Composite phosphate-solubilizing bacteria agent based on biochar carrier and preparation method and application thereof

Abstract

The present invention relates to a composite phosphate-solubilizing bacteria agent based on a biochar carrier, and a preparation method and application thereof. The composite phosphate-solubilizing bacteria agent of the present invention can effectively improve the phosphorus absorption and utilization rate of plants during their growth, and more significantly improves the phosphorus absorption capacity and growth performance of plants, especially rapeseed, by fixing inorganic phosphorus-dissolving bacteria on different types of biochar carriers. The composite phosphate-solubilizing bacteria agent has broad agricultural application prospects, and is particularly useful for improving the effectiveness of phosphorus in poor soils, reducing dependence on chemical phosphorus fertilizers, and improving the effectiveness of soil phosphorus and phosphorus absorption of crops. The composite phosphate-solubilizing bacteria agent is a method for achieving more environmentally friendly and sustainable agricultural production.

Description

Technical Field

[0001] The invention belongs to the technical field of agriculture, and specifically relates to a composite phosphate-solubilizing bacteria agent based on a biochar carrier, and a preparation method and application thereof. Background Art

[0002] Phosphorus (P) is a key macronutrient required for plant growth, yet it is a non-renewable resource. To meet the growing agricultural demand, the mining of phosphate rock and the use of phosphate fertilizers have increased significantly. However, phosphate fertilizers in the soil are easily passivated due to their strong fixation with soil particles, making them difficult for plants to absorb. This unavailability of phosphorus not only limits crop growth, but also leads to a large amount of waste of phosphate fertilizers.

[0003] Traditionally, the phosphorus content in the soil is supplemented by increasing the application of phosphorus fertilizers, but this method is not only costly, but also causes eutrophication of soil and water bodies, bringing environmental pollution problems. Therefore, it is urgent to develop new and environmentally friendly phosphorus utilization strategies.

[0004] Microorganisms play an important role in the biogeochemical cycle of soil phosphorus, among which inorganic phosphorus solubilizing bacteria are an important driving force for the release of phosphorus in soil. These bacteria can effectively solubilize inorganic phosphorus in the soil by releasing organic acids or protons, thereby improving the phosphorus absorption rate of plants. In addition, inorganic phosphorus solubilizing bacteria are also widely accepted as plant growth-promoting rhizobacteria (PGPR) and can be used as bioinoculants to improve phosphorus absorption and enhance plant productivity.

[0005] However, the direct application of inorganic phosphorus-dissolving bacteria in the soil faces a major problem, namely, the low survival rate of the bacterial community, which makes it difficult to maintain the inoculation effect of inorganic phosphorus-dissolving bacteria. In order to solve this problem, researchers have tried to use biochar as a carrier of the bacterial community to improve the survival rate and functionality of inorganic phosphorus-dissolving bacteria. Although biochar has a significant effect on increasing the abundance and diversity of inoculated microorganisms, biochar prepared from different raw materials has different effects on maintaining inorganic phosphorus-dissolving bacteria due to differences in their physical and chemical properties. Therefore, how to find a suitable biochar carrier is crucial to the inoculation effect of inorganic phosphorus-dissolving bacteria.

[0006] At present, there is no universal biochar carrier that can show good performance in all soil environments. Therefore, studying the effects of different types of biochar on the survival and function of inorganic phosphorus-solubilizing bacteria and optimizing the application strategy of composite phosphorus-solubilizing agents is a research hotspot in the current field. Summary of the invention

[0007] In view of the problems existing in the prior art, the present invention provides a composite phosphate-solubilizing bacteria agent based on a biochar carrier and a preparation method and application thereof, which can be effectively used to improve the effectiveness of soil phosphorus and the phosphorus absorption of crops.

[0008] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is:

[0009] A composite phosphate-solubilizing bacteria agent based on a biochar carrier, the composite phosphate-solubilizing bacteria agent comprising a plurality of inorganic phosphorus-solubilizing bacteria strains of different bacterial genera, the bacterial genera being Agrobacterium ( Agrobacterium ), Pseudomonas ( Pseudomonas ), Ralstonia ( Ralstonia ), Burkholderia spp. Burkholderia ), Bacillus ( Bacillus ); one or more strains from each of the above-mentioned different genera are selected and mixed, and then fixed on a biochar carrier prepared from rice straw, rice husk, soybean straw, corn cob, peanut shell or wood. By fixing a variety of inorganic phosphorus-dissolving bacteria on different types of biochar carriers, the phosphorus absorption efficiency of plants can be improved.

[0010] Preferably, the selected strains are Agrobacterium LYS-n49 ( Agrobacterium sp. LYS-n49), the deposit number is CGMCC No.32845; Pseudomonas LYS-11 ( Pseudomonas sp. LYS-11), the deposit number is CGMCC No.32841; Ralstonia LYS-14 ( Ralstonia sp. LYS-14), the deposit number is CGMCC No.32842; Ralstonia LYS-16 ( Ralstoniasp. LYS-16), the deposit number is CGMCC No.32843; Pseudomonas LYS-p19 ( Pseudomonassp. LYS-p19), the deposit number is CGMCC No.32846; Burkholderia LYS-21 ( Burkholderiasp. LYS-21), the deposit number is CGMCC No.32844; Bacillus LYS-p36 ( Bacillus sp. LYS-p36), the deposit number is CGMCC No.32847.

[0011] Preferably, the rRNA gene sequence of each strain is SEQ ID NO.1 to SEQ ID NO.7.

[0012] The composite phosphate-solubilizing bacteria agent of the present invention can promote plant growth by increasing the content of available phosphorus in the soil.

[0013] The present invention selects multiple inorganic phosphorus-dissolving bacteria strains of different categories with high calcium phosphate dissolving ability, and combines them with biochar carriers to generate a composite bacterial agent. The biochar carrier can not only provide a protective habitat for these inorganic phosphorus-dissolving bacteria and prolong their survival time in the soil, but also enhance the phosphorus dissolving ability of the bacterial community through its unique pore structure, thereby improving the effectiveness of phosphorus in the soil and promoting phosphorus absorption by crops.

[0014] Preferably, the biochar carrier is a biochar carrier prepared from rice straw or soybean straw.

[0015] Different types of biochar carriers have different effects on the survival rate and functionality of inorganic phosphorus-dissolving bacteria. Studies have shown that biochar prepared from rice straw and soybean straw is the best strain carrier in the present invention, which can maximize the survival rate and functionality of inorganic phosphorus-dissolving bacteria. The application of this composite microbial agent can improve the phosphorus absorption capacity and growth performance of rapeseed, and has broad agricultural application prospects, especially for improving the effectiveness of phosphorus in poor soils, reducing dependence on chemical phosphorus fertilizers, and thus achieving a more environmentally friendly and sustainable agricultural production method.

[0016] Preferably, the strains are mixed in equal volumes.

[0017] The present invention also provides a method for preparing a composite phosphate-solubilizing bacteria agent based on a biochar carrier, comprising the following steps:

[0018] (1) Screening and identification of strains

[0019] The inorganic phosphorus-dissolving bacteria isolated from the soil sample were inoculated into the inorganic phosphorus culture medium for purification, and the purified colonies were classified and identified;

[0020] (2) Selection of strains

[0021] From the multiple phosphate-solubilizing bacteria identified in step (1), seven inorganic phosphate-solubilizing bacterial strains of multiple different genera are selected according to their calcium-phosphate solubilizing ability greater than 13 μg / mL, iron-phosphate solubilizing ability greater than 12 μg / mL, and aluminum-phosphate solubilizing ability greater than 7 μg / mL;

[0022] (3) The inorganic phosphorus-solubilizing bacterial strains of the above-mentioned different bacterial genera are mixed in proportion and fixed on a biochar carrier to form a composite phosphate-solubilizing bacterial agent based on a biochar carrier.

[0023] Preferably, the inorganic phosphorus culture medium consists of: 10 g / L glucose, 0.5 g / L ammonium sulfate, 0.2 g / L sodium chloride, 0.1 g / L magnesium sulfate, 0.002 g / L manganese sulfate monohydrate, 0.2 g / L potassium chloride, 0.002 g / L ferrous sulfate heptahydrate, 1 g / L hydroxyapatite, 15 g / L agar powder, pH 7.0-7.2, and 0.01 mM bromocresol purple.

[0024] Preferably, the biochar carrier is prepared by the following method: raw materials are pyrolyzed in a muffle furnace at 450°C, equipped with an automatic nitrogen flow, to produce biochar at 450°C; the raw materials include rice straw, rice husk, soybean straw, corn cob, peanut shell and wood.

[0025] Preferably, the concentration of inorganic phosphorus-solubilizing bacteria in the composite phosphate-solubilizing bacteria agent is 10 9 ~10 10 CFU / mL.

[0026] The present invention also provides an application of the composite phosphate-solubilizing bacteria agent based on a biochar carrier, wherein the composite phosphate-solubilizing bacteria agent is inoculated into soil where plants grow, so as to improve the phosphorus absorption efficiency of the plants.

[0027] Preferably, the soil treatment includes soil treatment before sowing or during the growing season, and the plant is rapeseed. The amount and frequency of application of the microbial agent can be determined according to the phosphorus content of the soil and the needs of the plant.

[0028] The present invention significantly improves the phosphorus absorption and utilization rate of plants, especially rapeseed, during the growth process by fixing seven representative inorganic phosphorus-dissolving bacteria on different types of biochar carriers.

[0029] The composite phosphate-solubilizing bacteria agent of the present invention can effectively improve the phosphorus absorption and utilization rate of plants during their growth process. By fixing inorganic phosphorus-solubilizing bacteria on different types of biochar carriers, the phosphorus absorption capacity and growth performance of plants, especially rapeseed, are more significantly improved. It has broad agricultural application prospects, especially for improving the effectiveness of phosphorus in poor soils, reducing dependence on chemical phosphorus fertilizers, and improving the effectiveness of soil phosphorus and phosphorus absorption of crops. It is a method for achieving more environmentally friendly and sustainable agricultural production. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a picture showing the morphological characteristics of inorganic phosphorus-solubilizing bacteria observed using a scanning electron microscope;

[0031] Figure 2 Figure 2 shows the growth characteristics of colonies in different nitrogen and phosphorus source culture media; (a) LB medium (b) NH4 + PKV medium with NO3 as nitrogen source and (c) -Colonies after 72 hours of cultivation on PKV medium with nitrogen source; Escherichia coli without phosphate solubilization ability was used as blank control;

[0032] Figure 3 The heat map of medium pH and organic acid anion leakage after 144 hours of cultivation of inorganic phosphate-solubilizing bacteria was plotted based on the average of three repeated experiments. DETAILED DESCRIPTION

[0033] The technical scheme of the present invention is further specifically described below through examples, which are provided for the purpose of explaining the present invention, not for limiting the present invention. Based on the examples in this application, all other examples obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0034] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0035] The 7 strains selected in the present invention were obtained from multiple sites and classified and identified by 16S rRNA sequencing and were identified as Agrobacterium ( Agrobacterium ), Pseudomonas ( Pseudomonas ), Ralstonia ( Ralstonia ), Burkholderia spp. Burkholderia ), Bacillus ( Bacillus ), and are classified as:

[0036] Agrobacterium ( Agrobacterium sp. ) strain LYS-n49 (Agrobacterium LYS-n49, Agrobacterium sp. LYS-n49), with the deposit number of CGMCC No.32845, was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on November 29, 2024, with the address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing;

[0037] Pseudomonas ( Pseudomonas sp. ) strain LYS-11 (Pseudomonas sp. LYS-11, Pseudomonas sp. LYS-11), with the deposit number of CGMCC No.32841, was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on November 29, 2024, with the address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing;

[0038] Ralstonia Ralstonia sp. ) strain LYS-14 (Ralstonia LYS-14, Ralstonia sp.LYS-14), with the deposit number of CGMCC No.32842, was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on November 29, 2024, with the address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing;

[0039] Ralstonia Ralstoniasp. ) strain LYS-16 (Ralstonia LYS-16, Ralstoniasp. LYS-16), with the deposit number of CGMCC No.32843, was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on November 29, 2024, with the address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing;

[0040] Pseudomonas ( Pseudomonassp. ) strain LYS-p19 (Pseudomonas sp. LYS-p19, Pseudomonassp. LYS-p19), with the deposit number of CGMCC No.32846, was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on November 29, 2024, with the address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing;

[0041] Burkholderia Burkholderiasp. ) strain LYS-21 (Burkholderia LYS-21, Burkholderiasp. LYS-21), with the deposit number of CGMCC No.32844, was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on November 29, 2024, with the address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing;

[0042] Bacillus ( Bacillus sp. ) strain LYS-p36 (Bacillus LYS-p36, Bacillus sp. LYS-p36), with the deposit number CGMCC No.32847, was deposited on November 29, 2024 at the General Microbiology Center of China Culture Collection Administration, with the address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0043] 1. Screening and identification of strains

[0044] Soil samples (0–15 cm) were collected from farmland soils in Hailun City (47°26′32.54″N, 126°38′43.26″E), Heilongjiang Province, China, Yingtan City (28°14′05.38″N, 116°54′25.12″E), Jiangxi Province, China, and Changshu City (31°32′11.93″N, 120°41′34.54″E). Inorganic phosphorus-dissolving bacteria were initially screened from soil samples from three collection sites and inoculated into modified inorganic phosphorus solid medium (glucose 10 g / L, ammonium sulfate 0.5 g / L, sodium chloride 0.2 g / L, magnesium sulfate 0.1 g / L, manganese sulfate monohydrate 0.002 g / L, potassium chloride 0.2 g / L, ferrous sulfate heptahydrate 0.002 g / L, hydroxyapatite 1 g / L, agar powder 15 g / L, pH 7.0-7.2, 0.01 mM bromocresol purple). After isolating a single colony, it was streaked again on an inorganic phosphorus solid medium plate for purification. The purified colonies were selected for 16S rRNA sequencing for taxonomic identification. The 16S rRNA gene was amplified using polymerase chain reaction (PCR), and the primers used were 24F / 1492R bacterial universal primers. The reaction system (50 μL) contained 1 μL of Premix Ex Taq Hot Start Version, 0.2 μM of each primer, and 1 colony. The amplification protocol was 95°C initial denaturation for 4 minutes, 95°C for 30 seconds, 58°C for 90 seconds, and 72°C for 30 seconds for 30 cycles, and finally 72°C for 5 minutes. The amplified products were sent to a sequencing company, and a total of 133 phosphate-solubilizing bacteria were obtained (57 strains from Changshu soil, and 76 strains from Hailun and Yingtan soils).

[0045] 2. Strain selection, characteristic analysis and community construction

[0046] From these 133 phosphate-solubilizing bacteria, 7 inorganic phosphate-solubilizing bacteria of different phyla with high efficiency in dissolving insoluble phosphates (calcium-phosphate, iron-phosphate, aluminum-phosphate) were selected based on their phosphorus-solubilizing ability and phylogenetic diversity. They were selected according to their ability to dissolve calcium-phosphate, iron-phosphate, and aluminum-phosphate. Specifically, seven inorganic phosphate-solubilizing bacteria strains of five different genera were selected according to their ability to dissolve calcium-phosphate greater than 13 μg / mL, iron-phosphate greater than 12 μg / mL, and aluminum-phosphate greater than 7 μg / mL; they were Agrobacterium LYS-n49 ( Agrobacterium sp. LYS-n49), Pseudomonas LYS-11 ( Pseudomonas sp. LYS-11), Ralstonia LYS-14 ( Ralstonia sp. LYS-14), Ralstonia LYS-16 ( Ralstoniasp. LYS-16), Pseudomonas LYS-p19 ( Pseudomonassp.LYS-p19), Burkholderia LYS-21 ( Burkholderiasp. LYS-21) and Bacillus LYS-p36 ( Bacillus sp. LYS-p36).

[0047] Specifically: weigh 0.1g farmland soil in a 2mL centrifuge tube, add 1mL sterile water, shake for 30 seconds and then stand for 5 to 10 minutes, take the supernatant, dilute 10,000 times to make a soil suspension. Take 0.1mL of the soil suspension diluted 10,000 times and inoculate it on an inorganic phosphorus solid culture medium plate, place it in a 30°C incubator and invert it for 3-4 days until colonies are formed. Select colonies with different shapes, sizes, and colors for purification and separation. The purified colonies are selected for 16S rRNA sequencing for classification and identification. A total of 133 strains of phosphate-solubilizing bacteria were isolated, and seven strains of the present invention were selected from the 133 strains of phosphate-solubilizing bacteria according to their efficient phosphate-solubilizing ability.

[0048] The rRNA gene sequences of each strain are SEQ ID NO.1 to SEQ ID NO.7, that is, the gene sequence SEQ ID NO.1 corresponds to the strain name Agrobacterium LYS-n49, the gene sequence SEQ ID NO.2 corresponds to the strain name Pseudomonas LYS-11, the gene sequence SEQ ID NO.3 corresponds to the strain name Ralstonia LYS-14, the gene sequence SEQ ID NO.4 corresponds to the strain name Ralstonia LYS-16, the gene sequence SEQ ID NO.5 corresponds to the strain name Pseudomonas LYS-p19, the gene sequence SEQ ID NO.6 corresponds to the strain name Burkholderia LYS-21, and the gene sequence SEQID NO.7 corresponds to the strain name Bacillus LYS-p36.

[0049] The biocompatibility between these inorganic phosphate-solubilizing bacteria (iPSB) strains was detected by cross-streaking on the same inorganic phosphorus solid culture medium plate. The test results showed that there was no antagonism between the seven strains selected in the present invention, that is, they could coexist harmoniously. The microscopic morphology of these strains was observed by scanning electron microscopy (SEM). Before observation, the strains were dehydrated with ethanol and metal-plated to obtain clear microstructural images, such as Figure 1 As shown. Figure 1 It can be seen that they have different cell sizes and shapes (0.5-10 μm, spherical or rod-shaped).

[0050] Different nitrogen sources can affect the phosphate solubilization ability of strains and the survival of inorganic phosphate-solubilizing bacteria. Therefore, the colony growth characteristics in LB medium (as a control for colony morphology), inorganic phosphate solid medium (ammonium sulfate as the only nitrogen source) and inorganic phosphate solid medium (0.5 g / L sodium nitrate instead of 0.5 g / L ammonium sulfate), that is, different nitrogen and phosphorus source media were observed; Escherichia coli BL21 was also cultured in these media as a reference strain, such as Figure 2 As shown in (a)-(c) in the figure. Figure 2 (a) in the figure uses LB medium, whose components are: 10 g / L tryptone, 5 g / L yeast extract powder, 10 g / L sodium chloride, and 15 g / L agar. Figure 2 (b) in the above analysis uses an inorganic phosphorus solid culture medium (ammonium sulfate as the only nitrogen source), whose components are: glucose 10 g / L, ammonium sulfate 0.5 g / L, sodium chloride 0.2 g / L, magnesium sulfate 0.1 g / L, manganese sulfate monohydrate 0.002 g / L, potassium chloride 0.2 g / L, ferrous sulfate heptahydrate 0.002 g / L, hydroxyapatite 1 g / L, agar powder 15 g / L, pH 7.0-7.2, and 0.01 mM bromocresol purple. Figure 2 (c) in the above culture medium uses an inorganic phosphorus solid culture medium (0.5 g / L sodium nitrate replaces 0.5 g / L ammonium sulfate), whose components are: glucose 10 g / L, sodium nitrate 0.5 g / L, sodium chloride 0.2 g / L, magnesium sulfate 0.1 g / L, manganese sulfate monohydrate 0.002 g / L, potassium chloride 0.2 g / L, ferrous sulfate heptahydrate 0.002 g / L, hydroxyapatite 1 g / L, agar powder 15 g / L, pH 7.0-7.2, and 0.01 mM bromocresol purple.

[0051] from Figure 2 As can be seen from (a)-(c) in the figure, these strains can survive in LB, inorganic phosphorus solid medium (ammonium sulfate as the only nitrogen source), and inorganic phosphorus solid medium (0.5 g / L sodium nitrate replaces 0.5 g / L ammonium sulfate).

[0052] Seven strains and seven bacteria were mixed in the same volume (10 9 CFU / mL) were placed in liquid inorganic phosphorus solid culture media with different phosphorus sources (hydroxyapatite, iron phosphate and aluminum phosphate) at 30℃ for 72 h, and the phosphorus concentration in the supernatant was measured. After 72 h, the solubility of the seven strains and the mixed community for various forms of phosphorus are shown in Table 1.

[0053] Table 1

[0054] ;

[0055] The mean ± SD of three replicates. Different letters in the column indicate significant differences at P < 0.05.

[0056] The liquid inorganic phosphorus medium with hydroxyapatite as the phosphorus source has the following components: glucose 10 g / L, ammonium sulfate 0.5 g / L, sodium chloride 0.2 g / L, magnesium sulfate 0.1 g / L, manganese sulfate monohydrate 0.002 g / L, potassium chloride 0.2 g / L, ferrous sulfate heptahydrate 0.002 g / L, hydroxyapatite 1 g / L, pH 7.0-7.2. The liquid inorganic phosphorus medium with iron phosphate as the phosphorus source has the following components: glucose 10 g / L, ammonium sulfate 0.5 g / L, sodium chloride 0.2 g / L, magnesium sulfate 0.1 g / L, manganese sulfate monohydrate 0.002 g / L, potassium chloride 0.2 g / L, ferrous sulfate heptahydrate 0.002 g / L, ferrous phosphate 1 g / L, pH 7.0-7.2. The liquid inorganic phosphorus culture medium with aluminum phosphate as the phosphorus source consists of: glucose 10 g / L, ammonium sulfate 0.5 g / L, sodium chloride 0.2 g / L, magnesium sulfate 0.1 g / L, manganese sulfate monohydrate 0.002 g / L, potassium chloride 0.2 g / L, ferrous sulfate heptahydrate 0.002 g / L, aluminum dihydrogen phosphate 1 g / L, pH 7.0-7.2.

[0057] As can be seen from Table 1, the seven selected strains have outstanding solubility for calcium-phosphorus, iron-phosphorus, and aluminum-phosphorus. The mixed bacterial community has further improved its solubility for calcium-phosphorus and iron-phosphorus, especially the calcium-phosphorus solubilizing ability of the mixed bacterial community is much greater than that of a single strain.

[0058] The seven strains were cultured for 12, 24, 48, 72, 96 and 144 hours, respectively. The anions of lactic acid, acetic acid, malic acid, succinic acid, oxalic acid and citrate were analyzed by ion chromatography. The contents of various organic acids were determined and the pH value of the culture medium was measured using a pH meter. The heat map of the culture medium pH value and the amount of organic acid anion leakage after 144 hours of cultivation of inorganic phosphate-solubilizing bacteria was drawn based on the average value of three repeated experiments. Figure 3 As shown. Figure 3 It can be seen that the pH value and the ability to secrete organic anions after 144 hours of culture are different among the strains, indicating that the seven strains have different abilities to release organic acids after 144 hours of culture, which indirectly reflects their different abilities to dissolve different insoluble phosphorus (calcium phosphorus, iron phosphorus, aluminum phosphorus). Making the seven strains into a composite bacterial agent can further enhance their advantages in dissolving calcium phosphorus, while making up for their weaknesses in dissolving iron phosphorus and aluminum phosphorus.

[0059] 3. Preparation method of biochar

[0060] The six raw materials were rice straw (RS), rice husk (RH), soybean straw (SS), corn cob (CC), peanut shell (PN) and wood (WD). All raw materials were slowly pyrolyzed in a muffle furnace at 450 °C with automatic N2 flow (0.5 L / min) to produce biochar at 450 °C.

[0061] The pH of the biochar was determined after shaking the suspension of biochar: deionized water (w / v) = 1:20 for 1 h using an XL60 pH meter (Fisher Scientific, Asheville, USA). The percentages of carbon and nitrogen were measured using an elemental analyzer. The content of available phosphorus was determined using sodium bicarbonate extraction. The Brunner-Emmett-Teller (BET) surface area was measured using a NOVA-2000E surface area analyzer. The pore size was measured using the N2 absorption-desorption isotherm using an Autosorb-1-C gas adsorption system (Quantachrome, Boynton Beach, USA). The physical and chemical properties of the obtained biochar are shown in Table 2.

[0062] Table 2

[0063] ;

[0064] Mean ± SD of 4 replicates. Different letters in the same column indicate significant differences, P < 0.05;

[0065] CC, corn cob; PN, peanut shell; RH, rice husk; RS, rice straw; SS, soybean straw; WD, wood.

[0066] Different biochars have different physical and chemical properties, which will affect which one is selected as the carrier of the microbial agent to better exert the phosphorus-solubilizing ability of the microbial agent. From the data of available phosphorus, it can be seen that rice straw and soybean straw biochar (RS and SS) have excellent promoting effects on inorganic phosphorus-dissolving bacterial communities, so they are selected as carriers of mixed colonies to make the composite microbial agent of the present invention.

[0067] 4. Preparation and application of composite phosphate-solubilizing bacteria agent

[0068] The selected 7 inorganic phosphate solubilizing bacterial strains were mixed in the same volume (10 9CFU / mL) and inoculated on 6 different sterilized biochar carriers to form a composite phosphate-solubilizing bacteria agent based on biochar carriers. This inorganic phosphorus-dissolving bacterial community was inoculated on 6 types of biochars, and four independent replicates were made (inoculated once every 7 days). Specific operation: After the biochar was sieved (0.2 mm), liquid inorganic phosphorus culture medium (glucose 10g / L, ammonium sulfate 0.5 g / L, sodium chloride 0.2 g / L, magnesium sulfate 0.1 g / L, manganese sulfate monohydrate 0.002 g / L, potassium chloride 0.2 g / L, ferrous sulfate heptahydrate 0.002 g / L, hydroxyapatite 1 g / L, pH7.0-7.2) was added at a weight ratio of 1:20 and sterilized at 121°C for 20 minutes. In the liquid inorganic phosphorus culture medium (8.6 × 10 8CFU / mL) incubated at 30°C for 72 h in an inorganic phosphate-solubilizing bacterial community was centrifuged at 5000 g, and the precipitate was resuspended in sterile liquid inorganic phosphate medium with biochar. The culture was shaken at 30°C and 200 rpm for 24 h. There were 7 treatments in the experiment: 6 biochar-modified treatments and 1 control (CK) without biochar. All experimental soils were sterilized by 25 kGy radiation before applying the composite phosphate-solubilizing bacteria inoculant. The experiment was designed in a completely randomized block design with 4 replicates. 1 kg of sterile soil and 3% (w / w) of a mixed inorganic phosphate-solubilizing bacterial community of 7 species fixed on biochar were added to cylindrical polyvinyl chloride (PVC) jars. Before sowing rapeseed seeds, the surface of the seeds was sterilized with 30% H2O2 for 10 min and then thoroughly washed with deionized water. The experiment was conducted in a greenhouse (humidity was controlled at 70 ± 5%) with 12 h of light and temperature (25 ± 2 °C during the day and 20 ± 3 °C at night) for 4 weeks. All pots were irrigated manually with deionized water to maintain the water content at 60%. After four weeks of growth, rapeseed plants were collected and carefully separated from the soil. Rhizosphere soil was defined as the soil attached to the roots and was collected by manually shaking the roots. The size of the bacterial community was determined by culturing and counting the inoculum colony forming units (CFUs) in serial dilutions of rhizosphere soil (from rapeseed roots but with plant tissue removed) in LB medium and incubating at 30 °C for 24 h. To calculate the survival rate of the inorganic phosphate solubilizing bacterial community, the biochar was rinsed with sterile water. Bacteria immobilized on each biochar were detached by ultrasonic treatment and then serially diluted and cultured in liquid inorganic phosphorus medium (glucose 10 g / L, ammonium sulfate 0.5 g / L, sodium chloride 0.2 g / L, magnesium sulfate 0.1 g / L, manganese sulfate monohydrate 0.002 g / L, potassium chloride 0.2 g / L, ferrous sulfate heptahydrate 0.002 g / L, hydroxyapatite 1 g / L, pH 7.0-7.2) to count CFUs. Survival was calculated by comparing the difference in CFU counts before and after four weeks of plant growth. pH, available phosphorus content, and total phosphorus content of rhizosphere soil were measured. Plant samples were washed with deionized water and dried in an oven at 70 °C for 72 h to analyze total plant biomass (grams per pot). Dried plant samples were ground and digested with sulfuric acid, and total phosphorus content (milligrams per gram) was determined using the molybdenum blue method. Phosphorus absorption by plants (mg per pot) = total phosphorus content (mg per gram) × plant biomass (gram per pot). The results of the effects of biochar on rhizosphere soil and plant growth during the four-week plant growth period after inoculation with inorganic phosphorus-dissolving bacteria are shown in Table 3.

[0069] Table 3

[0070] ;

[0071] Mean ± SD of 4 replicates. Different letters in the same column indicate significant differences, P < 0.05;

[0072] CK, blank; RS, rice straw; SS, soybean straw; RH, rice husk; CC, corn cob; PN, peanut shell; WD, wood; ND, not detected.

[0073] The data of total phosphorus and available phosphorus in rhizosphere soil should be viewed together. The lower the total phosphorus in rhizosphere soil and the higher the corresponding available phosphorus, the better the effect of the composite microbial agent. The phosphorus content of plants is also significantly higher than that of the blank group. From Table 3, it can be seen that in the present invention, all biochar raw materials are beneficial to the inorganic phosphorus solubilizing bacterial community, but rice straw and soybean straw biochar (RS and SS) perform best in increasing the abundance of inorganic phosphorus solubilizing bacterial community and improving its phosphorus solubilizing ability.

[0074] In order to distinguish and quantify the effects of biochar and inorganic phosphate-solubilizing bacteria community on plant growth and phosphorus absorption, we carried out four treatments in rapeseed growth: control without biochar and inorganic phosphate-solubilizing bacteria (R-CK), adding 3% RS without inorganic phosphate-solubilizing bacteria community (RB), inoculating only inorganic phosphate-solubilizing bacteria culture solution without adding biochar (RP), and inorganic phosphate-solubilizing bacteria inoculated with RS biochar amendment (R-BP). All growth conditions were set the same as above. The available phosphorus concentration and total phosphorus content of the rhizosphere soil after rapeseed growth, the biomass of the plant, the total phosphorus content of the plant, and the phosphorus absorption of the plant were measured. The results are shown in Table 4:

[0075] Table 4

[0076] ;

[0077] Mean ± SD of 4 replicates. Different letters in the same column indicate significant differences, P < 0.05;

[0078] R-CK, control without biochar and inorganic phosphate-solubilizing bacteria; RB, 3% rice straw was added without inorganic phosphate-solubilizing bacteria community; RP, only inorganic phosphate-solubilizing bacteria culture solution was inoculated without biochar; R-BP, inorganic phosphate-solubilizing bacteria were inoculated with rice straw biochar amendment.

[0079] As can be seen from Table 4, the effective phosphorus content in the rhizosphere soil, the phosphorus content and the phosphorus absorption of the plants in the R-BP group are significantly higher than those in the other three groups, indicating that the addition of biochar alone or inorganic phosphate-solubilizing colonies alone does not promote rapeseed growth as well as the composite bacterial agent of the present invention, proving the superiority of the composite bacterial agent of the present invention.

[0080] The present invention significantly improves the phosphorus absorption and utilization rate of plants, especially rapeseed, during their growth by fixing seven representative inorganic phosphorus-dissolving bacteria on different types of biochar carriers. The present invention selects seven inorganic phosphorus-dissolving bacterial strains with the ability to efficiently dissolve calcium phosphate from different bacterial genera, and combines them with biochar carriers to generate a composite bacterial agent. There is no antagonism between the seven bacterial strains, and the effect of 1+1>2 can be achieved after being used together. The biochar carrier used in the present invention can not only provide a protective habitat for these inorganic phosphorus-dissolving bacteria and prolong their survival time in the soil, but also enhance the phosphorus-dissolving ability of the bacterial community through its unique pore structure, thereby improving the effectiveness of phosphorus in the soil and promoting phosphorus absorption by crops.

[0081] The present invention selects seven representative inorganic phosphorus-dissolving bacterial strains and fixes them in the soil through biochar carriers, which significantly improves the absorption and utilization rate of phosphorus in plants, especially rapeseed during growth; among them, rice straw and soybean straw biochar are the best strain carriers, which can maximize the survival rate and functionality of inorganic phosphorus-dissolving bacterial populations. The composite bacterial agent of the present invention has a wide range of application prospects in agriculture, especially for improving the effectiveness of soil phosphorus and crop phosphorus absorption.

[0082] Finally, it should be noted that the above are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. A composite phosphate-solubilizing bacteria agent based on biochar carrier, characterized in that: The composite phosphate-solubilizing bacteria agent comprises a plurality of inorganic phosphorus-solubilizing bacteria strains of different bacterial genera, wherein the bacterial genera are Agrobacterium ( Agrobacterium ), Pseudomonas ( Pseudomonas ), Ralstonia ( Ralstonia ), Burkholderia spp. Burkholderia ), Bacillus ( Bacillus ); one or more strains from each of the above-mentioned different genera are selected and mixed, and fixed on a biochar carrier made of rice straw, rice husk, soybean straw, corn cob, peanut shell or wood; The selected strains were Agrobacterium ( Agrobacterium sp. )LYS-n49, the deposit number is CGMCCNo.32845; Pseudomonas ( Pseudomonas sp. )LYS-11, the deposit number is CGMCC No.32841; Ralstonia ( Ralstonia sp. )LYS-14, the deposit number is CGMCC No.32842; Ralstonia ( Ralstonia sp. )LYS-16, the deposit number is CGMCC No.32843; Pseudomonas ( Pseudomonas sp. ) LYS-p19, the deposit number is CGMCC No.32846; Burkholderia ( Burkholderia sp. )LYS-21, the deposit number is CGMCC No.32844; Bacillus ( Bacillus sp. )LYS-p36, deposit number is CGMCC No.32847; The rRNA gene sequences of the strains are SEQ ID NO.1 to SEQ ID NO.

7.

2. The composite phosphate-solubilizing bacteria agent based on biochar carrier according to claim 1, characterized in that: The biochar carrier is a biochar carrier prepared from rice straw or soybean straw.

3. A method for preparing the composite phosphate-solubilizing bacteria agent based on biochar carrier according to claim 1, characterized in that: Inorganic phosphorus-solubilizing bacterial strains of different genera are mixed in proportion and fixed on a biochar carrier to form a composite phosphate-solubilizing bacterial agent based on a biochar carrier.

4. The method for preparing the composite phosphate-solubilizing bacteria agent based on biochar carrier according to claim 3, characterized in that: The biochar carrier is prepared by the following method: raw materials are pyrolyzed in a muffle furnace at 450° C., equipped with an automatic nitrogen flow, to produce biochar at 450° C.; the raw materials include rice straw, rice husk, soybean straw, corn cob, peanut shell and wood.

5. The method for preparing the composite phosphate-solubilizing bacteria agent based on biochar carrier according to claim 3, characterized in that: The concentration of inorganic phosphorus-dissolving bacteria in the composite phosphate-dissolving bacteria agent is 10 9 ~10 10 CFU / mL.

6. An application of the composite phosphate-solubilizing bacteria agent based on biochar carrier according to claim 1, characterized in that: The composite phosphate-solubilizing bacteria agent is inoculated into the soil where plants grow to improve the phosphorus absorption efficiency of the plants.

7. The use of the composite phosphate-solubilizing bacteria agent based on biochar carrier according to claim 6, characterized in that: When the soil is treated, it includes soil treatment before sowing or during the growing season, and the plant is rapeseed.

Images