Study on the effect of magnetic field treatment of newly isolated Paenibacillus sp.
© Li et al.; licensee Springer. 2015
Received: 5 April 2014
Accepted: 13 January 2015
Published: 30 January 2015
Symbiotic nitrogen fixation in plants occurs in roots with the help of some bacteria which help in soil nitrogen fertility management. Isolation of significant environment friendly bacteria for nitrogen fixation is very important to enhance yield in plants.
In this study effect of different magnetic field intensity and treatment time was studied on the morphology, physiology and nitrogen fixing capacity of newly isolated Paenibaccilus sp. from brown soil. The bacterium was identified by 16S rDNA sequence having highest similarity (99%) with Paenibacillus sp as revealed by BLAST. Different magnetic intensities such as 100mT, 300mT and 500mT were applied with processing time of 0, 5, 10, 20 and 30 minutes. Of all these treatment 300mT with processing time of 10 minutes was found to be most suitable treatment. Results revealed that magnetic treatment improve the growth rate with shorter generation time leading to increased enzyme activities (catalase, peroxidase and superoxide dismutase) and nitrogen fixing efficiencies. High magnetic field intensity (500mT) caused ruptured cell morphology and decreased enzyme activities which lead to less nitrogen fixation.
It is concluded that appropriate magnetic field intensity and treatment time play a vital role in the growth of soil bacteria which increases the nitrogen fixing ability which affects the yield of plant. These results were very helpful in future breading programs to enhance the yield of soybean.
Paenibacillus genus of bacteria was first included in Bacillus genus and then reclassified to a separate genus in 1993 (Ash et al. ). These bacteria found in variety of environments like soil, water, forage, rhizosphere, insect larvae, vegetable matter and in clinical samples (McSpadden Gardener , (Montes et al. ; Ouyang et al. ; Lal & Tabacchioni )). These bacteria are of prime importance in agriculture for nitrogen fixation and industrial importance due to production of antibiotics and enzymes (Mavingui & Heulin ; Von der Weid et al. ). These bacteria produce plant growth hormones, suppress phytopathogens and solubilize organic phosphate (Mavingui & Heulin ; Lebuhn et al. ; Pires & Seldin ).
Nitrogen is very essential nutrient for the growth of plants. So, these bacteria fix nitrogen from the air and provide this nitrogen to plants in the form of ammonium ions or other nitrogenous compounds essential for growth. From this symbiotic association, plant provides some organic compounds synthesized from photosynthesis (Sawada et al. ). These bacteria not only fix the nitrogen but also enrich the soil fertility, increase plant production, and improve the quality, degrade organic pollutants and production of vitamin B series compounds (Sierra et al. ; Agus et al. ). The nitrogen deficiency was recovered by these rhizobia (Fisher & Long ). In this process, plant produced some reactive oxygen species including the hydrogen peroxide and hydroxyl radicals and superoxide anion by defence reaction (Lamb & Dixon ; Santos et al. ). So it was necessary to study the rhizobia catalase, peroxidase and superoxide dismutase active changes.
A lot of research showed that the magnetic treatments have certain stimulative effect on crop production and development and, it also affect the genetic quality of seeds ((Zhu et al. ; Liu et al. ; Yan et al. ; He et al. ; Mao et al. ); Jia et al. ; (Liu et al. )). Enzyme as protein with catalytic activity has an important role in the life process, and as a catalyst it was increasingly being attention (Cheng et al. ). Magnetic field on the influence of the enzyme activity has been reported (He et al. ; Li et al. ; Hua et al. ), and this area now attracts more and more people’s attention, but most of these studies focused on animals, plants and very little research on bacteria. So this study was aimed to check the effect of magnetic field on soybean rhizobia isolated from brown soil and their enzyme activities (peroxidase, catalase and superoxide dismutase) under the influence various intensity of magnetic treatment.
The Brown soil samples were collected from Shenyang Agriculture University, Shenyang Liaoning P.R. China. The samples were kept in sterile plastic bags and transferred aseptically to the lab.
Isolation of Paenibacillus
The Paenibacillus sp. were isolated using standard procedures, and were purified by repeatedly streaking the bacteria on yeast extract-mannitol agar (YMA) medium (Vincent ) and stored at 4°C.
Molecular identification of Paenibacillus
Genomic DNA of the newly isolated bacterial strain was extracted by method as described by Ausubel et al. (). The DNA was amplified using universal primers 27 F:5′ -GAGAGTTTGATCCTGGCTCAG-3′ and 1492R:5′ -GGYTACCTTGTTACGACTT-3′. PCR reactions were performed in 50 l volume containing 1 μL template DNA, 4 μL MgCl2 (25 mmol/L), 5 μL 10× PCR buffer (Mg2+free), 4 μL dNTP(10 mmol/L), 1 μL of each primer (10 μmol/L), 0.5 μL of TaqDNA polymerase (5u/μL) and 33.5 μL ddH2O. PCR amplification conditions as follows: Initial denaturation at 94°C for 5 min followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension 72°C for 1 min, final extension at 72°C for 10 min. Amplification products were separated by 1 · 0% agarose gel electrophoresis and visualized under UV light after staining with ethidium bromide. The amplified 16S rRNA gene was sequenced using ABI 3730xl DNA Analyzer (Applied Biosystems, USA). The sequences were identified based on similarity using the Basic Local Alignment Search Tool (BLAST) program National Centre for Biotechnology Information (NCBI) online standard (http://www.ncbi.nlm.nih.gov/).
Magnetic treatment of soil
The soil was treated by magnetic field in 100 mT, 300 mT and 500 mT for 5 min, 10 min, 20 min and 30 min respectively. Soybean was planted in the treated soil samples using phosphate and potash fertilizers (75 mg kg−1P2O5;75 mg kg−1 K2O). After harvestation the plants and soil was used to determine the soybean nodulation and nitrogen fixation capacities.
Magnetic treatment of Paenibacillus sp.
The Paenibacillus sp. was inoculated in 100 mL of YMA medium, incubated at 28°C for 36 h with agitation speed 200 rpm. The cell growth was measured by taking OD at 520 nm. After the cell growth, 25 mL of Paenibacillus sp. cell suspension was taken in a test tube and treated it with different magnetic fields like 100, 300 and 500 mT with different time period such as 0, 5, 10, 20 and 30 min. Each experiment was conducted in triplicates and Paenibacillus sp. without magnetic treatment was taken as control.
The Paenibacillus sp. broth was centrifuge at 5000 × g, 4°C for 10 min. After centrifugation the supernatant was discarded and the pellet was suspended in 50 mmol L−1 phosphate buffer (pH 7.0) and then subjected to sonication. The homogenate solution was centrifuged for 10 min at 10000 × g, 4°C. After centrifugation, the supernatant was used for determination of peroxidase (POD), superoxidase dismutase (SOD) and catalase (CAT) activities. Catalase activity was assay of hydrogen peroxide based on the formation of its stable complex with ammonium molkbdate and the OD was measured at 405 nm (Fang et al. ). One unit of catalase activity was defined as the decomposition of 1 μ mol of hydrogen peroxide per minute under standard assay conditions. Peroxidase activity was determined by hydrogen peroxide-dependent oxidation of guaiacol. Samples were mixed with guaiacol solution (20 mmol/L guaiacol in 0.1 mol/L phosphate buffer (pH 6.8) and 0.03% (v/w) hydrogen peroxide) (Bergmeger et al. ). Increase in absorbance at 470 nm was recorded using UV-visible spectrophotometer. One unit of POD activity was defined as the change in absorbance of 0.01 per minute at room temperature. Total SOD activity was assayed by the inhibition of the photochemical reduction of pyrogallol (PAPG) by following the photo reduction of nitroblue tetrazolium (Cai et al. ). One unit of SOD activity was defined as amount of enzyme producing a 50% suppression of PAPG reduction. All the Enzyme specific activity is expressed as U/ml.
Total nitrogen determination
Total plant nitrogen (N) concentration was analysed with Kjeldahl determination and colorimetric method as described by Baethgen and Alley (Baethgen & Alley ). Nitrogen fixed was calculated as the total plant nitrogen content at harvest, minus the total nitrogen content at the start of the treatments.
The data obtained after experimentation was statistically evaluated using ANOVA at significance level of p < 0.05 by using computer based programme SPSS.
Results and discussion
Molecular identification of Paenibacillus sp.
Effect of magnetic field treated soil soybean nodular and nitrogen fixation
Effect of magnetic field treated soil on soybean nodular and nitrogen content
Bacterial dry weight
Effective number of root nodule
Magnetic field (mT)
Weight (g dry wt pl−1)
Percentage change (%)
Percentage change (%)
Percentage change (%)
7 ± 1.23
3.23 ± 0.12
24 ± 3.47
3.46 ± 0.13
18 ± 1.76
3.51 ± 0.12
17 ± 1.84
3.35 ± 0.11
16 ± 1.34
3.25 ± 0.12
37 ± 1.63
4.33 ± 0.17
28 ± 2.95
4.55 ± 0.15
24 ± 2.58
3.58 ± 0.13
13 ± 1.72
3.51 ± 0.16
7 ± 1.25
3.34 ± 0.13
7 ± 1.13
3.25 ± 0.12
6 ± 1.13
3.17 ± 0.11
7 ± 1.12
3.14 ± 0.13
Effect of magnetic treatment on generation time of Paenibacillus sp.
The magnetic treatment of soybean purification number and generation of rhizobium time influence
Magnetic field (mT)
Generation ofPaenibacillussp. (h)
Generation ofPaenibacillussp. (h)
Effect of magnetic treatment on morphology of the Paenibacillus sp.
Effect of magnetic field treatment on enzyme activity of Paenibacillus sp.
In conclusion the magnetic treatment significantly enhances the bacterial population with shorter generation time. This increased population of Paenibacillus sp. would increase the nitrogen fixing efficiency thus leading to greater yield. The enzyme activities were also increased under the influence of magnetic treatment. Increased magnetic field intensity and longer magnetic processing time resulted ruptured bacterial cell which leads to cell death, thus reduction in nitrogen fixation efficiency. To achieve the better yield, appropriate magnetic field intensity and magnetic processing time is very important for this whole process.
This study was supported by the National Natural Science Foundation of China (Grant No. 40771111) and the Shenyang agricultural university youth fund (Grant No. 20070136).
- Agus JE, Steven DG, Brian ND: Isolation and characterization of 2,3-dichloro-Ipropanol-edgrading. Appl Environ Microbiol 2000,66(7):2882–2887. 10.1128/AEM.66.7.2882-2887.2000View ArticleGoogle Scholar
- Ash C, Priest FG, Collins MD: Molecular identification of rRNA group 3 Bacilli (Ash, Farrow, Wallbanks and Collins) using a PCR probe test. Proposal for the creation of a new genus Paenibacillus . Antonie Van Leeuwenhoek 1993, 64: 253–260. 10.1007/BF00873085View ArticlePubMedGoogle Scholar
- Ausubel M, Brent R, Kingston RE, Moore DD, Smit JA, Seidman JC, Struhl KS: Current protocols in molecular biology, section 2.4. John Wiley and Sons, New York; 1994.Google Scholar
- Baethgen WE, Alley MM: A manual colorimetric procedure for measuring ammonium nitrogen in soil and plant. Commun Soil Sci Plant Anal 1989,20(9&10):961–969. 10.1080/00103628909368129View ArticleGoogle Scholar
- Barker AV, Bryson GM: Nitrogen. In Handbook of Plant nutrition. Edited by: Barker AV, Pilbeam DJ. CRC Press, Boca Raton; 2007:21–50.Google Scholar
- Bergmeger H, Bergmeyer J, Grabl M: Methods of enzymatic Analysis. 3rd edition. Verlag Chemie Press, Weinheim; 1983.Google Scholar
- Cai Y, Lai Z, Shen J: Detection of superoxide dismutase activities in different organsim Phyllanthus emblica L. Chin J Trop Crops 2006,27(4):29–33.Google Scholar
- Celik Ö, Büyükuslu N, Atak C, Rzakoulieva A: Effects of magnetic field on activity of superoxide dismutase and catalase in Glycine max (L.) Merr. Roots. Pol J Environ Stud 2009,18(2):175–182.Google Scholar
- Cheng X, Yi Y: Effect of Magnetic Treatment on Amount and Generation Time of Slow-Growing Rhizobium (USDA110) and Fast-growing Rhizobium (USDA191). Southwest Chin J Agri Sci 2009,22(5):1400–1403.Google Scholar
- Cheng X, Yi Y, Du A: Effects of Magnetic treatment on the Azotobacter contents in brown Earth. Chin J Soil Sci 2007,38(5):1025–1027.Google Scholar
- Fadel MA, Wael SM, Mostafa RM: Effect of 50 Hz, 0.2 mT magnetic fields on RBC properties and heart functions of albino rats. Bioelectromagnetics 2003, 24: 535–545. 10.1002/bem.10134View ArticleGoogle Scholar
- Fang F, Li Y, Du GC: Themo-alkali stable catalase Themoascus aurantiacus and its potential use in textile bleaching process. Chin J Biotechnol 2004,20(3):423–428.Google Scholar
- Fisher RF, Long SR: Rhizobium-plant signal exchange. Nature 1992,357(6380):655–660. 10.1038/357655a0View ArticlePubMedGoogle Scholar
- Gaafar EA, Hanafy MS, Tohamy EY, Ibrahim MH: Stimulation and control of E. coli by using an extremely low frequency magnetic field. Rom J Biophys 2006,16(4):283–296.Google Scholar
- Hagedorn F, Bucher JB, Schleppi P: Contrasting dynamics of dissolved inorganic and organic nitrogen in soil and surface waters of forested catchments with Gleysols. Geoderma 2001, 100: 173–192. 10.1016/S0016-7061(00)00085-9View ArticleGoogle Scholar
- He H, Zhu Y, Zhong K: Effect of magnetic field on celhlase activity and conformation. J Jishou Univ 1998,19(1):42–46.Google Scholar
- He H, Zhu Y, Fan Q: The Effect of Magnetic Field on Escherichia Coli and Glutamic Acid Decarbolxylase. J Jishou Univ 1999,20(3):26–29.Google Scholar
- Hua H, Shen Y, Wu W: Effects of magnetic field on seed quality, POD and SOD of Pinus massoniana. J Nanjing For Univ 2008,32(3):39–42.Google Scholar
- Jia Y, Ma Y, Wang Z: The enzymatic activity of tomato seeds with magnetic field treatment. Biotechnology 2002,10(2):14–17.Google Scholar
- Jing Y, Zhang B, Wang Y, Lin X: Effect of magnetic field on symbiotic nitrogen fixation of soybean nodules. Acta Botanica Sinica 1992,34(5):364–368.Google Scholar
- Lal S, Tabacchioni S: Ecology and biotechnological potential of Paenibacillus polymyxa: a minireview. Indian J Microbiol 2009, 49: 2–10. 10.1007/s12088-009-0008-yPubMed CentralView ArticlePubMedGoogle Scholar
- Lamb C, Dixon RA: The oxidative burst in plant disease resistance. Annu Rev Plant Physiol Plant Mol Biol 1997, 48: 251–275. 10.1146/annurev.arplant.48.1.251View ArticlePubMedGoogle Scholar
- Lebuhn M, Heulin T, Hartmann A: Production of auxin and other indolic and phenolic compounds by Paenibacillus polymyxa strains isolated from different proximity to plant roots. FEMS Micr Eco 1997, 22: 325–334. 10.1111/j.1574-6941.1997.tb00384.xView ArticleGoogle Scholar
- Li J, Jiao Y, Yi Y: Effects of magnetic field on catalase and peroxidase activities in brown earth. J Shenyang Agric Univ 2007,38(1):70–74.Google Scholar
- Liu XY, Yi YL, Xia LH: Effect of magnetic field on enzyme activities in main soils of Northeast China. Pedosphere 1996,6(4):341–348.Google Scholar
- Liu YH, Xu LH, Tang X: The Effect of Magnetic Field Treatment on Artificially Aging Pepper Seeds. J Shandong Inst Build Mat 2003,17(3):286–288.Google Scholar
- Mao N, Huang Y, Zhang Z: Study on Biological Effect of Magnetic Resonance and Magnetized Water on Agricus bisporus Strain 176. J Fujian Teachers Univ (Nat Sci) 2002,18(3):61–65.Google Scholar
- Marschner H: Mineral Nutrition of Higher Plants. Academic Press Limited, San Diego, London; 1995.Google Scholar
- Mavingui P, Heulin T: In vitro chitinase antifungal activity of a soil, rhizosphere and rhizoplane populations of Bacillus polymyxa . Soil Biol Biochem 1994, 26: 801–803. 10.1016/0038-0717(94)90277-1View ArticleGoogle Scholar
- McSpadden Gardener BB: Ecology of Bacillus and Paenibacillus spp. in Agricultural Systems. Phytopathology 2004, 94: 1252–1258. 10.1094/PHYTO.2004.94.11.1252View ArticlePubMedGoogle Scholar
- Mohamed AA, Ali FM, Gaafar EA, Magda HR (1997) Effects of magnetic field on the biophysical, biochemical properties and biological activity of Salmonella typhi., Master thesis submitted for Biophysics department, Faculty of science, Cairo University, Egypt.Google Scholar
- Montes MJ, Mercade E, Bozal N, Guinea J: Paenibacillus antarcticus sp. nov., a novel psychrotolerant organism from the Antarctic environment. Int J Syst Evol Microbiol 2004, 54: 1521–1526. 10.1099/ijs.0.63078-0View ArticlePubMedGoogle Scholar
- Ouyang J, Pei Z, Lutwick L, Dalal S, Yang L, Cassai N, Sandhu K, Hanna B, Wieczorek RL, Bluth M, Pincus MR: Case report: Paenibacillus thiaminolyticus : a new cause of human infection, inducing bacteremia in a patient on hemodialysis. Ann Clin Lab Sci 2008, 38: 393–400.PubMed CentralPubMedGoogle Scholar
- Owen AG, Jones DL: Competition for amino acids between wheat roots and rhizosphere microorganisms and the role of amino acids in plant N acquisition. Soil Biol Biochem 2001, 33: 651–657. 10.1016/S0038-0717(00)00209-1View ArticleGoogle Scholar
- Pires MN, Seldin L: Evaluation of Biology system for identification of strains of Paenibacillus azotofixans . Ant Van Leeu 1997, 71: 195–200. 10.1023/A:1000128314946View ArticleGoogle Scholar
- Santos R, Herouart D, Sigaud S, Touati D, Puppo A: Oxidative burst in alfalfa-Sinorhisobium meltloti symbiotic interaction. Mol Plant Micobe Interact 2001,14(1):86–89. 10.1094/MPMI.2001.14.1.86View ArticleGoogle Scholar
- Sawada H, Kuykendall LD, Young JM: Changing concepts in the systematics of bacterial nitrogen-fixing legume symbionts. J Gen Appl Microbiol 2003,49(3):155–79. 10.2323/jgam.49.155View ArticlePubMedGoogle Scholar
- Sierra S, Rodelas B, Martinez-Toledo MV, Pozo C, González-López J: Production of B-group vitamins by two Rhizobium stains in chemically defined media. J Appl Microbiol 1999, 86: 851–858. 10.1046/j.1365-2672.1999.00765.xView ArticleGoogle Scholar
- Vincent JM: A Manual for the Practical Study of Root-Nodule Bacteria. Blackwell Scientific, Oxford; 1970.Google Scholar
- Von der Weid I, Alviano DS, Santos ALS, Soares RMA, Alviano CS, Seldin L: Antimicrobial activity of Paenibacillus peoriae against a broad spectrum of phytopathogenic bacteria and fungi. J Appl Microbiol 2003, 95: 1143–1151. 10.1046/j.1365-2672.2003.02097.xView ArticlePubMedGoogle Scholar
- Yan L, Zhu Y, He S, Cao Z: Effect of static magnetic field on activity of immobilized a-amylase. Chinese Sci Bull 1997,42(2):127–130. 10.1007/BF03182784View ArticleGoogle Scholar
- Zhu Y, Zhong K, He S: Efect of Magnetic Field on Activity of Immobilized LDH. J Hunan Univ 1996,23(5):57–61.Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.