Biochemical and Molecular Identification of Azospirillum brasilense Bacteria and Evaluation of Their Efficiency in Producing Hormones, Dissolving Phosphorus, and Fixing Nitrogen

Authors

  • Naseer Jawad

    Department of Soil Science and Water Resources, College of Agriculture, Al-Qadisiyah University, Ad Diwaniyah P.O. Box 1881, Iraq

  • Jawad Abdul Kadhim Kamal

    Department of Soil Science and Water Resources, College of Agriculture, Al-Qadisiyah University, Ad Diwaniyah P.O. Box 1881, Iraq

DOI:

https://doi.org/10.30564/jees.v6i3.6871
Received: 11 June 2024 | Revised: 16 July 2024 | Accepted: 26 July 2024 | Published Online: 12 September 2024

Abstract

The study aimed to isolate A.brasilense bacteria from the soil of the rhizosphere of different plants and different locations in Al-Diwaniyah Governorate. They were identified in two ways. The first was the routine method, which included studying the microscopic and cultural characteristics and biochemical tests of the isolates. The second method was molecular, using polymerase chain reaction (PCR) technology and using primers. It also included testing the efficiency of these isolates in dissolving tricalcium phosphate (TCP) on Pikovskaya agar medium, fixing nitrogen in the liquid nutrient medium (N.B), and measuring the amount of hormone production using an HPLC device. The results of isolation and regular and molecular identification the presence of ten isolates of bacteria bearing the characteristics of A. brasilense bacteria, out of fifteen local bacterial isolates, took the following symbols and sequences (Az2, Az3, Az5, Az6, Az7, Az9, Az11, Az12, Az13, Az14), as the results showed confirmation of the identification of the bacterial isolates identified by biochemical tests. Using a specialized primer to amplify the 462bp fragment of the 16S ribosomal RNA gene, the results of testing the efficiency of the bacteria in dissolving phosphate (TCP) showed that the isolate (Az13) outperformed the highest value in its effectiveness in dissolving metallic phosphorus through the diameter of the clear zone around the colony, which was effective in dissolving phosphate of up to 3.89 mm. As for the nitrogen fixation efficiency test, the isolate (Az3) excelled in the amount of fixed nitrogen reaching 12.44 mg.L-1. As for the amount of its production of growth regulators for Auxins, Gibberellins, and Cytokinins, the isolate (Az3) recorded the highest value in the amount of Auxins production ( Indol-3-acetic acid) its concentration reached 34.4 µg.ml-1, and as for the production of the hormone Gibberellins (GA3), the isolate (Az3) recorded the highest value amounting to 34.7 µg.ml-1, as for the production of the hormone Cytokinins (CK), The isolate (Az11) recorded the highest value, amounting to 28.8 µg.ml-1.

Keywords:

A. brasilense; PCR; dissolving tricalcium phosphate Auxins; Gibberellins; bacterial isolates

References

[1] Drancourt, M., Bollet, C., Carlioz, A., et al., 2000. 16S ribosomal DNA sequences analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. Journal of Clinical Microbiology. 38, 3623–3630.

[2] Xu, Y., Chen, W., Chunping , Zhenmin Liu, 2017. Development of a Multiplex PCR Assay for Detection of Pseudomonas fluorescens with Biofilm Formation Ability. Journal of Food Science. 82(10), 2337–2342. DOI: https://doi.org/10.1111/1750-3841.13845.

[3] Kalam, S., Das, S.N., Basu, A., et al., 2017. Population densities of indigenous Acidobacteria change in the presence of plant growth promoting rhizobacteria (PGPR) in rhizosphere. Journal of Basic Microbiology. 57, 376–385.

[4] Stamenković, S., Beškoski, V., Karabegović, I., et al., 2018. Microbial fertilizers: A comprehensive review of current findings and future perspectives. Spanish Journal of Agricultural Research. 16(1), e09R01. DOI: https://doi.org/10.5424/sjar/(2018)161-12117 .

[5] Spaepen, S., Vanderleyden, J., 2015. Auxin signaling in Azospirillum brasilense: a proteome analysis. In: de Bruijn, F.J. (ed.). Biological nitrogen fixation. Wiley: Hoboken. pp. 937–940. https://doi.org/10.1002/9781119053095.ch91

[6] Vendruscolo, E.P., De Lima, S.F., 2021. The Azospirillum genus and the cultivation of vegetables. A review. Biotechnology, Agronomy and Society and Environment. 25(4), 236–246.

[7] Jehani, M.D., Singh, S., Archana, T.S., et al., 2023. Azospirillum—a free-living nitrogen-fixing bacterium. In Rhizobiome. Academic Press: Cambridge, MA, USA. pp. 285–308.

[8] Baldani, V.L.D., Dobereiner, J. 1980. Host-plant specificity in the infection of cereals with Azospirillum spp. Soil Biology and Biochemistry. 12, 433–439.

[9] Palleroni, N.J., 1984. Gram negative aerobic rods and cooci family: Pseudomonas daceae. In: Krieg, N.R., Holt, J.G. (eds.). Bergey ’s Manual of Systematic Bacteriology. Williams and Wilkins: Baltimore. Vol. 1, pp. 141–199.

[10] Atlas, R.M., Parks, L.C., Brown, A.E., 1995. Laboratory manual of experimental microbiology. Mosby Year Book: El Dorado, KS, USA.

[11] Collee, J.G., Miles, R.S., Watt, B., 1996. Tests for the identification of Bacteria. In: Colle, J.G., Fraser, A.G., Marmion, B.P., et al. (eds.). Mackie and McCartney practical medical Microbiology. 14th ed. Churchill Livingstone: Singapore. pp. 131–149.

[12] Cruickshank, R., Duguid, J.P., Marmion, B.P., et al., 1975. Medical Microbiology. 12th ed. Churchill Livingstone: London, UK. Vol. 2. DOI: https://doi.org/10.36899/JAPS.4,0117

[13] Pikovskaya, R.E., 1948. Mobilization of phosphorus in soil in connection with vital activity of some microbial species. Mikrobiologiya. 17, 362–370.

[14] Bremner, J.M., 1965. Total nitrogen. In: Black, C.A., Evans, D.P., Ensminger, L.E., et al. (Eds.). Methodes of soil analysis, part 2. American Society of Agronomy: Madison, WI, USA.

[15] Tien, T.M., Gaskins, M.H., Hubbel, D.H., 1979. Plant growth substances produced. by Azospirillum brasilense and their effect on the growth of pearl millet (Pennisetum americanum L.). Applied and Environmental Microbiology. 37, 1016–1024.

[16] Rouillon, R., Gay, G., Bernillon, J., et al., 1986. Analysis by HPLC–mass spectrometry of the indole compounds released by ectomycorrhiza. 1 fungus Hebeloua hiemale in pure culture. Canadian Journal of Botany. 64, 1893–1897.

[17] Abbas, M.F., Jasim, A.M., Ibrahim, A.O., 1995. Effect of pollen endogenous hormones on the fruit of the date palm (Phoenix daetylifera L.) cv. Hillawi – Basrah Journal of Agricultural Sciences. 8, 33–41.

[18] Audus, L.J., 1972. Plant growth substance. Phsiology and Biochemistry, 3rd ed. leonard Hill: London. p. 553; Volume 1.

[19] Macmillan, J., 1983. Gibberellins in higher plants. Biochemical Society Transactions. 11, 524–533.

[20] Sivagamasundari, U., Gandhi, A., 2018. Isolation, identification and characterization of endophytic bacteria-Azospirillum sp. and Pseudomonas sp. from Brinjal (Solanum melongena L.). International Journal of Life Sciences. A11, 11–16.

[21] Al-Zubaie, A.Z., Hammadi, A., 2021. The effect of a combination of biofertilizers (Azospirillum brasilense and Trichoderma harzianum) and organic and mineral fertilizers on the activity of the enzyme urease and alkaline phosphatase and on the growth and yield of sunflower plants [PhD thesis]. Baghdad: College of Agriculture - University of Baghdad.

[22] Al-Jayashi, S.Z.S., 2022. The effect of bacterial inoculum and organic fertilizer on irrigation efficiency, the readiness of some nutrients, and the growth and yield of wheat [PhD thesis]. Baghdad: College of Agriculture - University of Baghdad.

[23] Gerace, E., Mancuso, G., Midiri, A., et al., 2022. Recent Advances in the Use of Molecular Methods for the Diagnosis of Bacterial Infections. Pathogens. 11(6), 663. DOI: https://doi.org/10.3390/pathogens11060663

[24] Stets, M.I., Alqueres, S.M.C., Souza, E.M., 2015. Quantification of Azospirillum brasilense FP2 Bacteria in Wheat Roots by Strain-Specific Quantitative PCR. Applied and Environmental Microbiology. 81(19), 6700–6709. DOI: https://www.researchgate.net/publication/280115809

[25] Kbibo, I., Badran, A., Hleibieh, M., 2021. Effect inoculation soil and sweet corn seeds with Pseudomonas fluorescens and Azospirillum lipoferum on solubilizing some forms of fixed phosphorus in soil. Biological Sciences Journal University Tishreen. 34(3).

[26] Rosenblueth, M., Ormeño-Orrillo, E., López-López, A., et al., 2018. Nitrogen fixation in cereals. Frontiers in Microbiology, 9, 1794.

[27] Martin, T.N., Bison Pinto, M., Tabaldi, L.A., et al., 2022. Soil Acidity Conditioning the Productivity and Physiology of Wheat Inoculated with Azospirillum brasilense. Communications in Soil Science and Plant Analysis. 53, 2082–2093.

[28] Hujaira, G., Nasser, O., Kreidi, N., 2023. Isolation of Strains of Atmospheric Nitrogen-Fixing Bacteria from Hamima Soil in Aleppo Governorate and Testing their Effectiveness in Fixing Atmospheric Nitrogen. Syrian Journal of Agricultural Research. 10(4), 279–272.

[29] EL-Komy, H.M.A., Shaieb, F.M.A., Mohamed, H.M.E., 2019. Quantitative estimation of complex phosphate - solubilization by immobilized or free Azospirillum Lipoferum (H3) as vital vaccine in Pikovskaya broth (PVK) medium. Misurata University Journal of Agricultural Sciences. 1(1), 166–173.

[30] Ikhajiagbe, B., Anoliefo, G.O., Olise, O.F., et al., 2020. Major phosphorus in soils is unavailable, yet critical for plant development. Notulae Scientia Biologicae. 12(3), 500–535.

[31] AlJanaby, Z.A.A., Obeed, Z.O., Hamza, L.M., et al., 2020. THE EFFECT OF COMPOST NP AND SALICYLIC ACID SPRAY FOR QUANTITY AND QUALITY OF LUXURY VARIETY GRAPES (VITIS VINIFERA L.). Plant Archives. 20(1), 2691–2694.

[32] Ahmed, A., Hasnain, S., 2020. Extraction and evaluation of indole acetic acid from indigenous auxin- producing rhizosphere bacteria. JAPS: Journal of Animal & Plant Sciences. 30, 1024–1036.

[33] Zaheer, M.S., Ali, H.H., Iqbal, M.A., et al., 2022. Cytokinin Production by Azospirillum brasilense Contributes to Increase in Growth, Yield, Antioxidant, and Physiological Systems of Wheat (Triticum aestivum L.). Frontiers in Microbiology. 13, 886041.

[34] AlJanabi, A.Z., Ameer, A.J., Israa, H., et al., 2019. Effect of adding different levels of organic manure and potassium fertilizer in the yield growth of wheat (Triticum aestivum L.). Plant archives. 19.

[35] Hassan, D., Thamer, T., Mohammed, R., et al., 2023. Calibration and Evaluation of AquaCrop Model Under Different Irrigation Methods for Maize (Zea mays L.) in Central Region of Iraq. In: Kallel, A., Barbieri, M., Rodrigo-Comino, J., et al. Selected Studies in Environmental Geosciences and Hydrogeosciences. CAJG 2020. Advances in Science, Technology & Innovation. Springer: Cham. DOI: https://doi.org/10.1007/978-3-031-43803-5_10

[36] Jafaar, A.A., Mohammed, R.J., Hassan, D.F., et al., 2023. Effect of Foliar Seaweed and Different Irrigation Levels on Water Consumption, Growth and Yield of Wheat. IOP Conference Series: Earth and Environmental Science. 1252, 012057.

[37] Hassan, D.F., Ati, A.S., Naima, A.S., 2023. EVALUATION OF THE PERFORMANCE OF THE AQUACROP MODEL UNDER DIFFRENT IRRIGATION AND CULTIVATION METHODS AND THAIR EFFECT ON WATER CONSUMPTION. IRAQI JOURNAL OF AGRICULTURAL SCIENCES. 54(2), 478–490.

[38] Jafaar, A.A., Mohammed, R.J., Hassan, D.F., 2022. FFECT OF PHOSPHORUS FERTILIZER AND IRRIGATION LEVEL ON DESERT SOIL MANAGEMENT AND POTATO YIELD. International Journal of Agricultural & Statistical Sciences. 18(2), 689.

[39] Ali, Z.A., Hassan, D.F., Mohammed, R.J., 2021. Effect of irrigation level and nitrogen fertilizer on water consumption and faba bean growth. IOP Conference Series: Earth and Environmental Science. 722, 012043.

[40] Akol, A.M., Nassif, N., Jaddoa, K.A., et al., 2021. EFFECT OF IRRIGATION METHODS, TILLAGE SYSTEMS AND SEEDING RATE ON WATER CONSUMPTION, BIOLOGICAL YIELD AND HARVEST INDEX OF WHEAT (TRITICUM AESTIVUM L.). International Journal of Agricultural & Statistical Sciences. 17.

[41] Mohammed, R.J., Hameed, I.A., Thamer, T.Y., 2022. Effect of Using Different Types of Well Water in Karbala Governorate on Soil and Plant. Ecological Engineering & Environmental Technology. 23.

[42] Jaafer, A.A., Mohammed, R.J., Hassan, D.F., 2020. Studying the thermodynamic parameters for the evaluation of potassium availability by adding organic matter. Biochemical & Cellular Archives. 20(1), 785.

[43] Mohammed, R.J., Abdulkadhim, K.A., Hassan, D.F., et al., 2019. Effect of wheat straw as organic matter and different water quality on some chemical soil properties and growth of pepper (Capsicum annuum). IOP Conference Series: Earth and Environmental Science. 344, 012034.

Downloads

How to Cite

Jawad, N., & Abdul Kadhim Kamal, J. (2024). Biochemical and Molecular Identification of Azospirillum brasilense Bacteria and Evaluation of Their Efficiency in Producing Hormones, Dissolving Phosphorus, and Fixing Nitrogen. Journal of Environmental & Earth Sciences, 6(3), 92–103. https://doi.org/10.30564/jees.v6i3.6871