KR20080105881A - Metal tolerating methylotrophic bacteria reduces cadmium toxicity and promotes plant growth of tomato - Google Patents

Abstract

A methylotrophic bacterium is provided to reduce the toxicity of cadmium in tomatoes and to promote the growth of plants. A methylotrophic bacterium is used to reduce the toxicity of cadmium and to promote the growth of plants. Preferably the methylotrophic bacterium is Methylobacterium oryzae sp. nov. (KACC 11585). Preferably the Methylobacterium oryzae sp. nov. is separated from the stem of rice and is a novel microorganism capable of producing ACC deaminase.

Description

Metal tolerating methylotrophic bacteria reduces cadmium toxicity and promotes plant growth of tomato}

1 is 80μM CdCl methanol magnetization bacteria strains in the presence of ₂ Methylobacterium oryzae strain CBMB20 (a and c) and Burkholderia sp. Accumulation graph of Cd ²⁺ in growth phase cells (a and b) and resting cells (c and d) of strain CBMB40 (b and d)

2 shows M.oryzae CBMB20 and Burkholderia sp. Graph showing ethylene production by tomato seedlings grown in the presence of Cd in growth pouches inoculated with CBMB40

3 is an analysis graph of Cd ²⁺ measured by ICP-OES in a pollen mixture of tomato plants grown in the absence and presence of ₂ mM CdCl ₂ .

Figure 4 shows M.oryzae CBMB20 and Burkholderia sp. Heavy Metal Binding Test Graphs in Tomato Stem and Roots Grown in Pollen Culture in the Presence or Absence of CBMB40 in the Presence of ₂ mM CdCl ₂

The present invention relates to the use of methanol magnetization bacteria to reduce the toxicity and growth of cadmium in tomato, more specifically as of January 6, 2006 A new microorganism, methylobacterial ducky sp, deposited with KACC, KACC. Knob. (Methylobacterium oryzae sp.nov.) (Aka CBMB20) KACC 11585)

 The characteristics of civilization, industrialization and technological advances have increased heavy metal emissions to the environment, which poses a significant threat to the environment and public health because of their toxicity, accumulation in the food chain, and permanence in nature. Botanical contaminated soil purification (Phytoremediation), which uses live plants to remove, destroy or sequester dangerous substances from the environment, is considered a promising way to deal with heavy metal contamination of soil (Cunningham et al., 1996). Some plants have the ability to accumulate metals with known and unknown biological functions and have been effectively used to improve heavy metal contamination of the soil. However, high concentrations of heavy metals are generally toxic to most plants, impairing plant metabolism and reducing plant growth. It is reported that the interface between microorganisms and plant roots (rhinosphere) can have a significant effect on both increased nutrient absorption and reduced metal toxicity (Smith, 1994). Thus, alternative ways to reduce the toxicity of heavy metals to plants using rhizosphere microorganisms have been investigated (Burd et al., 1998; Rajkumar et al., 2006; vivas et al., 2006). The improvement of the heavy metal toxicity of plants by microorganisms may be due to the reduction of metal uptake by plants (Vivas et al., 2006), or the reduction of the amount of harmful stress ethylene induced by heavy metals with no effect on heavy metal uptake. , 1998; Rajkumar et al., 2006). On the other hand, metal-resistant microorganisms having plant growth promoting properties can improve the availability of heavy metals in plant absorption through solubilization or mobilization, and can be effectively used for botanical contaminated soil purification (Abou-Shanab et al., 2003; Shing and Xia, 2006).

 Among heavy metals, cadmium (Cd) is considered to be phytotoxic. Cd is an element of unknown biological function in plants, which is a strong phytotoxic agent that causes growth inhibition and even plant death, altered free radical metabolism and cell disturbances (Sandalio et al., 2001). Changes in photosynthesis, leaf blight, bleaching and disturbances of macronutrient and micronutrient uptake and distribution have also been reported to be associated with Cd toxicity in plants (Gussarson et al., 1996; Toppi and Gabbrielli, 1999). Therefore, there is a need to study the resistance to these two metals of methanolotropic strains having plant growth promoting properties.

 Methanol magnetization can grow on 1-carbon compounds such as methanol or formaldehyde, which is their only carbon source, most of which is a member of the genus Methylobacterium, a pink-coloring random methanolysis (PPFM). They often combine with carnivorous and aquatic plants and colonize their roots, leaf surfaces and other parts.

The combination of the methyllobacterium species and the plant is believed to be a symbiotic relationship where bacteria use methanol, a lung metabolite from the plant, and then produce compounds that promote plant growth. Plant growth hormones such as indole-3-acetic acid (IAA), the production of cytokinin, the presence of ACC deaminase involved in the regulation of ethylene levels, and the promotion of plant growth by methyllobacterium have been investigated (Madhaiyan et al. 2006; Madhaiyan et al., 2007). Also, methyllobacterium species are known to metabolize a range of toxic organic chemicals and toxic explosives (see, eg, McDonald et al., 2001; Van Aken et al., 2004). . Moreover, methyllobacterium has been reported to be resistant to extremely high concentrations of heavy metals such as cadmium, chromium, mercury and lead (Marco et al., 2004).

Therefore, an object of the present invention is to demonstrate that bacterial microbial growth and heavy metal binding by methanol magnetizing bacteria in the presence of high levels of Cd, the new microorganism CBMB20 can reduce metal toxicity and promote plant growth in tomato in the presence of heavy metals. The purpose is to provide.

The present invention for the purpose of the present invention as described above to reduce the toxicity and growth of nickel and cadmium in tomato, microbial methylobacterial duckjae sp. The use of the knob strain CBMB20 (KACC 11585) is provided.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments.

Example 1 Bacteria Strain and Growth Conditions.

Methylobacterium Oryzae strain CBMB20 ^T (KACC 11585) and Burkholderia spp. Strain CBMB40 (KACC 91210P) are pink colored and non-colored methanol magnetizing bacteria isolated from rice tissue, respectively. Typically in the present invention, strain CBMB20 was cultured in minimal ammonium mineral salt (AMS) medium with 0.5% methanol (Whittenbury et al., 1970) and strain CBMB40 was cultured in Tris-buffered low-phosphate (TLP) medium.

TLP medium contains Trizma base, 6.06; NaCl, 4.68; KCl, 1.49; NH ₄ Cl, 1.07; Na ₂ SO ₄ 0.43; MgCl ₂ ˜6H ₂ O, 0.2; CaCl ₂ O ₂ H ₂ O, 0.03; Na ₂ HPO ₄ -12H ₂ O, 0.23; Fe (III) (NH ₄ ) citrate, 0.005, containing trace metals (1 mL of trace metal stock solution per liter, stock solution containing FeSO ₄ ㅇ 7H ₂ O, 0.2; ZnSO ₄ ㅇ H ₂ O, 0.01; MnCl ₂ ㅇ 4H ₂ O, 0.003; CoCl ₂ 6H ₂ O, 0.02; CuCl ₂ 6H ₂ O, 0.001; NiCl ₂ 6H ₂ O, Na ₂ MoO ₄ ○ 2H ₂ O, 0.5; H ₃ BO ₃ , 0.03 ) And 0.2% sodium gluconate (unit g / L). 2% (wt / vol) Bacto Agar (Difco Laboratories, Detroit, Mich.) Was added as a solid medium. For seed inoculation, bacterial strains were grown to maturation in a medium supplemented with 30 ° C., 0.1 mM NiCl ₂ / CdCl ₂ (Sigma-Aldrich Chemical Co., St. St. Louis, Missouri, USA). The cells were pelleted by centrifugation (25,000 × g, 10 min) and washed twice with sterile distilled water and then adjusted to a cell concentration of 1 × 10 ⁶ (OD _{600 nm} 0.2) with distilled water, which adjusted the predetermined calibration curve (γ = 600 nm). The concentration necessary for the seed immersion treatment was obtained.

Example 2 Heavy Metal Resistance by Bacterial Strains

The growth capacity of the strains was tested by increasing the concentration of CdCl ₂ by plate and liquid medium assays. To determine the minimum inhibitory concentration (MIC), strains were seeded on agar plates supplemented with CdCl ₂ from 1.0 to 10 mM and their growth was checked after appropriate incubation. In the liquid medium assay, pre-cultures in medium free of heavy metals were used to inoculate medium containing CdCl ₂ at different concentrations (0.1-1.0 mM). The growth after incubation for 48 hours at 30 ° C. under vibration (200 rpm) was recorded by measuring absorbance at 600 nm.

Example 3 Heavy Metal Binding in Growing Cells and Resting Cells

1 mL of each culture previously incubated in a medium free of heavy metals was inoculated into a series of 25 mL media and grown for 24 to 48 hours under vibration (30 ° C.). When the OD ₆₀₀ of the culture reached about 0.6 (medium exponential growth), 80 μM CdCl ₂ / NiCl ₂ (final concentration) was added to each flask. Samples were analyzed for Cd binding at different intervals after compound addition.

 By inductively coupled plasma photoluminescence spectroscopy (ICP-OES, Optima 5300DV, Perkin Elmer, USA) analysis (APHA, 1992), the cell density of the sample at each time point was measured and the heavy metal bound to the cells was measured. Three samples were taken from each flask at each data point.

In order to measure heavy metal binding in resting cells, the pre-culture prepared as above was inoculated in 100 mL minimal medium not supplemented with heavy metals. The cells were grown for 48 hours, cooled in ice for 20 minutes, and then centrifuged at 4 ° C. and 3,000 rpm for 30 seconds.

The cell pellet was washed with 50 mM Tris Chloride Buffer (pH 7.4) and suspended again in the same buffer. CdCl ₂ (80 μM) was added to the cell suspension and incubated without vibration (Wu et al., 2006a). Samples were taken at different intervals and the heavy metals bound to the cells were analyzed as above.

Example 4 Non-bacterial Non-Infected Plant Analysis for Root Elongation and Ethylene Determination

Tomato seeds (Lycopersicon esculentum L. cv Mairoku, Sokata Korea, Seocho-dong, Seoul, Korea) were sterilized approximately 0.2 g per treatment with 70% ethanol for 1 minute and 2% sodium hypochlorite (NaOCl) for 10 minutes and then sterilized with distilled water. Rinse more than times. This seed was then incubated for 4 hours at room temperature in sterile distilled water (control) or bacterial suspension.

Seeds were transferred to a growth pouch (125x157 mm) (CYG ™ seed germination pouch, Mega International Manufacturer, USA) filled with 20 mL of distilled water supplemented with different concentrations of CdCl ₂ or aseptically without any heavy metals, followed by 30 minutes at 121 ° C. Autoclaved. Pouches (10 pouches / treatment) were incubated with a 12/12 hour dark / light cycle in a growth chamber maintained at 20 × 1 ° C. At 15 days of growth the length of the main roots and stems was measured and the results are expressed as resistance index TI. TI = RL _m / RL _c , where RL _m is the root length of a plant grown in the presence of the specific metal added, RL _c is the root length of a plant grown in the absence of the metal (Wilkins, 1978)

On day 15, seedlings of about 50 to 60 seed weights (about 50 to 60 seedlings) were placed in a rubber stoppered 120 mL vial and incubated for 4 hours, after which 1 mL of the upper space was sampled from each vial, and equipped with a flame ionization detector. Ethylene content was measured in gas chromatography (GC) (DS6200, Donam Instruments Inc., South Korea) packed with Poropak-Q column at 70 ° C. The carrier gas was N ₂ at 60 mL / min flow rate, the combustion gas was H ₂ at 50 mL / min flow rate, and the combustion-assistant gas was air at 500 mL / min flow rate. By simulation, air containing 0.2 nmol ethylene / L was passed through the column for 60 minutes to check the ethylene capture column capacity. Under these conditions, 100% ethylene was captured.

Example 5 Greenhouse Experiment

Plants were placed in plastic pots (upper diameter, 220 mm; bottom diameter, 130 mm; height 240 mm) filled with approximately 1.5 kg of air-dried Wonju-Mix top soil and vegetable cultivation growth medium (Nong-Kyung Co., Ltd, Jincheon, Chungbuk, Korea). Planted; Growth media were 65-75% cocoa peat, 15-20% zeolite, 10-15% pearlite and macronutrients (mg / L) NH ₄ -N 150-200; NO ₃ -N, 100-150; Contains effective P ₂ O ₅ , 200-300; pH 5.7-7.0; Water content 40wh 5%; Water retention capacity 30 ㅁ 5kPa. 250 mL distilled water or 2 mL CdCl ₂ solution was added to the pot and grown for 10 days. Each pot was placed in a polyethylene bag to maintain the soil's moisture content. 20-day-old seedlings were planted in pots in a fully randomly designed greenhouse to repeat each treatment four times. At the time of transplanting, the inoculum was applied to soil near the root area at a rate of 10 mL per pot (10 ⁷ cfu / mL), and then applied twice more at 15 day intervals. Crops samples were taken at root 45 days after transplantation and recorded.

Example 6 Heavy Metal Analysis by ICP-OES

To analyze the heavy metals bound to the bacterial cells, the samples were centrifuged for 5 minutes at maximum speed (13,000 rpm) to discard the supernatant. The cell pellet was washed three times with 0.8% sodium chloride in 5 mM HEPES buffer (pH 7.1), dried for 24 hours in an oven set at 65 ° C., and then immersed in 2 mL of concentrated nitric acid for at least 48 hours. 18 mL of deionized water was added to the soaked cell pellet and reconstituted to 20 mL (Milli-Q Plus system, Millipore), which was used for ICP-OES analysis. Data was normalized to nmol number of Cd (mg cell dry weight).

After soaking in perchloric acid, sulfuric acid and distilled water (10: 1: 2) on a hot plate, the absorption of Cd in the roots and stems of the tomatoes was measured (500 mg sample). For soil analysis, 10 g of dry fine soil (using a 2 mm sieve) was mixed with 50 mL of 0.1 N HCl and vibrated at 200 rpm for 30 minutes. After immersion, the sample was filtered twice with filter paper (No. 6, Advantec ToYo), the volume was made up to 100 mL in a volumetric flask, and 10 mL of the sample was used for ICP-OES analysis. A Quality Control Standard 21 stock solution (5% HNO ₃ /tr.Tart./tr.HF, 100 μg / mL) from Perkin Elmer Life and Analytical Sciences (06484 Shelton Bridgeport Avenue 710, California, USA) was used. . A working solution was prepared as a primary stock solution using deionized distilled water.

Example 7 Bacteria Population Analysis

In order to extract microorganisms from the rhizosphere soil, soil (10 g) attached to the root was collected and suspended in 90 mL sterile distilled water. Continuous on NB (Difco Laboratories, Detroit, Mich.) Modified with agar and AMS agar (Whittenbury et al., 1970) 15 g / L containing cycloheximide (10 μg / mL) and 0.5% (v / v) methanol, respectively Dilution plates were used to count total organotrophic and methanolic bacteria. To count Burkhloderia, soil suspensions were plated on selective Pseudomonas cepacia azela tritamine (PCAT) agar (Burbage and Sasser, 1982) and incubated for 3 hours. Burkholderia colonies showed a characteristic morphology on PCAT medium, small (about 1 mm in diameter) and white with clear borders.

Statistics method

Using the SAS package (version 9.1) from SAS Institute Inc. (Cary, USA), the square deviations of the data were analyzed and averaged by Duncan's Multiple Range Test (DMRT) at P ≦ 0.05.

result

[Measurement of Minimum Inhibitory Concentration of Cadmium]

Methanol magnetizable strains M. oryzae CBMB20 and Burkholderia sp. CBMB40 was resistant to CdCl ₂ up to 3 mM, showed growth in plants, and growth was not observed above this concentration. Thus, the concentration of CdCl _{2 in} the liquid medium was changed while increasing from 0.1 to 1.0 mM, and the culture growth was measured by measuring the absorbance at 600 nm at 48 hours. Both strains were resistant to the concentration of added heavy metals, but growth was reduced when compared to cultures without the addition of heavy metals. Considering growth in the presence of heavy metals, no change was observed in the added heavy metals for both strains (data not shown).

[Binding of Heavy Metals by Methanol Growing Cells and Resting Cells]

Experiments were performed with the listed cultures to examine the binding of Cd ²⁺ to the cells.

In the growing phase of M. oryzae CBMB20 cells, Burkholderia sp. More amount of Ni / Cd was accumulated compared to CBMB40 (FIGS. 1A and 1B). Cd could be detected 12 hours after adding CdCl ₂ . The amount of Cd bound to cells increased as the culture grew, and the concentrations in M. oryzae CBMB20 increased from 0.012 to 0.43 and 0.687 to 7.45 nmol (mg cell dry weight) ^-1 for Cd, respectively ( 1a). Burkholderia sp. CBMB40 cells bound from 263.69 to 447.01 pmol Cd ²⁺ (mg cell dry weight) ⁻¹ (FIG. 1B).

Methanol magnetization bacteria strain Figure 1 in the accompanying drawings is in the presence of CdCl ₂ Methylobacterium oryzae strain CBMB20 (a and c) and Burkholderia sp. In growth cells (a and b) and resting cells (c and d) of strain CBMB40 (b and d) And Cd ²⁺ accumulation in these cells at different time points after heavy metal solution was added to show accumulation of Cd ²⁺ was measured by ICP-OES. Data is presented as the average of three replicates (P = 0.05, n = 3).

In addition, heavy metal binding experiments were performed in resting cells to simulate the slow growth properties of bacterial strains in the rhizosphere range.

The concentration of Cd ²⁺ ranged from 0.271 to 1.20 and 13.25 to 27.68 nmol (mg cell dry weight) ⁻¹ in M. oryzae CBMB20 (FIG. 1C). Burkholderia sp. CBMB40 ranged from 0.384 to 1.452 and 0.984 to 1.857 nmol (mg cell dry weight) ⁻¹ for Cd ²⁺ , respectively (FIG. 1D). In general, the total amount of bound Cd ²⁺ was greater in resting cells than in growing cells.

[Tolerance Indices and Root Elongation of Tomato Seedlings in Non-Infectious Non-Infectious Conditions]

In the analysis of non-microbial non-infectious state analysis using growth pouches, TIs of tomato seedlings with and without bacterial inoculation in the presence of different concentrations of CdCl ₂ were measured. As a result, tomatoes were very sensitive to Cd, and it was found that TI decreased as the CdCl ₂ concentration was increased. At higher concentrations (CdCl _{2 10} mM), seedlings did not settle with or without bacterial inoculation, and this result was not considered. The TI shown in Table 1 was calculated from the obtained root lengths, and similar results were also obtained when compared to the stem lengths (results not shown). Inoculation of bacterial strains to tomato seeds increased the TI for Cd at different concentrations (Table 1).

TABLE 1 M.oryzae CBMB20 or Burkholderia sp. On the Tolerance Index of Tomatoes to Different Cds in Infected Growth Pouches of Known Bacteria. Effect of CBMB40

 Heavy metal concentration (mM) Immunity Index (TI) CdCl ₂ No bacteria CBMB20 0 1.0 1.0 0.01 0.88 1.15 0.10 0.79 1.14 0.50 0.75 0.86 1.0 0.44 0.45 5.0 NG 0.33 10.0 NG NG

^• TI = RL _m / RL _c , where RL _m is the root length of the plant grown in the presence of the specific metal added, and RL _c is the root length of the plant grown in the absence of the metal.

It is evident that bacterial inoculation increased root elongation in tomatoes as compared to non-inoculated or heavy metal improved controls. When 0.01 and 0.1 mM CdCl ₂ were added, the inoculation of M. oryzae CBMB20 recorded longer root lengths than the unvaccinated and uninoculated, unimproved controls. Burkholderia sp. Inoculation of CBMB40 increased tomato root elongation in the presence of CdCl ₂ as compared to no inoculation. However, in the presence of 0.5 and 1 mM concentrations of CdCl ₂ , root growth was much more inhibited and bacterial inoculation increased root elongation compared to no inoculation (Table 2). Similarly, M. oryzae CBMB20 and Burkholderia sp. Inoculation of CBMB40 increased the biomass of tomato seedlings under conditions known to be non-bacterial (Table 2).

TABLE 2 M. oryzae CBMB20 or Burkholderia sp. On seedling growth when different concentrations of Cd were present in non-bacterial growth pouches. Impact of CBMB40

 Heavy metal concentration (mM) Root height (cm) CdCl ₂ No bacteria CBMB20 CBMB40 0 9.27 ± 0.21 11.78 ± 0.38 10.21 ± 0.29 0.1 8.12 ± 0.40 10.59 ± 0.40 9.53 ± 0.32 0.10 7.30 ± 0.33 10.63 ± 0.33 8.02 ± 0.34 0.50 6.92 ± 0.14 7.92 ± 0.14 7.74 ± 0.26 1.0 4.10 ± 0.13 4.10 ± 0.13 7.31 ± 0.17 5.0 - 3.05 ± 0.16 1.25 ± 0.53 10.0 - - - Dry matter † 0 96.02 ± 4.95 113.81 ± 8.73 98.52 ± 3.99 0.01 81.44 ± 9.49 107.44 ± 9.49 93.16 ± 4.71 0.10 74.43 ± 3.71 101.43 ± 3.71 90.00 ± 5.20 0.50 48.68 ± 3.28 95.68 ± 3.28 78.42 ± 4.86 1.0 38.64 ± 4.99 68.64 ± 4.99 54.00 ± 5.20 5.0 55.35 ± 2.51 - 10.0 34.55 ± 2.05 -

^† Average dry weight of 60 seedlings: Seeds that did not germinate on day 2 and roots grown from these seeds were not measured. Data are expressed as mean W SE. The average root length of 15-day-old tomato seedlings was assessed by root elongation analysis from the measurement of 60 seedlings (6 seeds per growth pouch; 10 growth pouches per time; 2 replicates).

[Ethylene production]

The addition of 0.01-10 mM CdCl ₂ as the growth solution increased the stress ethylene in tomato seedlings. Bacterial inoculation reduced stress ethylene levels in the presence of heavy metals. However, when higher concentrations of CdCl ₂ (5 and 10 mM) were present, the seedlings could not settle under both uninoculated and inoculated conditions. M. oryzae CBMB20 and Burkholderia sp. When the seeds were treated with CBMB40 suspension for analysis without extramicrobial status, when treated with different concentrations of CdCl ₂ , the amount of ethylene released ranged from 0.77-2.51 pmol / h (gFw) ⁻¹ . oryzae CBMB20 [0.47-2.35 pmol / h (gFw) ⁻¹ ] or Burkholderia sp. Decreased when inoculated with CBMB40 [0.572-1.136 pmol / h (gFw) ⁻¹ ].

[Heavy metal accumulation and plant growth promotion in pot culture]

In pollen experiments, higher concentrations of CdCl ₂ (2 mM) were required to significantly inhibit the root and stem lengths of tomatoes than in growth pouch experiments, and the results were variable. However, biomass production (both stem and root) was greatly reduced in the presence of heavy metals, and application of bacteria by soil application significantly increased the biomass of tomatoes in the presence of heavy metal solutions (Table 3). The total organotrophic populations treated with different treatments ranged from 10 ⁵ to 10 ⁶ cfug ⁻¹ in soil, with the largest population present in M. oryzae CBMB20 applied soil. The total bacterial population decreased with heavy metals and increased with bacterial strains (Table 3). More PPFM populations were maintained in treatments involving the application of M. oryzae CBMB20, and a significant number could be observed in CBMB40 treatments. When observed on a PCAT agar plate, Burkholderia sp. Only a significant number of Burkholderia were present at CBMB40 treatment (Table 3).

The amount of Cd present in the soil at tomato harvest was measured for different treatments. Inoculation of bacteria by soil application significantly reduced the concentration of heavy metals in the soil. The amount of Cd ²⁺ was found in M. oryzae CBMB20 and Burkholderia sp. CBMB40 inoculation decreased from 6.808 mg / kg to 1.755 and 2.078 mg / kg, respectively.

Table 3 Effect of methanol magnetization on plant growth and Cd accumulation of tomato in pollen culture conditions in the presence of heavy metals and different treated rhizosphere populations

process Plant height Dry matter yield Soil population stem Root stem Root Total Organic Nutrients PPFMs Burkholderia Control 79.35 ± 4.52 ba 48.33 ± 11.78 ba 8.13 ± 0.88 bc 1.25 ± 0.22 de 5.87 ± 0.21 cb - - CBMB20 alone 81.83 ± 4.17 ba 65.00 ± 2.31 ba 9.30 ± 2.07 bac 1.31 ± 0.15 dec 6.19 ± 0.11 a 4.03 ± 0.07 a - CBMB40 alone 84.33 ± 3.08 a 69.33 ± 13.37 ba 8.70 ± 1.44 bac 1.42 ± 0.31 bdec 5.62 ± 0.13 e 3.10 ± 0.06 b 4.13 ± 0.08 CdCl2 alone 74.67 ± 3.27 b 52.27 ± 7.77 ba 7.25 ± 0.35 c 1.24 ± 0.01 de 5.73 ± 0.13 cde - - CBMB20 + CdCl2 84.33 ± 1.76 a 69.67 ± 12.39 ba 10.81 ± 2.15 a 2.01 ± 0.12 ba 5.81 ± 0.24 cd 3.96 ± 0.09 a - CBMB40 + CdCl2 80.00 ± 1.73 ba 60.67 ± 12.55 ba 8.50 ± 0.89 bac 1.81 ± 0.11 bdac 5.85 ± 0.26 cd - 4.10 ± 0.07 LSD (P = 0.05) 9.30 30.36 2.45 0.62 0.18 0.20 -

Values are mean W SE of 3 replicates. The values in each column and the same letter following it are statistically insignificant (P> 0.05) according to Fisher's least significant difference test. -Not measured.

Uptake of Cd ²⁺ by tomato roots and stems in inoculated and non-inoculated conditions in the presence of heavy metals was measured by ICP-OES analysis. The amount of Cd ²⁺ was kept more in the roots than the stems, while the tomato roots maintained about 75.95 mg (kg dry weight) ⁻¹ of Cd ²⁺ . Application of bacterial strains significantly reduced heavy metals accumulated in tomato roots and stems, and M. oryzae CBMB20 was found in Burkholderia sp. Better reduction than CBMB40 (see FIG. 4). These results suggest that methanol magnetization strains impart a protective effect on the presence of heavy metals by reducing the sequestration of Cd ²⁺ in tomato stems and roots.

According to the International Health Organization, Cd is one of the most immediate metals involved in the environment (Zatar et al., 2006). Since Cd is also phytotoxic, it has been reported to inhibit plant growth and disrupt nutrient absorption and other metabolic and physiological processes (Toppi and Gabbrielli, 1999; Molas, 2002). In the present invention, M. oryzae CBMB20 and burkholderia sp. It has been found that strain CBMB40 can very effectively protect tomato plants from growth inhibition caused by high concentrations of Cd.

Bacterial strains M. oryzae CBMB20 and Burkholderia sp. CBMB40 strain has a resistance to high concentrations of CdCl ₂ in the presence of growth medium. Heavy metal resistance in microorganisms generally includes metal binding by cell walls or proteins and extracellular polymers, insoluble metal sulfide formation, volatilization and enhanced diffusion from cells (Huges and Poole, 1989). The strain used in the present invention binds a large amount of Cd ²⁺ cells with both resting and growing conditions. Moreover, these strains also produce plant growth hormone and ACC diaminase, which properties help to promote plant growth (Madhaiyan et al., 2006; Poonguzali et al., 2007). With respect to these inherent properties, microorganisms with surfaces that interact strongly with metal ions in solution could play a significant role in the metal availability of absorbing and immobilizing toxic ions from the soil solution. The purpose of this study was to investigate whether tomato plants might contribute to the phytotoxic effects of Cd.

 In the non-microbial uninfected condition analysis, bacterial application to tomato seeds prior to growing tomatoes in the growth pouch promoted root elongation and increased TI for various concentrations of Cd under non-microbial conditions. However, as in previous reports (Safronova et al., 2006), the inoculation effect varied with the bacterial strains inoculated. The presence of heavy metals is a commonly encountered stress, and plants respond to different environmental stresses by synthesizing higher levels of ethylene (Abeles et al., 1992), resulting in reduced plant growth. Nevertheless, when plant growth promoting bacteria contain the ACC deaminase enzyme, it can act to regulate the level of ethylene in the plant.

 In a previous study, we proposed a model involving the production of plant growth hormone and ACC deaminase in Methylobacterium and lowering ethylene levels in canola.

Bacteria with ACC deaminase activity act as a precursor to ACC, an early precursor of ethylene in plants, and by converting ACC to α-ketobutyrate and ammonia to reduce levels of ACC and ethylene, Inhibitory effects were reduced (Madhaiyan et al., 2006). It is well documented that plant growth promoting bacteria with ACC deaminase activity promote plant growth even in the presence of heavy metals by reducing stress ethylene synthesized in plants (Burd et al., 1998; Safronova et al., 2006; Rajkumar et al., 2006). Simultaneously with these results, in the present invention, the increase in ethylene release in the presence of heavy metals was reduced due to the inoculation of bacteria in the non-microbial uninfected state experiment.

 In contrast, in the present invention, M. oryzae CBMB20 and Burkholderia sp. CBMB40 was used to reduce heavy metal uptake in the roots and stems of tomatoes, and also to reduce their availability in the soil. Similarly, the inoculation of plant growth promoting bacteria reduced Cd uptake in barley plants. , It is probably because Cd is bacteria fixed in the rhizosphere. Moreover, inoculation of plant growth promoting bacterial strains into plants can also contribute to reducing the phytotoxic effect of metals by sharing metal loads due to the biosorption and bioaccumulation of these strains (Zaidi and Mussarat, 2004).

 According to the present invention, a population consistent with the inoculated strain is observed in the rhizosphere soil, and increased resistance to heavy metals could provide a competitive advantage in contaminated environments. Microorganisms improved on metals appear to have several strategies for colonizing their soils (Meharg and Cairney, 2000). Inoculated bacterial strains reduce the level of Cd in the soil by sharing metal loads with the plant roots, and thus the plant settles during the early growth phase. Moreover, after seedlings have settled in the soil, these strains also help the plants acquire sufficient plant growth hormone and other nutrients for optimal plant growth in the presence of heavy metals (Zaibi et al., 2006). The decrease in absorption in tomato roots and stems is likely due to bacterial strains biosorbing these metals and preventing them from translating to plants.

According to the present invention, eventually, the inoculation of selected bacteria not only reduces stress ethylene, but also improves plant growth and reduces the harmful effects of heavy metals by reducing the uptake by plants and potential transfer to plants, Suggests a decrease in Cd toxicity.

The results are also the first to show that plant growth-promoting Methylobacterium and Burkholderia have been able to alleviate heavy metal stress induced in plants.

Claims (3)

Methanol magnetization bacteria, characterized in that it is used to reduce cadmium toxicity and promote plant growth. The method according to claim 1, wherein the methanol magnetization bacteria is methyl duck duck SP. Methanol magnetization bacteria characterized in that the knob (Methylobacterium oryzae sp. Nov .; KACC 11585). 4. The methanol magnetization bacterium according to claim 3, wherein the methylobacterial duck sp. Knob is isolated from the stem of rice and is a novel microorganism capable of producing ACC diminase.

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