LU502717B1 - Lysinibacillus fusiformis and its application in ecological reconstruction of mining area reclamation - Google Patents

Abstract

The invention discloses a kind of Lysinibacillus fusiformis, and its application in ecological reconstruction of mining area reclamation. The preservation number of Lysinibacillus fusiformis is CGMCC No.9799. Lysinibacillus fusiformis can improve the nutrient content of the mine reclamation soil, and has significant effect on improving the content of organic matter, alkali-hydrolyzable nitrogen, available potassium and available phosphorus of reclaimed soil in mining area; furthermore, further experiments show that Lysinibacillus fusiformis can also increase the crop biomass, which can increase the crop biomass in the reclaimed soil of the mining area, whether for the cultivation of wheat or brassica campestris; in particular, the use of Lysinibacillus fusiformis can effectively and significantly change the community structure of soil microorganisms, improve the diversity of soil microorganisms, effectively increase the number of microorganisms, thus speeding up the restoration of soil microbial communities.

Description

DESCRIPTION LU502717

LYSINIBACILLUS FUSIFORMIS AND ITS APPLICATION IN

ECOLOGICAL RECONSTRUCTION OF MINING AREA RECLAMATION

TECHNICAL FIELD

The invention belongs to the technical field of the ecological reconstruction, and in particular to a kind of Lysinibacillus fusiformis and its application in ecological reconstruction of mining area reclamation.

BACKGROUND

Coal plays a decisive role in energy production and consumption in China, and its proportion has always remained at about 70%. While supporting the social and economic development, the exploitation of coal resources has also caused lasting and serious negative impacts on land resources and ecological environment. The 2007 China Geological Environment Bulletin pointed out that the area of land occupied and damaged by the mining development is 1,658,000 hectares, including 909,000 hectares of tailings, 522,000 hectares of open pits, 203,000 hectares of mining subsidence; in addition, about 4 million mu of land is newly damaged or destroyed every year, of which more than 60% is cultivated land or other agricultural land. The contradiction between man and land in coal mine area is aggravating, which is a serious threat to the food security and sustainable development of social economy in our country.

Actively carrying out land reclamation and ecological reconstruction in mining areas is a systematic project with strategic significance. In 1988, China promulgated the Provisions on Land

Reclamation and clearly stipulated the responsibilities and obligations of land reclamation in the process of resource development, the implementation of this regulation can not only reduce land loss but also protect the ecological environment. It is a systematic project that benefits the country and the people and has important economic and social significance. However, at present, the land reclamation rate in mining areas in China is less than 10%. Land reclamation is not only the simple recovery and utilization of damaged or disturbed land, but also the ecological restoration. The ecological reclamation is the ultimate goal of land reclamation in mining areas. The key to ecological restoration is the restoration of ecosystem functions and the construction of a reasonable structure, as well as the restoration of vegetation and the construction of animal and plant communities and microbial communities. The key to ecological restoration is the restoration of ecosystem functions and the construction of a reasonable structure, as well as the restoration {15027 17 vegetation and the construction of animal and plant communities and microbial communities.

At present, in the research of land reclamation in coal mining subsidence area in our country, the traditional fertilization measures are mainly adopted in most places. The main problems lie in the absence of living organisms, the lack of microorganisms in the soil, the slow transformation of material and energy, the slow improvement and recovery of soil ecological system such as the physical properties and biological properties of the soil, and the long reclamation period. At present, many countries adopt advanced microbial reclamation technology to reclaim land. Microbial reclamation technology is a biotechnological measure to comprehensively treat and improve the soil in reclaimed areas by taking advantage of microbial inoculation. By inoculating some beneficial and growth-promoting microorganisms to newly planted plants, utilizing the life activities of microorganisms and rhizosphere microorganisms themselves, it digs up the potential fertility of reclaimed substrate, restores and establishes its symbiotic system, promotes plant growth, accelerates the improvement of substrate of reclaimed soil and the sustainable development of ecosystem, improves the quality of ecological management in mining areas, and thus shortening the reclamation period. However, in the production practice, the shortage of microbial strains with remarkable effects has become the bottleneck problem in the microbial bioremediation technology.

SUMMARY

The invention aims to overcome the defects of long reclamation period and poor structure of reclaimed soil in the existing mining area, and provides a kind of Lysinibacillus fusiformis with the functions of solubilizing phosphorus, solubilizing potassium and fixing nitrogen and an application thereof in the ecological reconstruction of the mining area reclamation, the invention can effectively improve the nutrient level and microbial community of the reclaimed soil in the mining area, promote the growth of crops and accelerate the ecological reconstruction speed.

The invention is realized by the following technical scheme:

A kind of Lysinibacillus fusiformis, which is Lysinibacillus fusiformis with the preservation number of CGMCC No.9799.

The Lysinibacillus fusiformis can improve the content of organic matter, alkali-hydrolyzable nitrogen, available phosphorus and available potassium in reclaimed soil of mining area.

The Lysinibacillus fusiformis can improve the biomass of crops growing in reclaimed soil of mining area. LU502717

The Lysinibacillus fusiformis can improve the diversity of microbial community structure in reclaimed soil of mining area.

The 16S rDNA sequence of Lysinibacillus fusiformis is shown in SEQ.ID.NO 1.

A microbial agent of Lysinibacillus fusiformis, which is prepared by the following steps: fully mixing the active ingredients obtained by culturing Lysinibacillus fusiformis in a mass ratio of 2: 10- 2: 20 with a carrier after the amplification culture, and storing at normal temperature; the carrier is a solid carrier or a liquid carrier, where the solid carrier is mineral material, plant material, polymer compound or organic material; the mineral material is one or more of clay, kaolin, montmorillonite, zeolite and fly ash; the plant material is one or more of corn flour, starch, soybean flour and peanut flour; the polymer compound is one or two of polyvinyl alcohol and polyglycol; the organic material is one or more of peat, humic acid and organic fertilizer; the active component is one or more of living cells obtained by enlarged culture, fermentation liquid containing cells and filtrate of cell culture; the microbial agent is prepared into that following dosage form: liquid agent, emulsion, suspending agent, granule, wettable powder or water dispersible granule.

An application of Lysinibacillus fusiformis in improving the reclamation soil of the mining area.

The Lysinibacillus fusiformis or the microbial agent prepared by Lysinibacillus fusiformis can improve the content of organic matter, alkali-hydrolyzable nitrogen, effective phosphorus and quick-available potassium in the mine area reclamation soil and improve the structural diversity of the mine area reclamation soil microbial community.

An application of Lysinibacillus fusiformis in preparing the microbial agent for improving the reclamation soil of the mining area.

An application of Lysinibacillus fusiformis in preparing the biological fertilizer for promoting the growth of reclamation soil plants of the mining area.

Compared with the prior art, the invention has the following beneficial technical effects:

The Lysinibacillus fusiformis provided by the invention is isolated and purified from soil collected from a reclaimed mining area, and then screened through an inorganic phosphorus selective culture medium, a solid silicate bacteria selective culture medium and a solid nitrogen- free culture medium in sequence, and the screened microorganism is determined to be a new microorganism, namely Lysinibacillus fusiformis, through biological identification, 16SrDNA 557 17 series analysis and determination of phosphate and potassium solubilizing capacity. Lysinibacillus fusiformis has extremely strong soil adaptability and is a high-efficiency microorganism with functions of solubilizing phosphorus, solubilizing potassium and fixing nitrogen; the experimental detection shows that it can improve the nutrient content of the mine reclamation soil, and has significant effect on improving the content of organic matter, alkali-hydrolyzable nitrogen, available potassium and available phosphorus of reclaimed soil in mining area; furthermore, further experiments show that Lysinibacillus fusiformis can also increase the crop biomass, which can increase the crop biomass in the reclaimed soil of the mining area, whether for the cultivation of wheat or brassica campestris; in particular, the use of Lysinibacillus fusiformis can effectively and significantly change the community structure of soil microorganisms, improve the diversity of soil microorganisms, effectively increase the number of microorganisms, thus speeding up the restoration of soil microbial communities, and can be applied to improve the reclaimed soil in mining areas and promote the growth of plants in the reclaimed soil in mining areas.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the morphology of Lysinibacillus fusiformis of the present invention cultured on solid beef paste peptone medium for 48 hours;

FIG. 2 shows the morphology of Lysinibacillus fusiformis cultured on solid nutrition agar medium for 24 hours, then stained by Gram stain under optical microscope oil microscope (10*100 times).

Preservation description

The Lysinibacillus fusiformis provided by the invention has been preserved in China General

Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC

No.9799.

DESCRIPTION OF THE INVENTION

The present invention will be further explained in detail with reference to the following specific examples, which are illustrative rather than restrictive.

The invention provides a kind of Lysinibacillus fusiformis, where, Lysinibacillus fusiformis with the preservation number of CGMCC No.9799.

The invention provides the application of Lysinibacillus fusiformis or the microbial agent in improving the reclaimed soil in mining areas and promoting the growth of plants.

The present invention will be further explained by the following specific examples. LU502717

Embodiment 1

This embodiment illustrates the screening and identification process of Lysinibacillus fusiformis (CGMCC No.9799) of the present invention. 1. Collecting samples

Collecting soil from typical reclaimed mining areas in Shanxi Province, respectively collecting 0-20cm soil samples from weeds (N 36°28'17.846", E 113°00'50.383") in coal mining subsidence reclamation area in Xishandi Village, Xiangyuan County, Changzhi, Shanxi Province, and preserving at 4°C; 2. Primary strain screening

Weighing 10g of collected mine reclamation soil, adding 90mL of sterilized 250mL triangular flask filled with glass beads and sterile water, shaking under a constant-temperature shaker at 30°C for 20min at 180r/min, standing for 5 min, then sucking 1mL of bacterial solution into 9mL of sterile water, and fully mixing (at this time, the dilution is 10), and then sucking 1mL of bacterial liquid from this test tube into another sterile water filled with 9mL, and mixing thoroughly; by that analogy, obtaining soil suspensions with different dilutions of 103, 10*, 10” and 10°. Finally, coating 0.1mL of bacterial suspension with dilution concentrations of 10, 10° and 10° on solid inorganic phosphorus selective medium (agar containing 10g/L glucose, 25g/L Caz(PO4),, 5 g/L magnesium chloride, 0.25g/L magnesium sulfate, 0.25g/L potassium chloride, 0.15g/L ammonium sulfate and 18 g/L agar), setting three repetitions for each dilution gradient of the soil suspension; after culturing at 30°C for 10 days, 8 strains which can form larger clear circle are screened, the ratios of the diameter (d) of pellucid circle and the diameter (d) of colony of these 8 strains are > 1.5.

Respectively inoculating the screened eight bacterial strains to a selective culture medium (Alexandrov medium, containing 5 g/L of sucrose, 0.5 g/L of magnesium sulfate, 0.2 g/L of

NazHPO4, 0.1 g/L of CaCOs, 0.005 g/L of FeCL3, 1.0 g/L of soil mineral and 18 g/L of agar) of the solid silicate bacteria by a scribing method, culturing for 4d at 28°C, selecting the colonies with large colonies, high transparent protrusions and elastic adhesion, scribing on the Alexandrov culture medium repeatedly, and performing microscopic examination until a pure culture is obtained, and obtaining four pure cultured bacterial strains in total.

Inoculating the screened four strains into a solid nitrogen-free culture medium ( Abei culture medium containing 10 g/L of glucose, 0.2 g/L. of potassium dihydrogen phosphate, 0.2 g/L Pfi5027 17 magnesium sulfate, 0.2 g/L of sodium chloride, 0.1 g/L of calcium sulfate, 5 g/L of calcium carbonate, 18 g/L. of agar, pH 7.0 and 18 g/L of agar) by a streaking method respectively, culturing at 30°C for 7 days to obtain a strain G-1207 with good growth, and storing the purified strain in an inclined plane of a beef paste protein aging culture medium 3. Re-screening

Testing the screened strain G-1207 for its ability to dissolve phosphorus and potassium. 3.1 Measuring phosphorus-solubilizing capacity:

Inoculating bacterial strain G-1207 into an inorganic phosphorus medium (containing 10g/L of glucose, 25 g/L of Cas(POa4),, 5 g/L of magnesium chloride, 0.25 g/L. of magnesium sulfate, 0.25 g/L of potassium chloride and 0.15 g/L. of ammonium sulfate) triangular flask, wherein the content of G-1207 is 10° cfu/mL, and placing on a shaker at 150 r/min for shaking culture at 30°C for 5 days; simultaneously inoculating Bacillus megaterium (ACCC11107) purchased from Agricultural

Culture Collection of China (ACCC) in the same method as a positive control; inoculating the inactivated strain G-1207 as a negative control, and repeating each treatment for three times; centrifuging the obtained fermentation broth at 5000 r/min for 30min, measure the effective phosphorus content in the supernatant by molybdenum antimony anti-colorimetric method, calculating the phosphorus-solubilizing rate, and measuring the pH value of the supernatant by the acidimeter (the detection results are shown in Table 1).

Table 1 The phosphorus-solubilizing rate of G-1207

Effective Phosphorus

Phosphorus-

Treatments pH Content Lo.

Solubilizing Rate (%) (mg/L)

ACCC11107 _. 4 75+0.06b 352.58+36.24b 7.28+0.75b

CK

. 6.75+0.05c 41.25+0.75a 1.09+0.03a

Note: Different lowercase letters in the same column show significant differences in p<0.05.

The test results showed that the strain had remarkable phosphorus-solubilizing effect, and the phosphorus-solubilizing rate reached 23.18%. 3.2 Measuring potassium-solubilizing capacity: 1) Preparation of seed liquid:

Selecting one ring of activated G-1207 slant fungus moss, inoculating into 30mL of seed502717 culture solution (containing 5.0 g/L of starch, 1.0 g/L. of yeast extract, 2.0 g/L. of K,HPO4, 0.5 g/L of MgSO4 7H,0, 0.1 g/L of CaCOs3, Sm g/L of FeCls-6H,O, pH 7.5-8.5), shaking culture at 28°C for 24h, the thallus content is about 2x10® cfu/mL. Inoculating the silicate bacteria (ACCC10013) purchased from Agricultural Culture Collection of China in the same way as the positive control. 2) The shaking flask culture of silicate bacteria

Adding 50 mL of prepared potassium-solubilizing culture medium (containing 10.0 g/L of sucrose, 0.5 g/L of MgSO4:7H,0, 0.2 g/L. of (NH4)>'SO4, 0.1 g/L of NaCl, 0.1 g/L. of CaCOs, 0.1g/50mL of soil mineral culture solution and pH 7.2-7.5) into 250mL conical flasks, sterilizing and cooling, and connecting 3mL of prepared seed liquid with each bottle; taking Silicate bacteria (ACCC10013) as a positive control, and at the same time adding the same amount of inactivated

G-1207 seed solution as a negative control. All the treatments are repeated for three times, and after inoculation, shake culture is carried out at 180r/min and 28°C for 5 days. 3) Studies on potassium-solubilizing rate of silicate bacteria

Pouring all fermentation broth into an evaporating dish, heating and concentrating to about mL in a water bath, adding 2.0 mL of H>O; solution, continue to evaporate, continuously stirring, and repeatedly adding 20% HzO, solution until the viscous substance is completely digested.

Centrifuging at 3500 r/min for 10 min, collect the supernatant into a 50 mL volumetric flask, then using distilled water to constant volume, and measuring the content of water-soluble potassium in the fermentation broth on a flame photometer. The detection results are shown in Table 2.

Table 2 The potassium-solubilizing rate of G-1207

Mee [ta content (mg/kg) potassium (%)

A 10013

CK

I EE | 8

Note: Different lowercase letters in the same column show significant differences in p<0.05.

The results show that the strain had remarkable potassium-solubilizing effect, and the increase rate of water-soluble potassium reached 74.26%. 4. Identification of strains

According to the experimental contents and methods recorded in Common Bacterial System

Identification Manual and Principles, Methods and Practice of Bacterial Systematics, the strain Fu 502717 1207 obtained by screening is analyzed and identified by its morphology, physiology and biochemistry and 16SrDNA.

The morphological characteristics of this strain are as follows: The colonies are round, colorless and transparent, with neat edges, protrusions in the middle, and stickiness. The colonies are rod-shaped (as shown in FIG. 1) under the electron microscope, and there are spores.

The further physiological and biochemical characteristics are shown in Table 3.

Table 3 Physiological and biochemical characteristics of G-1207

Catalase | + | brgalactosidase | — | D-sorbitol | +

Oxidase — | + | Urease | | D-mannitol | + rie =| Arme dvd | =] Para | +]

MR-test | — | Lysincdchydroxylase | — | Inositol | +

Nitrate reduction | — | Omithine decarboxylase | + | L-arginine | —

Gelatin liquefaetion | =] Ghormum | + | Line | — ; + | L-aspartate | +

Amylohydrolysis D-glucose

HiSproduction | — | Drgalactose | + | L-glutamate | +

Indole production | —| D-fructose | + | 50°C growth —

Citic acid growth | + | Dmammose [+] 7%NaClgrowth | + Dmaiose [+]

Physiological and biochemical determination results of the strain: Gram staining is positive (the staining results are shown in Figure 2), catalase is positive, oxidase is positive, acetyl methyl alcohol (V. P.) is negative, methyl red (M. R.) is negative, nitrate reduction is negative, gelatin liquefaction is negative, amylohydrolysis is negative, hydrogen sulfide production is negative, indole test is negative, citric acid growth is positive, 7%NaCl growth is positive, b-galactosidase, urease, arginine dihydrolase and lysine dehydroxylase are negative, Ornithine decarboxylase is positive, Glycerinum, D-glucose, D-galactose, D-fructose and D-mannose are fermented to produce acid, which are positive, D-sorbitol, D-mannitol, D-arabitol, Inositol are fermented to produce acid, which are positive, L-arginine, L-alanine are fermented to produce acid, which are negative, L-aspartate, L-glutamate are fermented to produce acid, which are positive.

Further carrying out biological 16SrDNA series analysis, extracting bacterial genome DNA, and then performing PCR amplification: 16SrDNA is amplified by 27F/1492R primers, and the primer sequence is as follows: 27F: S'-AGAGTTTGATCCTGGCTCAG-3', 1492R: 5'-GGTTACCTTGTTACGACTT-3".

PCR reaction system (25ul): Template < lpg, Primer1(10uM) 1ul , Primer2(10uM) yl 50271 7 2xPfu PCR MasterMix 12.5ul, the distilled water is sterilized and supplemented to 25 ul;

PCR reaction conditions: Pre-denaturing at 93°C for 5min, denaturing at 94°C for 18s, annealing at 56°C for 15s, stretching at 72°C for 78s, cycling for 30 times and stretching at 72°C for 7min. After the reaction, taking SL of the reaction product, and detecting the amplification product by agarose gel electrophoresis. PCR sequencing is completed by Institute of Microbiology,

Chinese Academy of Sciences.

Sequencing the 16S rDNA sequence (as shown in SEQ.ID.NO.1), comparing the BLAST homology of NCBI, and combining morphological observation and physiological and biochemical test results, it is finally identified Lysinibacillus fusiformis.

The strain is deposited in China General Microbiological Culture Collection Center (CGMCC) of Microbiology Chinese Academy of Sciences on November Sth, 2014 (address: NO.1 West

Beichen Road, Chaoyang District, Beijing 100101,China), with the preservation number of

CGMCC No0.9799.

Embodiment 2

This embodiment illustrates the cultivation of Lysinibacillus fusiformis and the preparation of microbial agents of the present invention.

A microbial agent of Lysinibacillus fusiformis, which is prepared by the following steps: fully mixing the active ingredients obtained by culturing Lysinibacillus fusiformis in a mass ratio of 2: 10- 2: 20 with a carrier after the amplification culture, and storing at normal temperature; the carrier is a solid carrier or a liquid carrier, where the solid carrier is mineral material, plant material, polymer compound or organic material; the mineral material is one or more of clay, kaolin, montmorillonite, zeolite and fly ash; the plant material is one or more of corn flour, starch, soybean flour and peanut flour; the polymer compound is one or two of polyvinyl alcohol and polyglycol; the organic material is one or two of peat, humic acid and organic fertilizer; the active component is one or more of living cells obtained by enlarged culture, fermentation liquid containing cells and filtrate of cell culture.

The microbial agent is prepared into that following dosage form: liquid agent, emulsion, suspending agent, granule, wettable powder or water dispersible granule.

Specifically, inoculating the Lysinibacillus fusiformis with the preservation number of

CGMCC NO.9799 into a broth medium (containing 5g/L of beef paste, 10g/L of peptone and 5g/L of NaCl), and culturing at 30°C and 200 r/min under shaking until the bacterial density ODypp 5027 17 value is 0.6-0.8 to obtain a seed bacterial liquid.

Adding the seed bacterial liquid into a 100L fermentation tank (the fermentation medium contains 10-30 mL of yellow serofluid, 10-50 g/L of groundnut meal, 40-80 g/L of saccharose, 2- 4 g/L of sodium chloride, 2-4 g/L of calcium carbonate and 3-6 g/L of ferrous sulfate) for culture at 30°C, pH 7.2 and 0.5vvm of the ventilation, sampling in the culture process and observing by a microscope direct counting method until the viable count of bacillus fusiforme in each gram of culture liquid is 10° cfu.

After the fermentation, adding the fermentation broth (active ingredients) into peat (carrier) according to the ratio of 2: 10- 2: 20, and storing at normal temperature to obtain the microbial agents of this embodiment.

Embodiment 3

The embodiment illustrates the effect of Lysinibacillus fusiformis agent of the invention on improving the nutrient content of the reclamation soil of the mining area.

Planting brassica campestris and spring wheat in pot for 60 days and 100 days respectively.

The experimental soil used for pot planting is from the reclaimed land in the coal mining subsidence area of Lu'an Group, located in the west foot of Taihang Mountain in the southeast of

Shanxi Province.

Brassica campestris soil contains organic matter 5.97g/kg, total nitrogen 0.50g/kg, total phosphorus 0.05g/kg, total potassium 20.00g/kg, alkali-hydrolyzable nitrogen 42.82mg/kg, available phosphorus 3.39mg/kg, available potassium 98.60mg/kg, pH 8.29.

Spring wheat soil contains organic matter 6.40g/kg, total nitrogen 0.40g/kg, total phosphorus 0.4g/kg, total potassium 19.80g/kg, alkali-hydrolyzable nitrogen 48.76mg/kg, available phosphorus 2.09mg/kg, available potassium 98. 5mg/kg and pH 8.37.

In this experiment, using the microbial inoculum in Embodiment 2, and selecting the commercially available ecological restoration microbial inoculum 1 (comparative example 1,

GeRuiDiBao bioremediation agent), ecological restoration microbial inoculum 2 (comparative example 2, Pathfinder Pioneer-Origen microbial inoculum) and the inactivated microbial inoculum in Embodiment 2 (comparative example 3) as controls, and adding them to brassica campestris soil and spring wheat soil respectively to compare their effects. The dosage of microbial inoculum is 10g microbial inoculum per kilogram soil, and the application method is that the microbial inoculum is mixed with the soil evenly, then put in a pot, and then plant crops. During planting, 5027 17 the soil moisture maintains 70% of the soil field capacity. The impacts on soil nutrient content after planting are shown in Table 4

Table 4 The effect of planting on soil nutrient content

Pot of brassica campestris Pot of wheat experiment

Treatments | Organi | Alkali- Available | Availabl | Organi | Alkali- Available | Availabl c hydrolyzab | phosphor | € c hydrolyzab | phosphor | € matter | le nitrogen | us potassiu | matter | le nitrogen | us potassiu (g/kg) | (mg/kg) (mg/kg) | m (g/kg) | (mg/kg) (mg/kg) | m (mg/kg) (mg/kg)

Embodime 100.37b 42.44c 155.33b | 10.24d 95.49b 6.72c 111.55c nt 9.41b

Comparati | 8.44a 89.29a 36.11ab 147.33a | 7.68b 74.01a 5.24ab 98.96b ve example 1

Comparati | 9.34b 89.42a 40.24bc 148.67a | 8.32c 76.07a 3.86a 98.96b ve example 2

Comparati | 8.2la 88.04a 32.98a 141.67a | 6.72a 70.92a 4 25ab 85.71a ve example 3

Note: Different lowercase letters in the same column show significant differences in p<0.05.

As can be seen from the results of table 4, Lysinibacillus fusiformis microbial inoculum provided by embodiment 2 has a significant effect in improving the contents of organic matter, alkali-hydrolyzable nitrogen, available potassium and available phosphorus in the reclamation soil of the mining area.

Embodiment 4

This embodiment illustrates the effect of the Lysinibacillus fusiformis agent of the present invention on improving crop biomass.

Planting brassica campestris and spring wheat in pot for 60 days and 100 days respectively.

The experimental soil used for pot planting is from the reclaimed land in the coal mining subsidence area of Lu'an Group, located in the west foot of Taithang Mountain in the southeast of

Shanxi Province.

Brassica campestris soil contains organic matter 5.97g/kg, total nitrogen 0.50g/kg, total phosphorus 0.05g/kg, total potassium 20.00g/kg, alkali-hydrolyzable nitrogen 42.82mg/kg,

available phosphorus 3.39mg/kg, available potassium 98.60mg/kg, pH 8.29. LU502717

Spring wheat soil contains organic matter 6.40g/kg, total nitrogen 0.40g/kg, total phosphorus 0.4g/kg, total potassium 19.80g/kg, alkali-hydrolyzable nitrogen 48.76mg/kg, available phosphorus 2.09mg/kg, available potassium 98.5mg/kg and pH 8.37.

In this test, the microbial inoculum in this embodiment 1s the microbial inoculum in

Embodiment 2, and the commercially available ecological restoration microbial inoculum is selected as a comparison; Comparative example 1, Comparative example 2 and Comparative example 3 are the same as Embodiment 3.

The dosage of microbial inoculum is 10g microbial inoculum per kilogram soil, and the application method is that the microbial inoculum is mixed with the soil evenly, then put in a pot, and then plant crops. During planting, the soil moisture maintains 70% of the soil field capacity. (The results of crop biomass are shown in Table 5.)

Table 5 The increase of crop biomass

D ight of brassi

Dry weight of wheat (g/pot) Wels © rassica campestris (g/pot)

Treatments

Comparative 6.78b 15.62b 17.57b 6.83c example 1 6.63b 15.05b 14.69a 2.76a example 2

Comparative 5.88a 11.84a 14.45a 5.74b example 3

Note: Different lowercase letters in the same column show significant differences in p<0.05.

As can be seen from the results of Table 5, the embodiments have a significant effect in increasing crop biomass of reclaimed soil in mining areas.

Embodiment 5

This embodiment illustrates the role of Lysinibacillus fusiformis in improving the diversity of microbial community structure in reclaimed soil of mining area.

Planting brassica campestris and spring wheat in pot for 60 days and 100 days respectively.

The experimental soil used for pot planting is from the reclaimed land in the coal mining subsidence area of Lu'an Group, located in the west foot of Taithang Mountain in the southeast of

Shanxi Province.

Brassica campestris soil contains organic matter 5.97g/kg, total nitrogen 0.50g/kg, total 502717 phosphorus 0.05g/kg, total potassium 20.00g/kg, alkali-hydrolyzable nitrogen 42.82mg/kg, available phosphorus 3.39mg/kg, available potassium 98.60mg/kg, pH 8.29.

Spring wheat soil contains organic matter 6.40g/kg, total nitrogen 0.40g/kg, total phosphorus 0.4g/kg, total potassium 19.80g/kg, alkali-hydrolyzable nitrogen 48.76mg/kg, available phosphorus 2.09mg/kg, available potassium 98.5mg/kg and pH 8.37.

In this test, the embodiment 1s Lysinibacillus fusiformis microbial inoculum of Embodiment 2, and the commercially available ecological remediation microbial inoculum is selected as a comparison. Comparative example 1, Comparative example 2 and Comparative example 3 are the same as Embodiment 3.

The dosage of microbial inoculum is 10g microbial inoculum per kilogram soil, and the application method is that the microbial inoculum is mixed with the soil evenly, then put in a pot, and then plant crops. During planting, the soil moisture maintains 70% of the soil field capacity.

Using phospholipid fatty acid method (PLFA) to characterize soil microbial biomass, the changes of soil microbial community structure after potted brassica campestris planting are analyzed (The results are shown in Table 6).

Table 6 Changes of soil microbial community structure after potted brassica campestris planting . | Gram-positive | Gram-negative . i

Treatments | Bacteria ; . Fungus | Actinomycetes | Protists bacterium bacterium

Embodiment 2.663b 0.647b 0.823b 1.842b 0.835c 0.045b example 1 2.017a 0.487a 0.394a 1.658a 0.503a 0.027a example 2 1.923a 0.491a 0.413a 1.45a 0.677b 0.016a example 3

Note: Different lowercase letters in the same column show significant differences in p<0.05.

The results in Table 6 show that the use of Lysinibacillus fusiformis can effectively and significantly change the community structure of soil microorganisms, improve the diversity of soil microorganisms, significantly increase the number of microorganisms, and accelerate the restoration of soil microbial communities.

The following applications are proposed based on the above actions of Lysinibacillus fusiformis and its microbial agents: LU502717

An application of Lysinibacillus fusiformis in improving the reclamation soil of the mining area.

An application of Lysinibacillus fusiformis in preparing the microbial agent for improving the reclamation soil of the mining area.

An application of Lysinibacillus fusiformis in preparing the biological fertilizer for promoting the growth of reclamation soil plants of the mining area.

The preferred embodiments of the present invention have been described in detail above, but the present invention 1s not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical scheme of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Sequence listing LU502717 <110> The College of Resources and Environment, Shanxi Agricultural

University;

Institute of Eco-environment and Industrial Technology, Shanxi Agricultural University <120> LYSINIBACILLUS FUSIFORMIS AND ITS APPLICATION IN

ECOLOGICAL RECONSTRUCTION OF MINING AREA RECLAMATION

<160> 1 <210> 1 <211> 1441 <212> DNA <213> Lysinibacillus fusiformis < 400> 1 agcgaacaga aaaggagctt gctcctttga cgttagegge ggacgggtga gtaacacgtg 60 ggcaacctac cctatagttt gggataactc cgggaaaccg gggctaatac cgaataatct 120 cttttgcttc atggtgaaag actgaaagac ggtttcgget gtcgtcatag gatgggcccg 180 cggcgcatta gctagttget gaggtaacgg ctcaccaagg cgacgatgcg tagccgacct 240 gagagggtga tcggecacac tgggactgag acacggecca gactectacg ggaggeagea 300 gtagggaatc ttccacaagg gcgaaagcct gatggagcaa cgecgegtga gtgaagaagg 360 ttttcggatc gtaaaactct gttgtaaggg aagaacaagt acagtagtaa ctggctgtac 420 cttgacggta ccttattaga aagccacggc taactacgtg ccagcagecg cggtaatacg 480 taggtggcaa gegttgtccg gaattattgg gegtaaageg cgegeaggeg gtectttaag 540 tctgatgtga aagcccatgg ctcaaccgtg gagggtcatt ggaaactggg ggacttgagt 600 gcagaagagg aaagtggaat tccaagtgta gcggtgaaat gegtagagat ttggaggaac 660 accagtggcg aaggcgactt tctggtetgt aactgacget gaggegegaa agegtgggga 720 gcaaacagga ttagataccc tggatgtcca cgccgtaaac gatgagtgct aagtgttagg 780 gggtttccge cecttagtge tgcagetaac gcattaagca ctecgectgg ggagtacggt 840 cgcaagactg aaactcaaag gaattgacgg gggcccgeac aageggtgga geatgtggtt 900 taattcgaag caacgcgaag aaccttacca ggtattgaca teccettgac cactgtagag 960 atatagtttc cccttcgggg gcaacggtga gagctgetec atggttgteg ttagetegtg 1020 tcgtgagatg ttgggttaag teccgcaacg agegeaaccc ttgatcttag ttgccatcat 1080 ttagttgggc actctaaggt gactgecggt gacaaaccgg aggaaggtgg ggatgacgte 1140 LU502717 aaatcatcat gecccttatg acctgggcta cacacctect acaatggacg atacaaaagg 1200 ttgccaacte gcgagaggga getaatccga taaagtggtt ctcagttegg attgtagget 1260 gcaactcgcec tacatgaage cggaatcgge agtaatcgcg gatcagcatg cegcggtgaa 1320 tacgtteccg ggecttgtac acaccgeccg tcacaccacg agagtttgta acacccgaag 1380 tcggtgaggt aaccttttgg agccagccgc cgaaggtggg atagatgatt gggatgaagt 1440 c 1441

Claims (10)

CLAIMS LU502717

1. À kind of Lysinibacillus fusiformis, characterized in that Lysinibacillus fusiformis with the preservation number of CGMCC No.9799.

2. The Lysinibacillus fusiformis according to claim 1, characterized in that Lysinibacillus fusiformis can improve the content of organic matter, alkali-hydrolyzable nitrogen, available phosphorus and available potassium in reclaimed soil of mining area.

3. The Lysinibacillus fusiformis according to claim 1, characterized in that Lysinibacillus fusiformis can improve the biomass of crops growing in reclaimed soil of mining area.

4. The Lysinibacillus fusiformis according to claim 1, characterized in that Lysinibacillus fusiformis can improve the diversity of microbial community structure in reclaimed soil of mining area.

5. The Lysinibacillus fusiformis according to claim 1, characterized in that the 16S rDNA sequence of Lysinibacillus fusiformis is shown in SEQ.ID.NO 1.

6. A microbial agent of Lysinibacillus fusiformis, characterized by being prepared by the following steps: fully mixing the active ingredients obtained by culturing Lysinibacillus fusiformis in a mass ratio of 2: 10- 2: 20 with a carrier after the amplification culture, and storing at normal temperature; the carrier is a solid carrier or a liquid carrier, where the solid carrier is mineral material, plant material, polymer compound or organic material; the mineral material is one or more of clay, kaolin, montmorillonite, zeolite and fly ash; the plant material is one or more of corn flour, starch, soybean flour and peanut flour; the polymer compound is one or two of polyvinyl alcohol and polyglycol; the organic material is one or more of peat, humic acid and organic fertilizer; the active component is one or more of living cells obtained by enlarged culture, fermentation liquid containing cells and filtrate of cell culture; the microbial agent is prepared into that following dosage form: liquid agent, emulsion, suspending agent, granule, wettable powder or water dispersible granule.

7. An application of Lysinibacillus fusiformis according to claim 1 in improving the reclamation soil of the mining area.

8. The application according to claim 7, characterized in that Lysinibacillus fusiformis or the microbial agent prepared by Lysinibacillus fusiformis can improve the content of organic matter, alkali-hydrolyzable nitrogen, effective phosphorus and quick-available potassium in the mine area reclamation soil and improve the structural diversity of the mine area reclamation soil microbial 5027 17 community.

9. An application of Lysinibacillus fusiformis according to claim 1 in preparing the microbial agent for improving the reclamation soil of the mining area.

10. An application of Lysinibacillus fusiformis according to claim 1 in preparing the biological fertilizer for promoting the growth of reclamation soil plants of the mining area.