AU2018402480A1

AU2018402480A1 - Endogenous bacillus megaterium BM18-2 with cadmium enrichment for promoting growth of hybrid pennisetum and application thereof

Info

Publication number: AU2018402480A1
Application number: AU2018402480A
Authority: AU
Inventors: Zhiwei Liu; Yumei PAN; Chen QIAN; Juanzi WU; Xiaoxian ZHONG
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2018-01-12
Filing date: 2018-12-07
Publication date: 2019-09-26
Anticipated expiration: 2038-12-07
Also published as: CN108102984A; AU2018402480B2; WO2019137122A1; CN108102984B

Description

ENDOGENOUS BACILLUS MEGATERIUM BM18-2 WITH CADMIUM ENRICHMENT FOR PROMOTING GROWTH OF HYBRID PENNISETUM AND APPLICATION THEREOF

I. Technical field

The present invention relates to a cadmium-enrichment and growth-promoting hybrid Pennisetum endophytic Bacillus megaterium BM18-2 and application thereof, belongs to the technical field of agricultural microbiology.

II. Background Art

According to statistical data, there are nearly 300 million mu of cultivated land that has been polluted to varying degrees in China, accounting for about 1/6 of the total area of cultivated land. According to the national Environmental Quality Standard for Soils (GB 15618-1995), when the pH of soil is < 7.5, in order to ensure agricultural production, the Cd limit value for maintaining human health in the soils shall be no more than 0.3 mg/kg. According to this standard, 43 soil samples of Zijiang River Basin and 72 soil samples of Xiangjiang River Basin in Hunan province were taken for testing in 2010. The results showed that the heavy metal Cd pollution is particularly serious, the Cd overstandard rate in the soil in Zijiang River Basin is 88.6% and that in Xiangjiang River Basin is 83.34% (Heavy metal pollution evaluation methodology for heavy metal risk assessment in the soils in Zijiang River Basin, Environmental Chemistry, 2011, 30(9); Heavy metal pollution in the soils in the Xiangjiang River Basin and its ecological environment risk assessment, Environmental Science, 2012, 33(1): 260-265). Different degrees of cadmium pollution in soils also exist in other provinces and cities.

American scientist Chaney et al. first proposed the assumption to remove heavy metal pollutants from soils using plants in 1983. Thereafter, the studies on the heavy metal pollution control have switched to phytoremediation technology gradually. Phytoremediation technology is a soil remediation technology that uses some plants and their coexisting microbial systems that can tolerate and hyperaccumulate toxic metal elements to remove pollutants. It is considered to be an important way to solve soil pollution problems fundamentally. Hybrid Pennisetum is a triploid distant hybrid of the diploid Pennisetum americanum sterile line and tetrapioid Pennisetum purpureum. It is a perennial C4 plant of the gramineous Pennisetum, with strong cadmium tolerance and high biological yield. It is a potential remediation crop for cadmium contaminated soils (Xingfeng Zhang, Hanping Xia, Zhian LiPing Zhuang Bo Gao. Potential of four forage grasses in remediation of Cd and Zn contaminated soils, 2010, Bioresource Technology, 2063-2066). In addition, as hybrid Pennisetum is not only the ideal raw material for vegetation composite products (wood/hybrid Pennisetum composite woodbased panel manufacturing method, ZL200610166308.9, melamine resin impregnated wood veneer soy gum hybrid Pennisetum wood-based panel, ZL 201420456683.7), but also an important biomass energy conversion raw material (Hou Xincun, Fan Xifeng, Wu Juying, Zhu Yi, Zhang Yongxia. Potential of herbaceous energy plants for phytoremediation of heavy metal contaminated soil, Chinese Journal of Grassland, 2012, 34(1): 59-63), it can avoid entering into the food chains and secondary pollution, to achieve remediation and improvement of cadmium-contaminated soils and high value-added industrial utilization. Therefore, the isolation and screening of cadmium-enrichment ·!·

James & Wells Ref: 312414AU and growth-promoting hybrid Pennisetum endophytic bacteria is of great significance for the application of hybrid Pennisetum to remediate cadmium contaminated soils and its industrial applications.

III. Contents of the Invention

It is an object of the present invention to obtain Bacillus megaterium BM18-2 with significantly improved cadmium tolerance by screening Bacillus megaterium BM18 strain in vitro for cadmium stress.

Another object of the present invention is to provide applications of this strain.

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

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

The hybrid Pennisetum cadmium-enrichment and growth-promoting strain agent prepared by the Bacillus megaterium BM18-2.

The hybrid Pennisetum cadmium-enrichment and growth-promoting strain agent is preferably obtained by shaking cultivation of the BM18-2 in an LB liquid medium at 30 °C, 200 rpm for 16~18h.

Use of the BM18-2 for promoting the growth of hybrid Pennisetum in cadmium-contaminated soils, preferably for promoting the growth of hybrid Pennisetum in the soil with cadmium concentration <75 mg/kg.

The application of the BM18-2 for enhancing the remediation efficiency of hybrid Pennisetum to heavy metal cadmium contaminated soils.

The method of the present invention is suitable for the remediation of heavy metal contaminated soil with Cd < 75 mg/kg by Pennisetum plants.

The application of the BM18-2 for enhancing the cadmium-enrichment ability of hybrid Pennisetum and promoting the growth of hybrid Pennisetum.

The present invention can achieve the following beneficial effects. The Bacillus megaterium BM182 can grow in LB solid medium with cadmium concentration of 24 μΜ; roots of Bacillus megaterium BM18-2-impregnated hybrid Pennisetum seed seedlings or stem seedlings are transplanted into cadmium-contaminated soils, and the cadmium content in leaves, stems and roots of plants, biological yield and total cadmium absorption are higher than those in the negative controls without endophytic bacteria. After being immersed with the strain, the hybrid Pennisetum is promoted to grow well in a cadmium-contaminated soil environment, and the cadmium content and biomass of hybrid Pennisetum is increased, and the remediation efficiency of heavy metal cadmium contaminated soil is enhanced.

IV. Description of Drawings

FIG. l.SEM image of BM18-2

FIG. 2 Phylogenetic tree analysis of 17 species of Bacillus megaterium

FIG. 3 Photos of determination of ammonia production, from the left to the right: blank, BM18 and •2·

James & Wells Ref: 312414AU

BM18-2

FIG. 4 Electron micrograph of the endophytic BM18-2 in roots

FIG. 5 Electron micrograph of the endophytic BM18-2 in young spike

FIG. 6 Hybrid Pennisetum plants growing in cadmium-contaminated fields; left: negative control, right: BM18-2 impregnated

Biomaterial deposit information

Bacillus megaterium BM18-2 is deposited in the China Center for Type Culture Collection on November 10, 2017, and the deposit address is Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M2017679.

V. Embodiments

Embodiment 1 (1) Availability of Cd-tolerance and nitrogen-fixing endophytic bacteria BM18-2

The inventors took the hybrid Pennisetum plants in booting stage from the field of the Xinyang Experimental Station in Yancheng, Jiangsu Province previously. The young spikes less than 5 cm were used as exophytes. After sterilized, young spikes were cut to 2-3 mm and inoculated into MS medium, and then an endophytic BM18 was screened from the regenerated plants cultured from young spikes in vitro. The strain was able to grow on a LB solid medium with a cadmium ion concentration of 18 μΜ. 10 pL of Bacillus megaterium BM18 bacteria solution stored in glycerol at a mass concentration of 50% at -18 °C was taken, inoculated into 25 ml of LB liquid medium, and cultured for 18h at 30°C, 200 rpm while shaking, to obtain activated bacteria in logarithmic growth phase; 50 pL of the above-mentioned activated bacteria solution was streak-inoculated on LB solid medium supplemented with different cadmium ion concentrations, and cultured at a constant temperature 30 °C for 24h to observe the colony growth of LB medium with different cadmium concentration; the vigorously growing colonies on LB solid medium with cadmium ion concentration of 18 μΜ were picked and inoculated into LB solid medium with 18 μΜ cadmium concentration, cultured at a constant temperature 30 °C for 24h, and then vigorously growing colonies were picked , and still inoculated into LB solid medium with 18 pM cadmium concentration; the vigorously growing colonies after being subcultured for 3 generations were picked and inoculated into LB solid medium with 20 pM cadmium ion concentration, cultured at a constant temperature 30°C for 24h, and vigorously growing colonies were picked and subcultured for twice in LB solid medium containing 20 pMcadmium concentration, and then inoculated into LB solid medium with 22 pM cadmium ion concentration, and vigorously growing colonies were picked and subcultured twice in LB solid medium containing 22 pM cadmium concentration, and then inoculated into LB solid medium with cadmium ion concentration of 24 pM. Vigorously growing colonies were picked and subcultured twice in LB solid medium containing 24 pM cadmium concentration, and then inoculated into LB solid medium with a cadmium ion concentration of 26 μΜ and subcultured, and found that colonies could not grow normally. The vigorously growing colonies in LB solid medium with 24 pM cadmium concentration were picked and streak-inoculated into cadmium-free LB solid medium, and subcultured for 3 generations; the vigorously growing pure strains were picked and streak-inoculated into the LB solid medium with 24 pM cadmium concentration. The vigorously growing strain BM182 was picked and inoculated into LB liquid medium, and cultured at 30°C, 200 rpm for 18h while •3·

James & Wells Ref: 312414AU shaking, 300 pL of glycerin at mass concentration of 50% was added to 700 pL of the bacteria solution in a 1.5 ml sterile centrifuge tube, and a total of 20 parts were stored in a freezer at -80 °C.

(2) Biological characteristics of endophytic bacteria BM18-2

BM18-2 grew well on LB medium, the optimum growth temperature was 30°C. The colony was round, white and opaque, Gram-positive, bacterial cells were in rod shape, while single cell was also in short-chain shape, 0.75-1.2 pm x 3-8 pm (FIG. 1).

(3) Whole genome resequencing analysis of endophytic bacteria BM18-2 pL of BM18-2 glycerol bacteria solution at 50% mass concentration that was stored at -80°C was taken and inoculated into 25 mL of LB liquid medium, and cultured at 30°C, 200 rpm for 16-18h while shaking to obtain activated bacteria in logarithmic growth phase; 50 pL of BM18-2 activated bacteria solution was streak-inoculated into a LB solid medium containing 24 pM cadmium ion concentration, monoclonal colonies were picked and inoculated into a 300 ml LB medium, and cultured at 30°C, 200 rpm for 16-18h while shaking until the ODeoo was about 0.5. The bacteria solution was transferred to a 50 ml sterile centrifuge tube under a sterile environment, centrifuged at 5,000 rpm for 5 minutes, and the supernatant was discarded in a sterile environment. The above steps were repeated for 6 to 7 times to collect BM18-2 bacteria cells, genomic DNA was extracted with genomic-Tip 500/G kit, and the product was sent to Beijing Novogene Technology Co., Ltd. for genome-wide resequencing. The whole-genome blast analysis was performed for the sequencing results in NCBI International Genome database, with the highest similarity as Bacillus megaterium QM B1551 (NC_014019.1) (84.25%). The analysis result showed that BM18-2 was bacillus megaterium. The phylogenetic tree analysis of 17 species of bacillus megaterium was shown in FIG.

2.

(3) Determination of nitrogen-fixing and ammonia-producing characteristics of bacillus megaterium BM18-2

Determination of nitrogen-fixing ability: The activated Bacillus megaterium BM18-2 bacteria solution was inoculated into an Ashby medium, and cultured in a 30°C shaker while shaking at 125 rpm for 7d, and 20 pL of the bacteria solution was taken, streaked and cultured into a Petri dish containing Jensen medium, repeated for 3 times, the inoculated sterile water as used as a control and placed for 7d at 30°C, and colonies growth in culture medium inoculated with bacteria solution was observed, indicating that BM18-2 had nitrogen fixation capacity; wherein, the Ashby medium components: glucose 10.0 g/L, K2HPO4 0.2 g/L, K2SO4 0.2 g/L, NaCl 0.2 g/L, CaCCfi 5.0 g/L, and MgSO4’7H2O 0.2 g/L; Jensen medium components: NaMoO4’2H2O 0.012 mg/L, CuSCUUHzO 0.012 mg/L, MgSO₄-4H₂O 0.5 mg/L, Na₂-EDTA 5.0 mg/L, FeSO₄-7H₂O 0.2 mg/L, and HBO3 5.0 mg/L. The pH of solution was adjusted to 5.8-6.0.

Determination of NFF-producing ability: The activated Bacillus megaterium BM18-2 bacteria solution was inoculated into peptone water, repeated for 3 times, the solution inoculated into sterile water was used as a control, and placed at 30°C for 48-72 h. When the bacteria solution became turbid, 0.5 ml of Nessler's reagent was added to 10 ml fermentation broth, when the solution became yellow (FIG. 3), it indicated that Bacillus megaterium BM18-2 had ammonia-producing ability; wherein, the components of peptone water: peptone 10.0 g/L, and NaCl 5.0 g/L.

Embodiment 2 Field evaluation of cadmium enrichment and growth promotion of Bacillus megaterium BM18-2 .4.

James & Wells Ref: 312414AU

On May 20, 2017, in planting structure-adjusted farmland in Hunan Province, the Cd content at 0-20 cm soil layer was 0.6564 mg/kg, pH value was 4.36; and the Cd content at 20-40 cm soil layer was 0.5625 mg/kg, pH value was 4.87, the roots of hybrid Pennisetum seed seedlings containing more than 8 leaves were soaked in BMI8-2 bacteria solution with ODeoo value of about 0.5 and sterile water for 2~3h, and then the seed seedlings were transplanted into Cd-contaminated farmland at a planting space of 40 cm x 50 cm, according to a randomized block design, each block was 18 square meters, 3 repeats for each treatment. Urea was applied at 75 kg/hm² during seedling stage, and after growing for 30d, the urea was applied at 150 kg/hm². On October 15, 2017, 2 individual plants were randomly cut in each block and their fresh weights were weighed. The roots, stems and leaves of the sampled individual plants were separated, and dried in an oven at 75 °C, and then the dry matter yield was calculated. The leaves, stems and roots samples were crushed separately, and then the cadmium contents in leaves, stems and roots were determined by graphite furnace atomic absorption spectrophotometry, to calculate the total amount of cadmium absorbed by leaves, stems and roots. The results showed that (Table 1), the average dry matter yield of hybrid Pennisetum inoculated with Bacillus megaterium BM18-2 was increased by 207.76%, 271.92% and 178.41% respectively, compared with the negative control hybrid Pennisetum without inoculating endophytic bacteria, the contents of Cd in leaves, stems and roots were increased by 74.24%, 15.95% and 25.39%, respectively. The average total amount of absorptions of Cd in leaves, stems and roots were increased by 428.98%, 249.89% and 234.67%, respectively. This indicated that Bacillus megaterium BMI8-2 enhanced the resistance of hybrid Pennisetum to cadmium stress and promoted the growth of hybrid Pennisetum and the cadmium absorption in different parts of the plants.

Table 1 Effect of immersion in endophytic bacteria BM18-2 on cadmium enrichment and growth promotion of hybrid Pennisetum

Item Name	BM18-2-hybrid Pennisetum	Hybrid Pennisetum (control)	Increase or decrease rate (%)
Dry matter yield (kg/666.7 m²)	Leaf	882.2275 b B±29.9198	286.6600 e E±34.7268	207.76
Stem	2,775.8500 a A±101.2530	746.3500 cC±44.1758	271.92
Root	487.8500 dD±89.5357	175.2300 fF±35.1198	178.41
Cd content (mg/kg)	Leaf	1.7150 a A±0.18064	0.9843 c C±0.2489	74.24
Stem	0.9175 cC±0.1783	0.7913 dD±0.2871	15.95
Root	1.7111 aA±0.1904	1.3646 b B±0.2454	25.39
Total amount of Cd accumulation (kg/666.7 m²)	Leaf	1.5039 bB±0.2551	0.2843 d D±0.0992	428.98
Stem	2.2708 a A±0.2994	0.6490 cC±0.1303	249.89
Root	0.7818 cC±0.2183	0.2336 dD±0.0500	234.67

Embodiment 3 Colonization identification of Bacillus megaterium BM18-2 in hybrid Pennisetum plants of hybrid Pennisetum at the booting stage that were immersed in Bacillus megaterium BMI 8- as described in Embodiment 2 and the negative controls without treatment were taken, and about 2 •5·

James & Wells Ref: 312414AU cm white roots and about 5 cm young spikes were cut and rinsed repeatedly for 3~4 times with pure water. Then the sample surfaces were repeatedly washed with 2 mol/L phosphate buffer. After the samples were immersed in 2~3% glutaraldehyde fixative in the darkness at 4°C for 2h, samples were immersed in 50%, 70%, 80%, 90% alcohol for 15 min respectively, and then immersed in 100% alcohol for Ih, repeated for 3 times, after the samples were completely dehydrated, they were observed by SEM, and Bacillus megaterium BM18-2 was found in roots and young spikes treated by endophytic bacteria (FIG. 4 and FIG. 5), but no endophytic bacteria were found in roots and young spikes of the negative controls.

Embodiment 4 Identification of cadmium tolerance and growth promotion of Bacillus megaterium BM18-2 in potted plants

The potted original soils were taken from the farmland soils of the headquarters of Jiangsu Academy of Agricultural Sciences. The soil bulk density was 1.32 g/cm³, the pH was 6.79, and the Cd content was 0.2004 mg/kg. The concentrations of Cd in the hybrid Pennisetum pot experiment established by artificial exposure to cadmium chloride were 25 mg/kg, 50 mg/kg, and 75 mg/kg, respectively, and were recorded as Cd25, Cd50, and Cd75. The potted original soil was used as control, and recorded as CdO. The test was carried out in the glass greenhouse of Jiangsu Academy of Agricultural Sciences. On August 31, 2017, the hybrid Pennisetum seed seedlings with 8-10 leaves were transferred to a pot, with soil layer thickness of 40 cm, 1 plant per pot, repeated for 3 times. On November 13, the aboveground parts of the plants were taken and weighed, after dried in ovens at 75 °C and weighed, the dry matter yield was calculated. The samples of aboveground parts of plants were crushed, and the cadmium content in the plant was determined by graphite furnace atomic absorption spectrophotometry. The total amount of cadmium absorbed in the aboveground parts of plants was calculated. Results showed that (Table 2), compared with the negative controls without inoculating endophytic bacteria, the dry matter yields of the aboveground parts of the endophytic bacteria BM182-immersed hybrid Pennisetum plants were increased significantly, and CdO, Cd25, Cd50 and Cd75 were increased by 112.22%, 64.49%, 26.20%, and 43.99% respectively. The cadmium concentrations in the aboveground parts of the plants were increased by 46.11%, 12.21%, 13.75% and 1.34%, and the total amount of cadmium absorptions of the aboveground parts of the plants were increased by 233.33%, 64.37%, 43.28% and 60.84%.

Table 2 Effect of endophytic bacteria BM18-2 immersion on the biomass, cadmium content and total amount of accumulation of hybrid Pennisetum

Item	Treatment	BM18-2-hybrid Pennisetum	Hybrid Pennisetum (control)	Increase or decrease rate (%)
Dry matter yield (g/ individual plant)	CdO	47.2975 aA±0.4900	22.2875 bB±0.9167	112.22
Cd25	19.6575 cC±1.0300	11.9508 dD±0.1734	64.49
Cd50	9.7775 e E±0.8900	7.7475 f FG±0.3400	26.20
Cd75	9.5575 e EF±0.6900	6.6375 f G±0.8100	43.99
Cd content (mg/kg)	CdO	0.2161 e E±0.0526	0.1479 eE±0.0070	46.11
Cd25	15.4503 cC±0.1132	13.7694 dD±0.6583	12.21
Cd50	16.9189 b B±0.2088	14.8738 c CD±0.2430	13.75

•6·

James & Wells Ref: 312414AU

	Cd75	20.3372 a A±0.9102	20.0677 a Ail.0492	1.34
Total amount of	CdO	0.0110 eD±0.0013	0.0033 e Di0.0002	233.33
Cd accumulation	Cd25	0.2699 a A±0.0221	0.1642 cCiO.0294	64.37
(mg/ individual	Cd50	0.1652 c CiO.0110	0.1153 dCi0.0069	43.28
plant)	Cd75	0.2152 bB±0.0313	0.1338 cd Di0.0232	60.84

•7·

Claims

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

2. A hybrid Pennisetum cadmium-enrichment and growth-promoting strain agent prepared by the Bacillus megaterium BM18-2 of claim 1.

3. The strain agent according to claim 2, wherein the hybrid Pennisetum cadmium-enrichment and growth-promoting strain agent is obtained by shaking cultivation of the BM18-2 of claim 1 in an LB liquid medium at 30°C, 200 rpm for 16~18h.

4. The application of the BM18-2 of claim 1 for promoting the growth of hybrid Pennisetum in cadmium-contaminated soils.

5. The application according to claim 4, wherein the application of the BM18-2 of claim 1 for promoting the growth of hybrid Pennisetum in the cadmium contaminated soil with cadmium concentration <75 mg/kg.

6. The application of the BM18-2 of claim 1 for enhancing the remediation efficiency of hybrid Pennisetum to heavy metal cadmium contaminated soils.

7. The application of the BM18-2 of claim 1 for enhancing the cadmium-enrichment ability of hybrid Pennisetum and promoting the growth of hybrid Pennisetum.