WO2019137122A1

WO2019137122A1 - Endogenous bacillus megaterium bm18-2 with cadmium enrichment for promoting growth of hybrid pennisetum and application thereof

Info

Publication number: WO2019137122A1
Application number: PCT/CN2018/119676
Authority: WO
Inventors: 钟小仙; 钱晨; 刘智微; 吴娟子; 潘玉梅
Original assignee: 江苏省农业科学院
Priority date: 2018-01-12
Filing date: 2018-12-07
Publication date: 2019-07-18
Also published as: CN108102984B; AU2018402480B2; AU2018402480A1; CN108102984A

Abstract

Disclosed in the present invention are an endogenous bacillus megaterium BM18-2 with cadmium enrichment for promoting the growth of hybrid pennisetum and an application thereof. The endogenous bacillus megaterium BM18-2 with cadmium enrichment for promoting the growth of hybrid pennisetum is preserved in China Center for Type Culture Collection in Wuhan University in Wuhan of China on November 10, 2017 with a preservation number of CCTCC NO: M2017679. Further disclosed is a hybrid pennisetum cadmium enrichment growth-promoting microbial inoculum prepared using the bacillus megatherium BM18-2. The BM18-2 according to the present invention can be used for promoting the growth of hybrid pennisetum in cadmium-contaminated soil. By invasion the hybrid pennisetum with the strain, the healthy growth of the hybrid pennisetum in a cadmium-contaminated soil environment can be promoted, thus improving the cadmium content and biomass of the hybrid pennisetum and enhancing remediation efficiency on heavy metal cadmium-contaminated soil.

Description

A cadmium-promoting hybrid Pennisetum endophytic Bacillus megaterium BM18-2 and application thereof

Technical field

The invention relates to a cadmium-promoting hybrid Pennisetum endophytic bacterium Bacillus BM18-2 and application thereof, and belongs to the technical field of agricultural microorganisms.

Background technique

According to statistics, the area of cultivated land that has been polluted to varying degrees in the country is close to 300 million mu, accounting for about one-sixth of the total area of cultivated land. According to the national "Soil Environmental Quality Standards" (GB15618-1995), when the soil pH is ≤ 7.5, in order to ensure agricultural production, the soil limit value for maintaining human health is Cd ≤ 0.3mg/kg. According to this standard, Hunan Province will be funded in 2010. The results of sampling and sampling of 43 soil samples in the river basin and 72 soils in the Xiangjiang River Basin are particularly prominent. The Cd pollution rate of the heavy metal cadmium is 88.6%, and the Cd over-standard rate of the Xiangjiang River Basin is 83.34%. Heavy metal risk in soils in the river basin, Environmental Chemistry, 2011, 30(9); Heavy metal pollution in the Xiangjiang River Basin and its ecological environment risk assessment, Environmental Science, 2012, 33(1): 260-265), other provinces and cities are also different Degree of cadmium contaminated soil.

American scientist Chaney et al. first proposed the use of plants to remove heavy metal pollutants from soil in 1983. Since then, people have gradually shifted the focus of research on heavy metal pollution control in soil to phytoremediation technology. Phytoremediation technology is a soil remediation technology that uses some plants that can tolerate and accumulate toxic metal elements and their coexisting microbial systems to remove pollutants. It is considered to be an important way to fundamentally solve soil pollution problems. Hybrid Pennisetum is a triploid distant hybrid of diploid P. sinensis and tetraploid grass, which is a perennial C4 plant of the genus Pennisetum, with strong cadmium tolerance and biological yield. High, a potentially rehabilitated crop of cadmium contaminated soil (Xingfeng Zhang, Hanping Xia, Zhilian LiPing Zhuang Bo Gao. Potential of four forage grasses in remediation of Cd and Zn contaminated soils, 2010, Bioresource Technology, 2063-2066). At the same time, because hybrid Hydrangea is not only the ideal raw material for grass and wood composite products (wood / hybrid pennisetum composite wood-based panel manufacturing method, ZL200610166308.9, melamine resin impregnated thin wood veneer bean gum hybrid penniseta artificial board, ZL 201420456683.7) It is also an important biomass energy conversion raw material (Hou Xincun, Fan Xifeng, Wu Juying, Zhu Yi, Zhang Yongxia. The potential of herbaceous energy for phytoremediation of heavy metal contaminated soil, Chinese Journal of Grassland, 2012, 34(1): 59-63), Avoid entering the food chain and secondary pollution, and achieve cadmium-contaminated soil remediation and improvement and high value-added industrial utilization. Therefore, the isolation and screening of endophytic bacteria that are rich in cadmium-promoting hybrid Pennisetum is of great significance for the application of hybrid Pennisetum to repair cadmium-contaminated soil and industrial application.

Summary of the invention

The object of the present invention is to obtain Bacillus megaterium BM18-2 (Bacillus megaterium BM18-2) which is significantly improved in cadmium tolerance by screening the Bacillus megaterium BM18 strain in vitro by cadmium stress.

It is an object of the invention to provide the use of this strain.

The object of the invention can be achieved by the following technical solutions:

A hybrid hybrid Pennisetum endophytic Bacillus megaterium BM18-2 was deposited with the China Center for Type Culture Collection on November 10, 2017. The deposit address is Wuhan University, Wuhan, China, with the accession number CCTCC NO: M2017679.

The hybrid Pennisetum prepared by the Bacillus megaterium BM18-2 is enriched with a cadmium promoter.

The hybridized Pennisetum enriched cadmium-promoting agent is preferably obtained by culturing the BM18-2 in an LB liquid medium at 30 ° C and shaking at 200 rpm for 16 to 18 hours.

The use of the BM18-2 of the present invention for promoting the growth of hybrid Pennisetum in cadmium-contaminated soil; preferably for promoting the growth of hybrid Pennisetum in soil with a cadmium concentration of ≤75 mg/kg.

The BM18-2 of the invention has the application of enhancing the repairing efficiency of hybrid hemerocallis to heavy metal cadmium contaminated soil.

The method of the invention is suitable for the repair of Cd≤75mg/kg heavy metal contaminated soil by Pennisetum plants.

The BM18-2 of the invention has the application of improving the cadmium-enriching ability of hybrid Pennisetum and promoting the growth of hybrid Pennisetum.

Beneficial effect: Bacillus megaterium BM18-2 is grown in LB solid medium with cadmium concentration of 22 μΜ; the roots of B. megaterium BM18-2-impregnated hybrid Pennisetum seedlings or seedlings are transplanted into cadmium-contaminated soil, plants The cadmium content, biological yield and total cadmium uptake of leaves, stems and roots were higher than those of non-inoculated endophytes. Using this strain to infect hybrid Pennisetum can promote its good growth in cadmium-contaminated soil environment, increase the cadmium content and biomass of hybrid Pennisetum, and enhance the repair efficiency of heavy metal cadmium contaminated soil.

DRAWINGS

Figure 1. SEM image of BM18-2

Figure 2 Phylogenetic tree analysis of 17 species of Bacillus megaterium

Figure 3. Photograph of ammonia production, blank from left to right, BM18 and BM18-2

Figure 4. Electron micrograph of the root of BM18-2

Figure 5. Electron micrograph of the young ear of BM18-2

Figure 6. Hybrid Pennisetum plants grown in cadmium-contaminated fields; left: negative control, right: BM18-2 infection

Biomaterial preservation information

Bacillus megaterium BM18-2, deposited on November 10, 2017, is a collection of typical culture collections in China. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M2017679.

Detailed ways

Example 1

(1) Acquisition of cadmium-fixing endophytic bacteria BM18-2

Inventors in the early stage from the Yancheng Xinyang Experimental Station in Yancheng, Jiangsu, took the booting stage of hybrid Pennisetum plants, with less than 5cm young ears as explants, the young ears were disinfected and cut to 2-3mm inoculated in MS medium, from young ears An endophytic BM18 was selected from the body culture regenerated plants, and the strain was able to grow on LB solid medium with a cadmium ion concentration of 18 μM. 10 μL of Bacillus megaterium BM18 deposited in glycerol at a concentration of 50% at -18 ° C, inoculated into 25 mL of LB liquid medium, and cultured at 30 ° C, shaking at 200 rpm for 18 h to obtain activated bacteria in logarithmic growth phase; 50 μL of the above-mentioned activated bacterial solution was streaked onto LB solid medium supplemented with different cadmium ion concentration, and cultured at 30 ° C for 24 h at constant temperature to observe the colony growth of LB medium with different cadmium concentration; LB solid culture with cadmium ion concentration of 18 μM was picked. The vigorously growing colonies were inoculated on LB solid medium with 18μM cadmium concentration, cultured at 30°C for 24h, and then picked up vigorous colonies. They were still inoculated into LB solid medium with 18μM cadmium concentration; picking subculture 3 The vigorously growing colonies were inoculated into LB solid medium with cadmium ion concentration of 25 μΜ, cultured at 30 ° C for 24 h, and the vigorously grown colonies were picked up and subcultured twice in LB solid medium containing 50 μM cadmium concentration, and then inoculated. In LB solid medium with cadmium ion concentration of 75 μΜ, the vigorously growing colonies were picked up and subcultured twice in LB solid medium containing 75 μM cadmium concentration, and then inoculated into LB solid culture with cadmium ion concentration of 100 μΜ. In the nutrient base, the vigorously growing colonies were picked up and subcultured twice in LB solid medium containing 100 μM cadmium concentration, and then inoculated into LB solid medium with a cadmium ion concentration of 125 μΜ, and found to be unable to grow normally. The vigorously growing colonies in LB solid medium with 100μΜ cadmium concentration were streaked into LB solid medium without cadmium for 3 times; the vigorously grown pure strains were selected and streaked at 100μΜ cadmium concentration. In the LB solid medium, pick the vigorous growth strain BM18-2, inoculate it in LB liquid medium, incubate at 30 ° C, shaking at 200 rpm for 18 h, add 700 μL of the bacterial solution to the mixture of 50 μl of glycerol at a concentration of 50%, and mix it at 1.5 ml. A total of 20 parts of the bacteria centrifuge tube were stored in a freezer at -80 °C.

(2) Biological characteristics of endophytic BM18-2

BM18-2 grows well on LB medium, the optimal growth temperature is 30 °C, the colony is round, the colony is white opaque, Gram-positive, the stem is rod-shaped, single or short-chain, 0.75-1.2μm× 3 to 8 μm (Fig. 1).

(3) Whole genome resequencing analysis of endophytic BM18-2

10 μL of BM18-2 glycerol bacteria solution stored at -80 ° C and 50% by mass was inoculated into 25 mL of LB liquid medium, and cultured at 30 ° C, 200 rpm for 16-18 h to obtain activated bacteria in logarithmic growth phase; Take 50 μL of BM18-2 activating bacterial solution and inoculate it in LB solid medium containing 100 μM cadmium ion concentration, pick up the monoclonal colonies and inoculate them in 300 ml of LB medium, and incubate at 30 ° C, 200 rpm for 16-18 h until the bacteria Incubate the solution to an OD ₆₀₀ of about 0.5; in a sterile environment, transfer the bacterial solution to a 50 ml sterile centrifuge tube, centrifuge at 5000 rpm for 5 minutes, discard the supernatant in a sterile environment, and repeat the above steps 6 to 7 times to collect BM18. -2 cells, genomic DNA was extracted with genomic-Tip 500/G kit, and the product was sent to Beijing Nuohe Zhiyuan Technology Co., Ltd. for genome-wide resequencing. The sequencing results were analyzed in the whole genome blast of NCBI International Genome database, with huge Bacillus megaterium QM B1551 (NC_014019.1) has the highest similarity and similarity of 84.25%. The analysis results show that BM18-2 is Bacillus megaterium, and the phylogenetic tree analysis of 17 species of B. megaterium is shown in Fig. 2.

(3) Detection of nitrogen fixation and ammonia production of Bacillus megaterium BM18-2

Nitrogen fixation test: The activated Bacillus megaterium BM18-2 was inoculated into Ashby medium, shaken at 30 ° C for 30 days at 125 rpm, and 20 μL of the bacterial solution was streaked into a Petri dish containing Jensen medium. Three times of repeated, inoculated sterile water as a control, placed at 30 ° C for 7 days, observed colonies in the culture medium inoculated with bacteria, indicating that BM18-2 has nitrogen fixation capacity; Among them, Ashby medium formula: glucose 10.0g /L, K ₂ HPO ₄ 0.2g / L, K ₂ SO ₄ 0.2g / L, NaCl 0.2g / L, CaCO ₃ 5.0g / L, MgSO ₄ · 7H ₂ O 0.2g / L; Jensen medium formula is : NaMoO ₄ · 2H ₂ O 0.012 mg / L, CuSO ₄ · 7H ₂ O 0.012 mg / L, MgSO ₄ · 4H ₂ O 0.5 mg / L, Na ₂ · EDTA 5.0 mg / L, FeSO ₄ · 7H ₂ O 0.2 Mg/L, HBO ₃ 5.0 mg/L. Adjust the pH to 5.8-6.0.

Production of NH ₃ capacity test: The activated Bacillus megaterium BM18-2 broth was inoculated into peptone water, repeated 3 times, inoculated with sterile water as a control, placed at 30 ° C for 48-72 h, observed that the bacterial liquid became cloudy, added to the naphthalene 0.5ml of sler reagent in 10ml fermentation broth, yellow color was observed (Fig. 3), indicating that B. megaterium BM18-2 has ammonia-producing ability; among them, peptone water formula is: peptone 10.0g / L, NaCl 5.0g / L.

Example 2 Evaluation of cadmium-promoting growth field of Bacillus megaterium BM18-2

On May 20, 2017, planting structure-adjusted farmland in Hunan Province, in which the Cd content of 0-20cm soil layer was 0.6564mg/kg, the pH value was 4.36, and the Cd content of 20-40cm soil layer was 0.5625mg/kg, and the pH value was 4.87, soak the roots of hybrid seedlings with more than 8 leaves in BM18-2 bacterial solution with OD ₆₀₀ value of 0.5 and 2-3h in sterile water, and transplant the plants to Cd-contaminated farmland at a distance of 40cm×50cm. Medium, random block design, each square 18 square meters, each treatment 3 repetitions, urea application 75kg / hm ² at the seedling stage, after 30d growth, urea application 150kg / hm ² , October 15, 2017, each Two single plants were randomly cast and weighed. The roots, stems and leaves of the sampled plants were separated, and dried at 75 °C in an oven. The dry matter yield was calculated and the leaves, stems and roots were crushed separately. The cadmium content in leaves, stems and roots was determined by atomic absorption spectrophotometry, and the total amount of cadmium absorbed by leaves, stems and roots was calculated. The results showed that (Table 1), the average dry matter yield of hybrid Pennisetum inoculated with Bacillus megaterium BM18-2 increased by 207.76%, 271.92% and 178.41%, respectively, compared with the negative control hybrid Pennisetum. The content of Cd in leaves, stems and roots increased by 74.24%, 15.95% and 25.39%, respectively. The average absorption of Cd in leaves, stems and roots increased by 428.98%, 249.89% and 234.67%, respectively, indicating that B. megaterium BM18-2 was enhanced. The resistance of hybrid Pennisetum to cadmium stress promoted the growth of hybrid Pennisetum and the absorption of cadmium in different parts of the plant.

Table 1 Effect of endophytic BM18-2 dip staining on cadmium-promoting growth of hybrid Pennisetum

Example 3 Colonization Identification of Bacillus megaterium BM18-2 in Hybrid Pennisetum

Take the B. megaterium BM18-2 and the untreated negative control for the booting stage of the hybrid Pennisetum plant in the same manner as in Example 2, and cut the white roots of about 2 cm and about 5 cm, and rinse them repeatedly with pure water. After ~4 times, the surface of the sample was repeatedly washed with 2 mol/L phosphate buffer. The sample was immersed in 2 to 3% glutaraldehyde fixative at 4 °C for 2 hours, and the samples were 50%, 70%, 80%, respectively. Soaked in 90% alcohol for 15 minutes, soaked in 100% alcohol for 1 hour, repeated 3 times, after the sample was completely dehydrated, and observed by electron scanning electron microscopy, it was found that there were Bacillus megaterium BM18 in the roots and ears of endophyte treatment. 2 (Fig. 4 and Fig. 5), no endophytes were found in the negative control roots and young ears.

Example 4 Identification of cadmium-promoting potted plants by Bacillus megaterium BM18-2

The potted original soil was taken from the farmland soil of the Jiangsu Academy of Agricultural Sciences. The soil bulk density was 1.32g/cm ³ , the pH was 6.79, and the Cd content was 0.2004mg/kg. The artificial soil of chlorinated cadmium was used to set the test soil of hybrid Pennisetum potted plants. The concentration of Cd was 25, 50, 75 mg/kg, which were recorded as Cd25, Cd50 and Cd75, respectively. The potted original soil was used as the control and recorded as Cd0. The test was carried out in the glass greenhouse of Jiangsu Academy of Agricultural Sciences and transplanted on August 31, 2017. 8-10 leaf hybrid wolftail seedlings to pots, soil thickness 40cm, 1 plant per pot, 3 replicates, on the 13th of November, the ground part of the plant was weighed, dried at 75 °C oven, weighing The dry matter yield was used to pulverize the aboveground part of the plant. The cadmium content in the plant was determined by graphite furnace atomic absorption spectrophotometry, and the total amount of cadmium absorbed in the aerial part of the plant was calculated. The results showed that (Table 2), compared with the negative control without endophytic bacteria, the dry matter yield of the aboveground part of the endophytic BM18-2-infected hybrid Pennisetum plant was significantly increased, Cd0, Cd25, Cd50, Cd75 respectively The increase of 112.22%, 64.49%, 26.20% and 43.99% increased the cadmium concentration of aboveground parts of plants by 46.11%, 12.21%, 13.75% and 1.34%, and the total cadmium uptake of plants increased by 233.33%, 64.37%, 43.28% and 60.84%. .

Table 2 Effect of Endophytic BM18-2 Dip on Biomass, Cadmium Content and Total Accumulation of Hybrid Pennisetum

Claims

A hybrid hybrid Pennisetum endophytic Bacillus megaterium BM18-2, which was deposited on November 10, 2017 in the China Center for Type Culture Collection, deposited at Wuhan University, Wuhan, China under the accession number CCTCC NO. :M2017679.
The hybrid pennisetum prepared by the Bacillus megaterium BM18-2 of claim 1 is a cadmium-promoting agent.
The bactericidal agent according to claim 2, wherein the BM18-2 according to claim 1 is cultured in an LB liquid medium at 30 ° C, shaking at 200 rpm for 16 to 18 hours to obtain the cadmium-enriched zein Growth fungi.
Use of BM18-2 of claim 1 for promoting the growth of hybrid Pennisetum in cadmium-contaminated soil.
The use according to claim 4, characterized in that the BM18-2 according to claim 1 is used for promoting the growth of hybrid Pennisetum in cadmium-contaminated soil having a cadmium concentration of ≤75 mg/kg.
The use of the BM18-2 of claim 1 for enhancing the repair efficiency of hybrid hemerocallis for heavy metal cadmium contaminated soil.
The use of BM18-2 according to claim 1 for enhancing the cadmium-enriching ability of hybrid Pennisetum and promoting the growth of hybrid Pennisetum.