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.
KeywordsMagnetic treatment Paenibaccilus sp Catalase Peroxidase Superoxide dismutase
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).
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