi H, Mncedi S, Tarus J. 2025. Discover soil unveiling the roles, mechanisms and prospects of soil microbial communities in sustainable agriculture. Discov Soil 2: 10. https://doi.org/10.1007/s44378-025-00037-4.
Koriyama T, Teranaka K, Tsuchida M, Kasai M. 2023. Effects of storage and roasting condition on the antioxidant activity of soybeans with different colors of seed coat. Foods 12: 92. https://doi.org/10.3390/foods12010092.
Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Mol Biol Evol 35 (6): 1547-1549. https://doi.org/10.1093/molbev/msy096.
Li H, Li L, Chi Y, Tian Q, Zhou T, Han C, Zhu Y, Zhou Y. 2020. Development of a standardized gram stain procedure for bacteria and inflammatory cells using an automated staining instrument. Microbiologyopen 9 (9): e1099. https://doi.org/10.1002/mbo3.1099.
Ministry of Agriculture of the Republic of Indonesia. 2021. Results of the National Working Meeting (Rakernas) of the Ministry of Agriculture, Jakarta. [Indonesian]
Moturu US, Nunna T, Avula VG, Jagarlamudi VR, Gutha RR, Tamminana S. 2023. Investigating the diversity of bacterial endophytes in maize and their plant growth-promoting attributes. Folia Microbiol 68: 369-379. https://doi.org/10.1007/s12223-022-01015-x.
Multazam Z. 2023. Kajian nilai pH tanah pada berbagai toposekuen dan kelas lereng yang berbeda pada lahan perkebunan karet rakyat di Kecamatan Pelepat Ilir, Kabupaten Bungo, Jambi. Jurnal Riset Rumpun Ilmu Teknik 2 (2): 179-188. https://doi.org/10.55606/jurritek.v2i2.2709. [Indonesian]
Nascimento F, Hernández A, Glick BR, Rossi MJ. 2019. Plant growth-promoting activities and genomic analysis of the stress-resistant Bacillus megaterium STB1, a bacterium of agricultural and biotechnological interest. Biotechnol Rep 25: e00406. https://doi.org/10.1016/j.btre.2019.e00406.
Nel T, Clarke CE, Hardie AG. 2023. Comparison of soil pH and exchangeable cation quantification by various wet methods with near- and mid-infrared spectroscopy prediction. Commun Soil Sci Plant Analysis 54 (17): 2425-2438. https://doi.org/10.1080/00103624.2023.2223657.
Nelson MS, Sadowsky MJ. 2015. Secretion systems and signal exchange between nitrogen-fixing rhizobia and legumes. Front Plant Sci 6: 491. https://doi.org/10.3389/fpls.2015.00491.
Nguyen HP, Miwa H, Obirih-Opareh J, Suzaki T, Yasuda M, Okazaki S. 2020. Novel rhizobia exhibit superior nodulation and biological nitrogen fixation even under high nitrate concentrations. FEMS Microbiol Ecol 96 (2): fiz184. https://doi.org/10.1093/femsec/fiz184.
Nirmal NP, Khanashyam AC, Mundanat AS, Shah K, Babu KS, Thorakkattu P, Al-Asmari F, Pandiselvam R. 2023. Valorization of fruit waste for bioactive compounds and their applications in the food industry. Foods 12: 556. https://doi.org/10.3390/foods12030556.
Omari RA, Yuan K, Anh KT, Reckling M, Halwani M, Egamberdieva D, Ohkama-Ohtsu N, Bellingrath-Kimura SD. 2022. Enhanced soybean productivity by inoculation with indigenous Bradyrhizobium strains in agroecological conditions of Northeast Germany. Front Plant Sci 12: 707080. https://doi.org/10.3389/fpls.2021.707080.
Paray AA, Singh M, Mir MA, Kaur A. 2023. Gram staining: A brief review. Intl J Res Rev 10 (9): 336-341. https://doi.org/10.52403/ijrr.20230934.
Rana N, Kumar R, Sharma G D, Sharma R P, Pareek B, Upadhyay RG. 2023. Performance of different farming practices in legume based cropping system under Mid-hills of H.P. Himalayas. Legume Res 46 (1): 80-84. https://doi.org/10.18805/LR-4722.
Räty M, Keskinen R, Yli-Halla M, Hyvönen J, Soinne H. 2021. Estimating cation exchange capacity and clay content from agricultural soil testing data. Agric Food Sci 30 (4): 131-145. https://doi.org/10.23986/afsci.111107.
Rilling JI, Acuña JJ, Nannipieri P, Cassan F, Maruyama F, Jorquera MA. 2019. Current opinion and perspectives on the methods for tracking and monitoring plant growth-promoting bacteria. Soil Biol Biochem 130: 205-219. https://doi.org/10.1016/j.soilbio.2018.12.012.
Santos ERS, Dubeux Jr JCB, Mackowiak C, Blount ARS, Jaramillo DM, Garcia L, Pereiro-Neto JD, Ruiz-Moreno M. 2019. Sward responses of Bahiagrass cultivars under no nitrogen fertilization. Crop Sci 59 (6): 2893-2902. https://doi.org/10.2135/cropsci2019.06.0387.
Sapalina F, Ginting EN, Hidayat F. 2022. Bakteri penambat nitrogen sebagai agen biofertilizer. Warta Pusat Penelitian Kelapa Sawit 27 (1): 41-50. https://doi.org/10.22302/iopri.war.warta.v27i1.80. [Indonesian]
Sari E, Flatian AN, Sari ZI, Sulaeman E. 2019. Isolasi dan karakterisasi Rhizobium dari Glycine max L. dan Mimosa pudica Linn. EKOTONIA: Jurnal Penelitian Biologi, Botani, Zoologi dan Mikrobiologi 3 (2): 55-62. https://doi.org/10.33019/ekotonia.v3i2.760. [Indonesian]
Shahid M, Zeyad MT, Syed A, Singh UB, Mohamed A, Bahkali AH, Elgorban AM, Pichtel J. 2022. Stress-tolerant endophytic isolate Priestia aryabhattai BPR-9 modulates physio-biochemical mechanisms in wheat (Triticum aestivum L.) for enhanced salt tolerance. Intl J Environ Res Public Health 19 (17): 10883. https://doi.org/10.3390/ijerph191710883.
Shivanna AM, Nagendrappa G. 2019. Chemical analysis of soil samples to evaluate the soil fertility status of selected command areas of three tanks in Tiptur Taluk of Karnataka, India. IOSR J Appl Chem 7 (11): 1-5. https://doi.org/10.9790/5736-071110105.
Souza F G, Campos Milton, Cunha J M, Martins Thalita, Assis J M, Filho Elilson, Brito Wildson, Silva D M P, Oliveira F P. 2023. Chemical attributes of amazon forest soil under conversion for different cultivation systems in the south of Amazonas, Brazil. Appl Ecol Environ Res 21: 1767-1787. https://doi.org/10.15666/aeer/2102_17671787.
Spaepen S, Vanderleyden J, Remans R. 2007. Indole-3-acetic acid in microbial and microorganism-plant signaling. FEMS Microbiol Rev (4): 425-448. https://doi.org/10.1111/j.1574-6976.2007.00072.x.
Srithaworn M, Jaroenthanyakorn J, Tangjitjaroenkun J, Suriyachadkun C, Chunhachart O. 2023. Zinc solubilizing bacteria and their potential as bioinoculant for growth promotion of green soybean (Glycine max L. Merr.). PeerJ 11: e15128. https://doi.org/10.7717/peerj.15128.
Susilowati DN, Setyowati M. 2016. Analisis aktivitas nitrogenase dan gen nifh isolat bakteri rhizosfer tanaman padi dari lahan sawah pesisir Jawa Barat. Al-Kauniyah 9 (2): 125-138. https://doi.org/10.15408/kauniyah.v9i2.4036. [Indonesian]
Szpunar-Krok E, Bobrecka-Jamro D, Pikuła W, Jańczak-Pieniążek M. 2023. Effect of nitrogen fertilization and inoculation with Bradyrhizobium japonicum on nodulation and yielding of soybean. Agronomy 13 (5): 1341. https://doi.org/10.3390/agronomy13051341.
Takimoto R, Tatemichi Y, Aoki W, Kosaka Y, Minakuchi H, Ueda M, Kuroda K. 2022. A critical role of an oxygen-responsive gene for aerobic nitrogenase activity in Azotobacter vinelandii and its application to Escherichia coli. Sci Rep 12: 4182. https://doi.org/10.1038/s41598-022-08007-4.
Verma KK, Song X-P, Li D-M, Singh M, Wu J-M, Singh RK, Sharma A, Zhang B-Q, Li Y-R. 2022. Silicon and soil microorganisms improve rhizospheric soil health with bacterial community, plant growth, performance and yield. Plant Signal Behav 17 (1): 2104004. https://doi.org/10.1080/15592324.2022.2104004.
Wang Y, Wang Y, Zhang Q, Fan H, Wang X, Wang J, Zhou Y, Chen Z, Sun F, Cui X. 2023. Saline-alkali soil property improved by the synergistic effects of Priestia aryabhattai JL-5, Staphylococcus pseudoxylosus XW-4, Leymus chinensis and soil microbiota. Intl J Mol Sci 24 (9): 7737. https://doi.org/10.3390/ijms24097737.
Weaver RW, Danso SKA. 2018. Dinitrogen fixation. In: Weaver RW, Angle S, Bottomley P, Bezdicek D, Smith S, Tabatabai A, Wollum A (eds). Methods of Soil Analysis: Part 2 Microbiological and Biochemical Properties. Soil Science Society of America, USA. https://doi.org/10.2136/sssabookser5.2.c43.
Yanti I, Kusuma YR. 2021. Pengaruh kadar air dalam tanah terhadap kadar c-organik dan keasaman (pH) tanah. Indones J Chem Res 6 (2): 92-97. https://doi.org/10.20885/ijcr.vol6.iss2.art5. [Indonesian]
Youseif S H, Fayrouz HAEM, Saleh SA. 2017. Improvement of faba bean yield using Rhizobium/Agrobacterium inoculant in low-fertility sandy soil. Agronomy 7 (1): 2. https://doi.org/10.3390/agronomy7010002.
Zahran HH. 1999. Rhizobium-legume symbiosis and nitrogen fixation under severe conditions and in an arid climate. Microbiol Mol Biol Rev 63 (4): 968-989. https://doi.org/10.1128/mmbr.63.4.968-989.1999.
Zhou R, Cai W, Xu B. 2017. Phytochemical profiles of black and yellow soybeans as affected by roasting. Intl J Food Properties 20 (12): 3179-3190. https://doi.org/10.1080/10942912.2017.1280678.