CN111172052A - Bacillus P75 for improving soil fertility of sandstone and application thereof - Google Patents
Bacillus P75 for improving soil fertility of sandstone and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of agricultural technology, environmental protection and ecological improvement application, and discloses bacillus P75 for improving the fertility of arsenicum sablimatum soil and application thereof. Bacillus halolerans P75 is used as a raw material and is prepared into bacterial suspension after being activated by bacteria. When the fertilizer is inoculated in the arsenopyrite, soil fertility indexes such as organic matters, quick-acting phosphorus, quick-acting potassium and the like of arsenopyrite soil can be obviously improved, and the growth of ryegrass and alfalfa is promoted.
Description
Technical Field
The invention belongs to the field of agricultural technology, environmental protection and ecological improvement application, and relates to bacillus P75 for improving the fertility of arsenicum soil and application thereof.
Background
The arsenic sandstone area in China is about 1.67 km2The method is mainly distributed in Jinshanmeng soil-covered areas with quasi-Geer flags in Erdos city, inner Mongolia as the center. Due to the fact that rock components of the arsenopyrite contain a large amount of hydrophilic montmorillonite, the arsenopyrite is very easy to expand and disperse when meeting water, water and soil loss is serious, and ecological environment improvement is urgently needed. The slope surface of the arsenicum sand region is extremely thin in soil layer and deficient in water resource, and vegetation is difficult to grow, so that the traditional biological measures interception and engineering measures are difficult to implement comprehensively.
Plant Growth Promoting Bacteria (PGPB) are bacteria which are beneficial to plant growth under certain conditions and freely live in soil, rhizosphere, root surface and phyllosphere. These bacteria are capable of fixing nitrogen, solubilizing phosphorus, releasing potassium, and producing plant hormones such as auxins, gibberellins, cytokinins, and ethylene. In addition, they can improve the stress resistance of plants, and resist drought, high salt and heavy metal poisoning.
Therefore, plant growth promoting bacteria are used for secreting plant growth hormone IAA and siderophores to promote plant growth, biofilm and extracellular polymers are produced to reduce water and nutrient loss in soil, soil fertility is improved, and the plant growth promoting bacteria and plants are combined to play a certain role in preventing and treating water and soil loss in the sandstone area.
Disclosure of Invention
The invention aims to provide bacillus P75 capable of improving soil fertility of sandstone and application thereof, aiming at the problems of bioremediation and engineering restoration of the sandstone area at present.
The purpose of the invention can be realized by the following technical scheme:
the Bacillus P75 for improving the soil fertility of arsenic sandstone is classified and named as Bacillus halotolerans P75, is preserved in China center for type culture collection, has the preservation date of 2018, 10 months and 17 days, and has the strain preservation number of CCTCC NO: m2018690. The colony is round, semi-transparent, moist and sticky. The thallus is short and rod-shaped, has spores, can secrete polysaccharide and generate a biological membrane. Aerobic, the optimum growth temperature is 25-30 ℃, and the optimum pH is 6.5-7.5.
The bacillus P75 disclosed by the invention is applied to improving the fertility of arsenicum sand soil and promoting the growth of ryegrass and/or alfalfa.
The bacillus P75 is applied to increasing organic matters in arsenopyrite soil and increasing the activities of quick-acting phosphorus, quick-acting potassium and sucrase in the arsenopyrite soil.
A biological agent prepared by the Bacillus (Bacillus halotolerans) P75.
As a preferred embodiment of the invention, the cell number of bacillus (Bacillus halotolerans) P75 in the biological agent reaches more than 5 hundred million CFU/ml.
The biological agent disclosed by the invention is applied to improving the fertility of arsenicum sablimatum soil and promoting the growth of ryegrass and/or alfalfa.
The biological agent is applied to increasing organic matters in the arsenicum sablimatum soil and increasing the activity of quick-acting phosphorus, quick-acting potassium and sucrase in the arsenicum sablimatum soil.
Advantageous effects
The invention screens a Bacillus halolelansP 75 strain capable of improving the fertility of arsenicum soil, which is preserved in China center for type culture collection with the preservation date of 2018, 10 months and 17 days and the preservation number of the strain is CCTCCNO: m2018690, and the bacillus can promote the growth of ryegrass and alfalfa on arsenicum sand, and increase plant biomass.
The Bacillus halotolerans P75 strain can improve soil fertility of arsenicum sablimatum, promote growth of ryegrass and alfalfa and increase plant biomass. Compared with the prior art, the method has the following advantages:
(1) the preservation number of the invention is CCTCC NO: m2018690 Bacillus halotolerans P75 can produce indoleacetic acid, and the highest yield of IAA can reach 23.9mg/L
(2) Bacillus halolerans P75 is capable of producing siderophores.
(3) The organic matter content of the arsenopyrite soil inoculated with Bacillus halolerans P75 is obviously increased by 24.3% (P < 0.05).
(4) The quick-acting phosphorus content of the arsenic sandstone soil inoculated with Bacillus halolelanes P75 is obviously increased by 11.9 percent (P < 0.05).
(5) The quick-acting potassium content of the arsenic sandstone soil inoculated with Bacillus halolelans P75 is obviously increased by 21.0 percent (P < 0.05).
(6) The sucrase content of the arsenic sand soil inoculated with Bacillus halolerans P75 is obviously increased by 58.9% (P < 0.05).
(7) Aerial biomass of alfalfa and ryegrass treated with inoculated Bacillus halolelans P75 increased significantly by 18.9% and 22.4% (P < 0.05).
(8) The root biomass of alfalfa and ryegrass treated with inoculated Bacillus halolelans P75 increased significantly by 55.1% and 58.7% (P < 0.05).
Biological sample preservation information
The plant growth promoting bacteria P75 are classified and named as Bacillus halotolerans, CCTCC M2018690 is preserved in China center for type culture collection, the preservation address is Wuhan university in Wuhan, China, the preservation date is 2018, 10 and 17 days, and the strain preservation number is CCTCC NO: m2018690.
Detailed Description
The following claims are hereby incorporated into the detailed description of the invention, with the understanding that the invention is not to be limited in any way, as any number of modifications may be made by one within the scope of the claims and the invention is still within the scope of the claims.
The following examples are, unless otherwise indicated, all of the routine experimentation and procedures known in the art.
Example 1
The plant growth promoting bacteria P75 strain (CCTCC NO: M2018690) for improving the fertility of arsenopyrite soil is obtained by separating and purifying alfalfa roots growing in inner Mongolia arsenopyrite areas, and the separation and identification method comprises the following steps:
the roots of alfalfa were rinsed clean with hot deionized water, sterilized with 75% alcohol followed by 2.5% NaClO, and rinsed several times with sterile water. Grinding 0.1g of the root with the surface sterilized in a sterile mortar to homogenate, adding 1mL of sterile water, mixing uniformly, and putting into a water bath kettle with the constant temperature of 85 ℃ for 15 min. And (3) coating 0.1mL of suspension on LB solid culture containing 5mg/L Cd, and coating 0.1mL of sterile water for the last time of immersion cleaning on an LB solid culture medium to detect whether the surface of the sample is disinfected completely. After culturing at 28 ℃ for 72h, if bacteria do not grow on the plates coated with sterile water, single colonies with good growth vigor are selected from LB resistant plates, and the single colonies are subjected to streaking separation and purification for multiple times and then are stored at 4 ℃.
Extracting total bacterial DNA by a conventional method, amplifying the bacterial 16S rDNA by PCR, comparing and analyzing an amplification product with a known 16S rDNA sequence in GenBank after sequencing, reaching 99.93 percent of similarity with the 16SrDNA sequence of Bacillus halolerans ATCC 25096(T), and identifying the strain by combining other bacterial physiological and biochemical experiments.
Example 2 activation of Strain P75 and preparation of a bacterial suspension
P75(CCTCC NO: M2018690) slant culture was inoculated in LB solid medium (tryptone 10.0g, yeast extract 5.0g, NaCl10.0g, distilled water 1000ml, agar 20g, pH 7.0) and cultured at 30 ℃ for 1 day. Then, a single colony of full and viscous P75 is selected and inoculated in an LB liquid culture medium (tryptone 10.0g, yeast extract 5.0g, Nacl10.0g, distilled water 1000ml, pH 7.0), shaking culture is carried out for 18-20h at 30 ℃ and 160rmp, and the cell number reaches more than 10 hundred million CFU/ml.
Example 3 ability of Strain P75 to produce IAA
According to the method of Gordon and Weber (1951), LB liquid medium was dispensed from tubes, 4mL of each tube was sterilized at 121 ℃ and 1mL of filter-sterilized 2.5mg/mL tryptophan solution was added to the tubes, so that the final concentration of tryptophan in the medium was 0.5 mg/mL. Inoculating the strain into the culture medium, and performing shake culture at 30 ℃ for 3 d. The fermentation broth was centrifuged at 12000r/min for 5min, 1mL of the supernatant was taken, 50. mu.L of 10mM orthophosphoric acid was added, 2mL of Sackowski's color developer was added, mixed well, developed at 25 ℃ in the dark for 30min, and the absorbance was measured at 530 nm. Sterile medium was treated as above for the same treatment as control zero. And (3) making a standard curve by using IAA standard solutions with the concentrations of 0, 5, 10, 20, 40 and 60mg/L according to the same method, and calculating the concentration of the IAA in the fermentation liquor. The result shows that P75 can produce indoleacetic acid, and the maximum yield of IAA can reach 23.9 mg/L.
Example 4 ability of Strain P75 to produce siderophores
Siderophores were determined according to wangping et al. The strain P75(CCTCC NO: M2018690) was inoculated in LB liquid medium and cultured with shaking at 30 ℃ and 150rpm for 48 hours. Centrifuging the fermentation liquid at 12000rpm for 5min, collecting supernatant, mixing with equal volume CAS detection liquid, developing for 1 hr, measuring light absorption value (A) at 630nm wavelength, and zeroing with deionized water as control. And (3) fully and uniformly mixing the same volume of the sterile LB culture medium and the CAS detection solution, and determining the light absorption value by the same method to obtain a reference value (Ar). A/Ar values <1, can be considered high yielding siderophores. The results show that the strain P75 can produce siderophores with high yield.
Example 5 Effect of the Strain P75 on increasing organic matter content in arsenicum Sablimatum soil
The P75(CCTCC NO: M2018690) bacterial suspension of example 2 was inoculated into arsenopyrite soil at an inoculum size of 2%, and allowed to stand for one month. And measuring the content of organic matters in the soil by adopting a potassium dichromate volumetric method-dilution heat method. The organic matter is oxidized by utilizing the heat generated when concentrated sulfuric acid and potassium dichromate are rapidly mixed to replace oil bath heating in an external heating method, and because the generated heat temperature is low and the oxidation degree of the organic matter is only 77 percent, the organic matter is multiplied by a correction coefficient to obtain the content of organic carbon in the soil, and then the content of the organic carbon in the soil is converted into the content of the organic matter in the soil according to the content of the organic carbon in the soil. As can be seen from Table 1, the content of organic matters in the arsenopyrite soil treated by the inoculation is significantly increased by 24.3% compared with the control.
Table 1 Strain Bacillus halotolerans P75 increasing organic matter content of arsenicum sand soil
Example 6 Effect of the Strain P75 on improving sucrase activity of arsenopyrite soil
The P75(CCTCC NO: M2018690) bacterial suspension of example 2 was inoculated into arsenopyrite soil at an inoculum size of 2%, and allowed to stand for one month. The colorimetric determination is carried out by adopting a 3, 5-dinitrosalicylic acid (DNS) colorimetric method and taking cane sugar as a substrate according to the amount of a colored compound 3-amino-5-nitro salicylic acid generated by the reaction of enzymatic product glucose and 3, 5-dinitrosalicylic acid. Each treatment was repeated 3 times while performing standard curve, no matrix control and no soil control tests. The sucrase activity is expressed in mg of 1g of soil glucose after 24 h. As can be seen from Table 2, the sucrase activity in the inoculated sandstone soil is significantly increased by 58.9% compared with the control.
TABLE 2 Effect of the Strain Bacillus halolerans P75 on sucrase activity in sandstone soils
Example 7 Effect of the Strain P75 on increasing the content of available phosphorus in arsenicum sablimatum soil
The P75(CCTCC NO: M2018690) bacterial suspension of example 2 was inoculated into arsenopyrite soil at an inoculum size of 2%, and allowed to stand for one month. With 0.5mol/LNaHCO3Method for measuring content of available phosphorus in soil, NaHCO of pH8.53Ca in solution2+、Al3+、Fe3+The activity of the plasma is very low, which is beneficial to the extraction of phosphorus, and OH in the solution-、HCO3 -、CO3 2-Plasma homoenergetic replacement of H2PO4 -And then, the phosphorus in the solution to be detected is developed by a molybdenum-antimony anti-reagent, and colorimetric determination is carried out to determine the content of the quick-acting phosphorus in the soil. The results in Table 3 show that the content of available phosphorus in the inoculated sandstone soil is remarkably increased by 11.9 percent compared with the control.
TABLE 3 influence of Bacillus halolerans P75 strain on the content of available phosphorus in sandstone soil
Example 8 Effect of the Strain P75 on increasing the content of quick-acting potassium in arsenicum sablimatum soil
Examples of the invention2P 75(CCTCC NO: M2018690) bacterial suspension is inoculated into arsenic sandstone soil according to the inoculation amount of 2 percent and is kept stand for one month. By NH4And (4) OAc leaching, and measuring the content of the quick-acting potassium in the soil by a flame photometry. The results in Table 4 show that the content of the quick-acting potassium in the arsenopyrite soil treated by the inoculation is obviously increased by 21.0 percent compared with the control.
TABLE 4 influence of Bacillus halolerans P75 strain on the content of potassium in quick-acting soil from sandstone
Example 9 Effect of Strain P75 in sandstone soil on promoting the growth of alfalfa and ryegrass
The soil is obtained from the soil in inner Mongolia arsenicum sablimatum area. The experiment adopts a pot experiment, 200g of soil is filled in each hole tray, and 20g of deionized water is added into the hole trays, so that the whole soil environment is kept at a certain drought degree. Respectively carrying out surface disinfection on ryegrass and alfalfa seeds to be tested by using 5% sodium hypochlorite solution for 20min, placing the seeds on sterile gauze, placing the sterile gauze in a sterile incubator at 30 ℃, keeping the humidity at 60% -80% for accelerating germination, and picking the seeds with the consistent sizes and exposed white and placing the seeds on a water culture bowl for culture. When the seed buds grow to about 1cm, the ryegrass and alfalfa seedlings with uniform growth vigor are selected and put into the P75(CCTCC NO: M2018690) bacterial suspension of the example 2, and the root dipping treatment is carried out for about 0.5h, and the inoculation-free treatment is used as a control. Then transplanting the seedlings into a plug tray, repeating the treatment three times each time, inoculating 20ml of bacterial suspension every half month for strengthening treatment, and carrying out scientific management for one month. The dry weight of the aerial parts and the dry weight of the roots of ryegrass and alfalfa were measured after one month to evaluate the growth promoting effect of the strains on them.
As can be seen from Table 5, the biomass of the aerial parts of alfalfa and ryegrass treated with inoculum P75 was significantly increased by 18.9% and 22.4% (P <0.05), and the biomass of roots was significantly increased by 55.1% and 58.7% (P <0.05) compared to the control.
TABLE 5 growth promoting action of the Strain Bacillus halolerans P75 on alfalfa and ryegrass
Claims (7)
1. A Bacillus (Bacillus halotolerans) P75 capable of improving the fertility of arsenicum sand soil is preserved in the China center for type culture collection with the preservation date of 2018, 10 months and 17 days, and the preservation number of the strain is CCTCC NO: m2018690.
2. Use of the Bacillus (Bacillus halotolerans) P75 in improving the fertility of sandstone soil, promoting the growth of ryegrass and/or alfalfa.
3. The use of Bacillus (Bacillus halotolerans) P75 in increasing organic matter in sandstone soil, increasing the activity of available phosphorus, available potassium and/or sucrase in sandstone soil.
4. A biological agent prepared from the Bacillus subtilis P75 according to claim 1.
5. The biological agent according to claim 4, wherein the number of Bacillus halotolerans P75 cells is 5 hundred million CFU/ml or more.
6. The use of the biological agent of claim 4 or 5 in improving fertility of arsenopyrite soil and promoting growth of ryegrass and/or alfalfa.
7. The application of the biological agent of claim 4 or 5 in increasing organic matters in arsenicum sablimatum soil and increasing activity of fast-acting phosphorus, fast-acting potassium and sucrase in arsenicum sablimatum soil.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110819565A (en) * | 2019-11-20 | 2020-02-21 | 青岛农业大学 | Salt-tolerant bacillus BW9 with plant pathogenic bacteria inhibiting effect and application thereof |
| CN111919533A (en) * | 2020-07-09 | 2020-11-13 | 亿利资源集团有限公司 | Comprehensive improvement method for desertification land |
| CN113913331A (en) * | 2021-10-12 | 2022-01-11 | 南京农业大学 | Saline-alkali-tolerant bacillus pumilus for producing polyglutamic acid and application thereof |
| CN114982413A (en) * | 2022-06-16 | 2022-09-02 | 南京农业大学 | Solid-state fermentation preparation and application of biochar-based bacillus inoculant |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110819565A (en) * | 2019-11-20 | 2020-02-21 | 青岛农业大学 | Salt-tolerant bacillus BW9 with plant pathogenic bacteria inhibiting effect and application thereof |
| CN110819565B (en) * | 2019-11-20 | 2021-04-13 | 青岛农业大学 | Salt-tolerant bacillus BW9 with plant pathogenic bacteria inhibiting effect and application thereof |
| CN111919533A (en) * | 2020-07-09 | 2020-11-13 | 亿利资源集团有限公司 | Comprehensive improvement method for desertification land |
| CN111919533B (en) * | 2020-07-09 | 2022-12-02 | 亿利资源集团有限公司 | Comprehensive improvement method for desertification land |
| CN113913331A (en) * | 2021-10-12 | 2022-01-11 | 南京农业大学 | Saline-alkali-tolerant bacillus pumilus for producing polyglutamic acid and application thereof |
| CN113913331B (en) * | 2021-10-12 | 2023-02-17 | 南京农业大学 | Saline-alkali-tolerant bacillus pumilus for producing polyglutamic acid and application thereof |
| CN114982413A (en) * | 2022-06-16 | 2022-09-02 | 南京农业大学 | Solid-state fermentation preparation and application of biochar-based bacillus inoculant |
| CN114982413B (en) * | 2022-06-16 | 2023-12-08 | 南京农业大学 | Solid state fermentation preparation and application of biochar-based bacillus inoculant |
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