Phosphorus-dissolving aluminum-tolerant microorganism, synthetic microbial flora and application thereof

Abstract

The present application relates to a kind of phosphorus-dissolving aluminum-tolerant microorganism, synthetic flora and its application, belong to the field of microbial technology.The present application provides a kind of phosphorus-dissolving aluminum-tolerant microorganism, the microorganism is Ochrobactrum teleogrylli05217 or Talaromyces fuscoviridis 20762.The present application also provides a kind of synthetic flora, the synthetic flora includes strain Ochrobactrum teleogrylli and strain Talaromyces fuscoviridis.The present application is by 2 strains of microorganism with phosphorus-dissolving aluminum-tolerant efficacy is collocated as synthetic flora, can further improve the efficiency of phosphorus aluminum conversion in soil, the phosphorus content in crop is improved, to realize the purpose of promoting crop growth.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a phosphorus-soluble aluminum-resistant microorganism, a synthetic microbial community, and its applications. Background Technology

[0002] The parent material of soils in Guangdong is mostly acidic, such as lateritic red soil and acidic sulfate soil, leading to phosphorus fixation and high aluminum toxicity. This results in the large-scale application of phosphorus fertilizers to vegetable fields, but with low phosphorus utilization and inhibited crop growth. Low phosphorus fertilizer utilization and aluminum toxicity are the two major nutrient limiting factors restricting crop production in acidic soils. Improper application of soil conditioners such as phosphate fertilizers and lime can cause soil compaction and high costs, making them unsuitable for large-scale agricultural practices. Microorganisms have become a focus of attention due to their environmental friendliness, low cost, and high biological efficiency. Phosphate-solubilizing microorganisms can convert insoluble phosphorus into a form that is readily absorbed by plants through the secretion of organic acids and chelation. However, whether phosphate-solubilizing microorganisms can simultaneously promote phosphorus utilization by plants and help reduce aluminum toxicity to plants in acidic soils remains unreported. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide a microorganism, synthetic flora and its application that has the effect of dissolving phosphorus and resisting aluminum, can transform phosphorus and aluminum in the soil to promote crop growth.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0005] In a first aspect, the present invention provides a phosphorus-soluble aluminum-resistant microorganism, wherein the microorganism is Ochrobactrumteleogrylli 05217 or Talaromyces fuscoviridis 20762;

[0006] The microorganism Ochrobactrum teleogrylli 05217 is deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 6, 2024, with accession number GDMCC No: 64715.

[0007] The microorganism Talaromyces fuscoviridis 20762 is deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 6, 2024, with accession number GDMCC No: 64713.

[0008] This invention screened microorganisms with phosphorus-solubilizing ability from the rhizosphere soil of vegetable fields. Experiments have confirmed that these two microorganisms not only have phosphorus-solubilizing ability, but also have a high tolerance to aluminum concentrations and can improve the crop growth inhibition caused by aluminum stress.

[0009] In a second aspect, the present invention provides a synthetic microbial community, which includes strains Ochrobactrumteleogrylli and Talaromyces fuscoviridis;

[0010] The strain Ochrobactrum teleogrylli is deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 6, 2024, with accession number GDMCC No: 64715.

[0011] The strain Talaromyces fuscoviridis is deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 6, 2024, with accession number GDMCC No: 64713.

[0012] This invention combines two microorganisms with phosphorus-solubilizing and aluminum-tolerant properties into a synthetic microbial community, which can further improve the efficiency of phosphorus-aluminum conversion in the soil, increase the phosphorus content in crops, and thus promote crop growth.

[0013] In a preferred embodiment of the synthetic microbial community described in this invention, the concentration ratio of Ochrobactrum teleogrylli to Talaromyces fuscoviridis is Ochrobactrum teleogrylli:Talaromyces fuscoviridis = 1:1.

[0014] Thirdly, the present invention provides a microbial agent, which includes the above-mentioned phosphorus-soluble and aluminum-resistant microorganisms or the above-mentioned synthetic microbial groups.

[0015] In a preferred embodiment of the microbial agent of the present invention, the bacterial concentration of strain Ochrobactrum teleogrylli in the microbial agent is 10. 7～9 CFU / mL;

[0016] The spore concentration of the microbial agent containing the strain *Talaromyces fuscoviridis* was 10. 6～7 CFU / mL.

[0017] Fourthly, the present invention provides the application of the above-mentioned phosphorus-soluble and aluminum-resistant microorganisms, synthetic microbial communities, or microbial agents in promoting the growth of Brassica crops.

[0018] Fifthly, the present invention provides the application of the above-mentioned phosphorus-soluble and aluminum-tolerant microorganisms, synthetic microbial communities, or microbial agents in promoting the conversion of phosphorus and aluminum in the rhizosphere of Brassica crops.

[0019] As a preferred embodiment of the application described in this invention, the Brassica genus crops include at least one of Chinese cabbage, mustard greens, bok choy, Chinese cabbage, and kale.

[0020] Sixthly, the present invention provides a method for promoting the growth of Brassica crops, comprising the following steps: applying the above-mentioned phosphorus-solubilizing aluminum-tolerant microorganisms, the above-mentioned synthetic microbial communities, or the above-mentioned microbial agents to the roots of the crops, wherein the cell concentration of the phosphorus-solubilizing aluminum-tolerant microorganisms, synthetic microbial communities, or microbial agents is 10. 6 ~10 9 CFU / mL.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] 1. This invention screens microorganisms with phosphorus-solubilizing ability from the rhizosphere soil of vegetable fields. Experiments have confirmed that these two microorganisms not only have phosphorus-solubilizing ability, but also have high tolerance to aluminum concentrations and can improve the crop growth inhibition caused by aluminum stress.

[0023] 2. This invention combines two microorganisms with phosphorus-solubilizing and aluminum-tolerant properties into a synthetic microbial community, which can further improve the efficiency of phosphorus-aluminum conversion in the soil, increase the phosphorus content in crops, and thus promote crop growth. Attached Figure Description

[0024] Figure 1 This is a phylogenetic tree of the phosphorus-soluble and aluminum-tolerant microorganism Ochrobactrum teleogrylli 05217 in Example 1 of the present invention;

[0025] Figure 2 This is a phylogenetic tree of the phosphorus-soluble and aluminum-tolerant microorganism Talaromyces fuscoviridis 20762 in Example 2 of the present invention;

[0026] Figure 3 This is a compatibility test diagram of Ochrobactrum teleogrylli and Talaromyces fuscoviridis in Example 1 of the present invention.

[0027] Figure 4 The effect of different bacterial suspensions on the growth of phosphorus-soluble aluminum-tolerant microorganisms is shown in Example 1 of the present invention.

[0028] Figure 5 This is a graph showing the effect of different bacterial suspensions on the growth of phosphorus-soluble aluminum-tolerant microorganisms in Example 1 of the present invention.

[0029] Figure 6 This is an example of the effect of different treatments on phosphorus forms in the rhizosphere soil of Chinese cabbage in Example 3 of the present invention.

[0030] Figure 7This is an example of the effect of different treatments on the aluminum speciation in the rhizosphere soil of Chinese cabbage in Example 3 of the present invention.

[0031] Figure 8 This is an example of the effect of different treatments on the abundance of phosphorus-soluble genes in the rhizosphere soil of Chinese cabbage in Example 3 of the present invention.

[0032] Figure 9 This is an example of the effect of different treatments on the abundance of aluminum-tolerant genes in the rhizosphere soil of Chinese cabbage in Example 3 of the present invention.

[0033] In the above figure, different lowercase letters represent significant differences between the two, P < 0.05. Detailed Implementation

[0034] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0035] Unless otherwise specified, “Ochrobactrum teleogrylli”, “O.teleogrylli”, and “05217” mentioned in the following examples and effect examples refer to Ochrobactrum teleogrylli 05217; and “Talaromyces fuscoviridis”, “T.fuscoviridis”, and “20762” refer to Talaromyces fuscoviridis20762 unless otherwise specified.

[0036] Unless otherwise specified, all materials and reagents used in the following examples and effect examples are commercially available.

[0037] NBRIP medium: glucose 10.0g, MgCl2·6H2O 5.0g, MgSO4·7H2O 0.25g, KCl 0.20g, (NH4)SO4 0.10g, Ca3(PO4)2 5.0g, agar 18.0g, distilled water 1000mL, pH 6.8-7.0, sterilized at 121℃ for 20-25min.

[0038] NA solid medium: Add 3g beef extract, 10g bacteriological peptone, 5g sodium chloride, and 18g agar powder to 1000mL distilled water. After complete dissolution, autoclave at 121℃ for 15min. Cool after sterilization for later use. Remove the agar powder from the above medium to prepare NB liquid medium, and sterilize at 121℃ for 30min.

[0039] LB liquid medium: Dissolve 10g tryptone, 5g yeast extract, and 10g sodium chloride in 1000mL distilled water. Autoclave at 121℃ for 30min, then cool before use. To prepare a solid medium, add 18g / L agar powder and autoclave at 121℃ for 30min.

[0040] PDA medium: Peel 200g of potatoes, cut them into 0.5cm cubes, add 1L of deionized water, boil for 30min, filter through 4 layers of gauze, add 20g of glucose and 20g of agar to the filtrate, bring the volume to 1L, and sterilize at 121℃ for 20min; if preparing PDB medium, do not add agar.

[0041] PKO Inorganic Phosphorus Medium: 10g glucose, 5g tricalcium phosphate, 0.5g ammonium sulfate, 0.2g sodium chloride, 0.2g potassium chloride, 0.3g magnesium sulfate heptahydrate, 0.03g manganese sulfate, 0.03g ferrous sulfate heptahydrate, 0.5g yeast extract, 1000mL distilled water. After complete dissolution, adjust the pH to 6.8-7.0 and sterilize at 121℃ for 30min. To prepare a solid medium, add 18g / L agar powder and sterilize at 121℃ for 30min.

[0042] In this invention, the Brassica genus crop can be any Brassica genus crop reported in the prior art. In the following embodiments and effect examples, Fukuda cabbage is used as a representative of Brassica genus crops for relevant experiments. Fukuda cabbage can be purchased commercially or grown by purchasing seeds.

[0043] Planting method of Futian cabbage: Select plump Futian cabbage seeds, disinfect them with 1v / v% sodium hypochlorite for 15 minutes, then rinse them 5-6 times with sterile deionized water to remove the residual disinfectant, and transfer them to seedling trays for seedling cultivation. When they grow to 3-4 leaves, transplant them into nutrient solution or soil according to their intended use.

[0044] The method for hydroponically growing Futian Chinese cabbage: Gently pull the seedlings with 3-4 leaves out of the substrate, and slowly wash the roots several times to remove the substrate attached to the roots. After washing, transfer the Chinese cabbage seedlings to a plastic box with small holes in the lid. Let the seedlings recover in 1 / 4 low phosphorus Hogland nutrient solution for 2-3 days, then transfer them to 1 / 2 low phosphorus Hogland nutrient solution for 2-3 days. Finally, switch to the normal concentration of Hogland nutrient solution for the experiment.

[0045] Planting method of Fukuda cabbage in soil: Transplant seedlings with 3-4 leaves from the substrate into flower pots with an inner diameter of 13.5cm and a height of 10.7cm. Plant 2 seedlings in each pot. Fill each pot with 1000g of soil. The soil used is red soil containing urea, superphosphate and calcium chloride. The ratio of urea: superphosphate: calcium chloride is 50mg / kg: 745mg / kg: 37.5mg / kg. Water normally and select seedlings with uniform growth for the experiment.

[0046] The Fast DNASPIN Kit for Soil was purchased from MP Biomedicals, Santa Ana, CA, USA.

[0047] The Universal DNAPurification Kit was purchased from TIANGEN, China.

[0048] Example 1

[0049] This embodiment provides a phosphorus-solubilizing and aluminum-tolerant microorganism, Ochrobactrum teleogrylli 05217, which was isolated from soil. The specific steps are as follows:

[0050] 1. Isolation and purification of phosphate-solubilizing microorganisms

[0051] Rhizosphere soil samples from Chinese cabbage, potato, winter melon, and tomato grown in vegetable planting bases in Huizhou, Foshan, and Maoming cities, Guangdong Province, were collected, mixed thoroughly, and stored at 4°C. Soil bacterial suspensions were prepared using sterile pure water. 2g of soil sample was weighed and placed in an Erlenmeyer flask containing 18mL of sterile water, with approximately 10-15 glass beads added. The flask was shaken at 160rpm for 2 hours. After 2 hours, 1mL of the supernatant was transferred to a test tube containing 9mL of ultrapure water and shaken well. At this point, the bacterial suspension was diluted 10-10. -2 Repeat the above steps to obtain a 10x dilution. -3 10 -4 A multiple of soil suspension.

[0052] Take the diluted 10 -3 10 -4100 μL of soil suspension was evenly spread on NBRIP medium supplemented with 30 mg / L actinomycete ketone to screen for phosphate-solubilizing bacteria. After the medium fully absorbed the soil bacterial suspension, the plates were inverted and incubated at 30°C for 5-7 days. Various phosphate-solubilizing colonies from the soil samples were then inoculated onto NBRIP plates. Only strains that continued to grow on NBRIP plates and produced phosphate-solubilizing zones after incubation were considered phosphate-solubilizing bacteria. These strains were then inoculated onto fresh NBRIP solid medium plates, and purification was repeated for at least three generations until a pure strain was isolated. The purified strains were classified, labeled, and cryopreserved in glycerol tubes at -80°C for later use. A rhizosphere bacterium, labeled 05217, was obtained through the above procedures.

[0053] 2. Identification of phosphate-solubilizing microorganisms

[0054] Single colonies of purified rhizosphere bacteria 05217 were picked and inoculated into 5 mL of LB broth. The cultures were incubated at 28°C for 24 h to obtain 05217 bacterial suspension. DNA from 05217 was extracted using a bacterial DNA extraction kit. Using universal primers for bacterial 16S rRNA amplification and 05217 DNA as a template, PCR amplification was performed using 2×PCR Mix. PCR amplification conditions are shown in Table 1. The PCR product of 05217 was obtained. The PCR product was detected by 1% agarose gel electrophoresis, purified by gel extraction, and then subjected to first-generation sequencing to obtain the nucleic acid sequence of the 05217 PCR product, which is shown in SEQ ID NO.1. The universal primers for bacterial 16S rRNA amplification were 27F (5'-AGAGTTTGATCCT-GGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The nucleic acid sequences obtained from sequencing were aligned in the EzBioCloud database (https: / / www.ezbiocloud.net / identify), and a phylogenetic tree was constructed using the Neighbor-joining method (see...). Figure 1 Based on the comparison results and phylogenetic tree results, 05217 was identified as Ochrobactrum teleogrylli.

[0055] Ochrobactrum teleogrylli 05217 is deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 6, 2024, with accession number GDMCC No: 64715. The address of the depository is Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

[0056] Table 1 PCR amplification conditions

[0057]

[0058] Example 2

[0059] This embodiment provides a phosphorus-solubilizing and aluminum-tolerant microorganism, *Talaromyces fuscoviridis* 20762, which was isolated from soil. The specific steps are similar to those in Example 1, with the following differences:

[0060] The NBRIP medium with 30 mg / L actinomycete ketone added used in the isolation (step 1) was replaced with NBRIP medium with 30 mg / L streptomycin sulfate and 70 mg / L Bengal red added to screen for phosphate-solubilizing fungi. The screened phosphate-solubilizing fungi were numbered 20762.

[0061] During identification (step 2), the universal primers for bacterial 16S rRNA amplification were used instead of the universal primers for fungal ITS. The nucleic acid sequence of the resulting 20762 PCR product is shown in SEQ ID NO.2, and the constructed phylogenetic tree is shown in [link to phylogenetic tree]. Figure 2 Based on the comparison results and phylogenetic tree results, 20762 was identified as *Talaromyces fuscovi ridis*. The universal primers for fungal ITS are ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3').

[0062] Talamyces fuscoviridis 20762 is deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 6, 2024, with accession number GDMCC No: 64713. The address of the depository is Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

[0063] Example 3

[0064] This embodiment provides a synthetic microbial community, which includes Ochrobactrumteleogrylli 05217 obtained in Example 1 and Talaromyces fuscoviridis 20762 obtained in Example 2.

[0065] The method for preparing the synthetic microbial community includes the following steps:

[0066] (1) Ochrobactrum teleogrylli 05217 was inoculated into LB liquid medium and cultured at 30℃ for 24 h. The culture was then transferred to centrifuge tubes and centrifuged at 4℃ and 8000 rpm for 10 min. The supernatant was removed, and sterile water was added to prepare a bacterial suspension. The concentration of the bacterial suspension was controlled at 10.7 CFU / mL, for later use;

[0067] (2) Inoculate *Talaromyces fuscoviridis* 20762 into PDA liquid medium and incubate at 30℃ for 3-5 days. Transfer to centrifuge tubes and centrifuge at 8000 rpm for 10 min at 4℃. Remove the supernatant and add sterile water to prepare a bacterial suspension, controlling the spore concentration to 10. 7 CFU / mL, for later use;

[0068] (3) Mix the Ochrobactrum teleogrylli 05217 bacterial suspension obtained in step (1) and the Talaromyces fuscoviridis 20762 bacterial suspension obtained in step (2) at a bacterial concentration ratio of 1:1 to obtain a synthetic bacterial group.

[0069] Example of effect 1

[0070] To verify the compatibility and phosphorus solubility / aluminum tolerance of Ochrobactrum teleogrylli 05217 obtained in Example 1 and Talaromyces fuscoviridis 20762 obtained in Example 2, the compatibility, soluble phosphorus content, and aluminum tolerance of the above strains were measured. The specific procedures are as follows:

[0071] 1. Compatibility testing

[0072] Activated *Talaromyces fuscoviridis* 20762 was used to create 6mm mycelial cakes using a sterile punch. The mycelial cakes were placed in the center of NBRIP medium. *Ochrobactrum teleogrylli* 05217 was streaked around the mycelial cakes at a distance of 2cm from the center using a sterile bamboo skewer. The inoculated medium was then incubated at 28℃ for 7 days, and the presence of inhibition zones was observed. The presence of inhibition zones indicated antagonism between the strains; the absence of inhibition zones indicated compatibility between the strains. Results are shown below. Figure 3 .

[0073] like Figure 3 As shown, Ochrobactrum teleogrylli 05217 and Talaromyces fuscoviridis20762 are compatible and can be combined to form a synthetic microbial community.

[0074] 2. Determination of soluble phosphorus content

[0075] Ochrobactrum teleogrylli 05217 was inoculated onto NA agar plates and incubated at 28°C for 24 hours. A small amount of the bacterial suspension was picked and inoculated into 2 mL of sterile water, shaken well, and the bacterial suspension was tested at 10 μL. 8 CFU / mL (controlling the initial concentration), take 0.1 mL of bacterial suspension and inoculate it into 9.9 mL of PKO inorganic phosphorus medium. Use 0.1 mL of sterile water added to the medium as a control. Each treatment is repeated three times and cultured at 28℃ and 160 rpm for 5 days. Then, centrifuge the culture medium at 4℃ and 4000 rpm for 15 min, collect the supernatant, and determine the soluble phosphorus content using the molybdenum antimony colorimetric method.

[0076] Talaromyces fuscoviridis 20762 was inoculated onto PDA medium and incubated upside down at 30°C for 7 days. Spores were then scraped from the medium into an Erlenmeyer flask using 10 mL of sterile water and filtered through 9 cm filter paper to obtain 10... 6 A bacterial suspension (i.e., spore suspension) of CFU / mL was prepared. 0.1 mL of the suspension was inoculated into 9.9 mL of PKO inorganic phosphorus medium, with 0.1 mL of sterile water added to the medium as a control. Each treatment was repeated three times, and cultured at 28°C and 160 rpm for 5 days. The culture was then centrifuged at 4°C and 4000 rpm for 15 min, and the supernatant was collected. The soluble phosphorus content was determined using the molybdenum antimony colorimetric method.

[0077] The results of the above determination of soluble phosphorus content are shown in Table 2.

[0078] Table 2. Results of soluble phosphorus content determination for different strains

[0079] Group Phosphate solubility value (mg / L) CK (bacteria) 1.10±0.00 05217 351.60±8.05 CK (Fungi) 3.86±1.41 20762 278.47±2.97

[0080] As shown in Table 2, both 05217 and 20762 have good phosphorus-dissolving properties.

[0081] 3. Determination of aluminum tolerance

[0082] Adjust the pH of LB medium to 7.8, add AlCl3·6H2O, and prepare Al... 3+ LB medium with final concentrations of 0, 0.1, 0.3, 0.5, 1.0, and 5.0 mM was prepared. The pH of the LB medium was adjusted to 4.0 with HCl, and the medium was aseptically filtered before use. Activated 05217 was inoculated into 5 mL of LB medium and cultured for 12-16 h to prepare 10... 8 A bacterial suspension of CFU / mL was used to inoculate 0.5 mL of the suspension onto 3.5 mL of solution containing different Al values. 3+ In LB medium at a concentration of [concentration], the cells were incubated at 30°C for 120 h, with OD600 measured every 12 h. Each treatment was repeated in triplicate. Results are shown below. Figure 4To avoid aluminum precipitation, the pH of the LB medium was pre-adjusted to 7.8 with KOH before adding AlCl3·6H2O, and the final pH was adjusted to 4.0 with HCl after adding AlCl3·6H2O.

[0083] Adjust the pH of the PDB medium to 7.8, add AlCl3·6H2O, and prepare Al... 3+ PDB medium with final concentrations of 0, 0.1, 0.3, 0.5, 1.0, and 5.0 mM was prepared for use. 6 mm mycelial discs were prepared in activated 20762 PDB plates, and the discs were inoculated onto plates containing different Al concentrations. 3+ The cells were cultured in PDB medium at a concentration of 1000 ppm at 28°C for 5 days. After centrifugation, the cells were washed with deionized water, dried at 80±1°C for 24 hours, and weighed to determine biomass. Each treatment was replicated in triplicate. Results are shown below. Figure 4 .

[0084] like Figure 4 As shown, both 05217 and 20762 have good aluminum resistance.

[0085] Example 2

[0086] To verify the effect of phosphorus-solubilizing aluminum-tolerant microorganisms on aluminum-stressed Chinese cabbage, aluminum stress was applied to the Chinese cabbage using Ochrobactrum teleogrylli 05217 obtained in Example 1 and Talaromyces fuscoviridis 20762 obtained in Example 2. The specific scheme is as follows:

[0087] 1. Preparation of bacterial culture

[0088] The Ochrobactrum teleogrylli 05217 obtained in Example 1 was inoculated into LB medium and cultured at 30°C for 24 h. After centrifugation at 8000 rpm for 10 min, the supernatant was removed, and the bacterial cells were resuspended in sterile water to prepare 10... 7 05217 bacterial suspension at CFU / mL.

[0089] The *Talaromyces fuscoviridis* 20762 obtained in Example 2 was inoculated into PDB medium and cultured at 30°C for 3–5 days. After centrifugation at 8000 rpm for 10 min, the supernatant was removed, and the cells were resuspended in sterile water to prepare 10... 7 20762 spore suspension at CFU / mL.

[0090] AlCl3·6H2O was mixed with water to prepare a solution for later use.

[0091] 2. Aluminum stress treatment of Futian Chinese cabbage

[0092] Gently pull the seedlings (3-4 leaves) from the substrate, wiping them thoroughly several times to remove any remaining substrate. After cleaning, transfer the seedlings to a plastic box with small holes in the lid. Soak the seedlings in a 1 / 4 low-phosphorus Hoagland solution for 2-3 days to allow them to recover, then transfer them to a 1 / 2 low-phosphorus Hoagland solution for another 2-3 days. Finally, switch to the normal concentration nutrient solution for the experimental treatment. Simultaneously, treat the seedlings with root drenching for fungal inoculation and aluminum stress (0, 0.1, 0.5 mM), with six replicates for each treatment. Each root drenching for fungal inoculation involved 10 mL of solution. Two to three weeks after planting, measure the fresh weight and root weight of the plants using an electronic balance. Scan the root system morphology and count the total root length using a root scanner. Change the nutrient solution every 3 days, maintaining a pH of 4. Results are shown in Tables 4-5.

[0093] Table 3 Aluminum stress treatment of Futian Chinese cabbage

[0094]

[0095] Table 4 shows the results at 0.1 mM Al. 3+ Effects of different treatments under stress on physiological indicators of Chinese cabbage

[0096]

[0097]

[0098] Table 5 shows the results at 0.5 mM Al. 3+ Effects of different treatments under stress on physiological indicators of Chinese cabbage

[0099]

[0100] As shown in Tables 4-5, compared with the CK1 / CK2 treatment groups, B1 / F1 / B2 / F2 all significantly improved various physiological indicators of Chinese cabbage, indicating that the phosphorus-solubilizing and aluminum-tolerant microorganisms Ochrobactrum teleogrylli05217 and Talaromycesfuscoviridis 20762 of the present invention can improve the phosphorus-solubilizing and aluminum-tolerant properties of Chinese cabbage. 3+ The phenomenon of poor growth of Chinese cabbage caused by stress showed that the plant height, root weight and total root length of B1 and F1 were significantly different from those of CK1, but the fresh weight was not significantly different because the fresh weight was measured as the fresh weight of the aboveground parts.

[0101] Example 3

[0102] To verify the effect of the synthetic microbial community on Chinese cabbage, aluminum stress was applied to the Chinese cabbage, and Ochrobactrum teleogrylli 05217 obtained in Example 1 and Talaromyces fuscoviridis 20762 obtained in Example 2 were used. The specific scheme is as follows:

[0103] Prepare 05217 bacterial suspension and 20762 spore suspension according to step 1 in Example 2 for later use.

[0104] The root irrigation method was used to apply 50 mL of 05217 bacterial suspension (RB1), 20762 spore suspension (RF1), or synthetic bacterial group (RB1+RF1) to the transplanted potted Chinese cabbage. The root irrigation was carried out once a week for 3 consecutive weeks. On the 7th day after transplanting, nitrogen fertilizer (90 mg / kg urea) and potassium fertilizer (67.5 mg / kg potassium chloride) were applied. On the 14th day after transplanting, nitrogen fertilizer (70 mg / kg urea) and potassium fertilizer (52.5 mg / kg potassium chloride) were applied. Sterile water was used as the control group (RCK). Each treatment was replicated 6 times.

[0105] The heart of the seedlings was collected on day 35 after transplanting, and the following indicators were measured:

[0106] (1) Plant height and root length were measured using a graduated ruler. Fresh weight of the above-ground parts and root weight were measured using an electronic balance. Root morphology, total root length, average root diameter, root surface area, and root volume were scanned using a root scanner. The results are shown in Table 6 and... Figure 5 .

[0107] Table 6. Effects of different treatments on the growth of Chinese cabbage.

[0108]

[0109] As shown in Table 6 and Figure 5 As shown, compared with RCK, RB1, RF1 and RB1+RF1 showed significantly increased plant height, fresh weight, total root length, root surface area and root volume, and the fresh weight of RB1+RF1 was significantly higher than that of other treatment groups, indicating that the phosphorus-solubilizing aluminum-tolerant microorganisms and synthetic flora of the present invention have excellent effects on promoting the growth of Futian Chinese cabbage.

[0110] (2) The total phosphorus content of the aboveground and underground plant tissues was determined by H2SO4-H2O2 digestion and vanadium molybdenum yellow colorimetric method. The results are shown in Table 6.

[0111] As shown in Table 6, compared with RCK, the phosphorus accumulation of RB1, RF1 and RB1+RF1 was significantly increased, with RB1+RF1 having the highest phosphorus accumulation, indicating that both phosphorus-solubilizing and aluminum-tolerant microorganisms and synthetic flora have the effect of increasing phosphorus accumulation in Fukuda cabbage.

[0112] (3) Soil pH was determined by potentiometric method with soil-liquid ratio of 1:2.5; soil organic matter (SOM) was determined by potassium dichromate-ferrous sulfate titration method; available phosphorus (AP) was determined by sodium bicarbonate extraction-molybdenum antimony colorimetric method; available nitrogen (AN) was determined by sodium hydroxide (NaOH) diffusion method; available potassium (AK) was determined by ammonium acetate extraction-flame photometry method. The results are shown in Table 7.

[0113] Table 7. Results of soil physicochemical properties determination for different treatment groups of Chinese cabbage.

[0114] project RCK RB1 RF1 RB1+RF1 pH 4.79±0.06b 4.88±0.02ab 5.05±0.06a 4.96±0.06ab organic matter g / kg 6.12±0.4bc 5.49±0.25c 7.66±0.19a 7.09±0.43ab Alkaline nitrogen mg / kg 58.48±1.32a 55.15±1.32a 62.12±3.41a 59.55±0.45a Available phosphorus mg / kg 26.82±0.67c 36.06±2.16b 33.13±2.8bc 54.38±0.61a Available potassium mg / kg 79.67±1.48c 87.17±1.48c 100±3.04b 149±3.69a

[0115] As shown in Table 7, the available phosphorus content in the RB1+RF1 soil was significantly higher than that in other treatment groups, indicating that the synthetic microbial community of the present invention can more effectively increase the available phosphorus content in the soil and promote the phosphorus-aluminum conversion in the soil.

[0116] (4) The Hedley continuous extraction method was used to determine the phosphorus speciation in the soil. 0.5 g of 100-mesh soil sample was placed in a 50 mL centrifuge tube. Seven phosphorus speciations (Water-Pi, NaHCO3-Pi, NaHCO3-Po, NaOH-Pi, NaOH-Po, HCl-Pi, and Residual-P) were extracted sequentially by centrifugation with deionized water, 0.5 mol / L NaHCO3 solution, 0.1 mol / L NaOH solution, and 0.5 mol / L HCl solution. The phosphorus concentration in the extract was determined using a molybdenum antimony reagent and a UV-Vis spectrophotometer. The phosphorus content of each speciation in the rhizosphere soil was calculated using a pre-constructed standard curve. The contents of NaHCO3-Po and NaOH-Po were obtained by subtracting the difference between the total phosphorus content and the corresponding inorganic phosphorus content. The results are shown in [Figure number missing]. Figure 6 .

[0117] like Figure 6 As shown, the contents of Water-Pi and NaHCO3-Pi of single bacterial strain RB1 and single fungal strain RF1 were higher than those of the control group, but the differences were not statistically significant. The contents of NaHCO3-Po of RB1 and RF1 in the treatment group were significantly higher than those of the control group. The contents of available phosphorus Water-Pi, NaHCO3-Po, and NaHCO3-Pi of the synthetic microbial community RB1+RF1 were all significantly higher than those of the control group, increasing by 82.4%, 127%, and 42.1%, respectively, compared with the control group. The contents of NaOH-Po and HCl-Pi of single bacterial and single fungal treatment groups were lower than those of the control group. The contents of slow-release phosphorus NaOH-Pi and HCl-Pi of the synthetic microbial community RB1+RF1 were significantly lower than those of the control group, and its NaOH-Po was lower than that of the control group, but not to a statistically significant level. The contents of Residual-P of RB1 in the bacterial treatment group were lower than those of the control group, but there was no statistically significant difference compared with the control group. Therefore, the results show that the bacterial treatment promoted the conversion of slow-release phosphorus and residual phosphorus into available phosphorus, with the synthetic microbial community showing the most significant effect.

[0118] (5) Determination of aluminum speciation: 3g (<2mm) of soil sample was added to a 50mL centrifuge tube, followed by 30mL of 1mol / L KCl (exchangeable form), shaking for 30min, and centrifugation. Then, 0.1mol / L CuCl2 + 0.5mol / L KCl (organically bound form) was added, shaking for 1h, and centrifugation was performed. Next, 1mol / L NH4OAc (pH 4.0) (adsorbed hydroxyl form) was added, shaking for 8h. After each extraction step, the extract was centrifuged. If necessary, it was filtered through Whatman No. 1 filter paper to remove floating debris. The Al content was analyzed by ICP-OES. The extracted Al fractions were exchangeable Al, organically bound Al, and adsorbed Al, respectively. The results are shown in […]. Figure 7 .

[0119] like Figure 7 As shown, compared with single-strain treatment, the trans-kingdom synthetic microbial community RB1+RF1 can effectively inhibit aluminum activation, and its reduced exchangeable Al (Ex-Al) may be converted into plant-free organically bound Al (Or-Al).

[0120] (6) Genomic DNA was extracted from rhizosphere soil using the Fast DNA SPIN Kit for Soil. Samples were homogenized using magnetic beads and then purified using the Universal DNA Purification Kit to remove humic substances and potential enzyme inhibitors. DNA concentration was determined using a Nano Drop ND-2000 spectrophotometer (Thermo Fisher Scientific, MA, USA), and all extracted DNA samples were stored at -80°C. The absolute abundance of phosphorus and aluminum-related functional genes in each soil was determined using qPCR with iTaq Universal SYBR Green Ultramix (Bio-Rad Laboratories, Hercules, CA, USA). Standard curves were generated using different gradient dilutions of recombinant plasmids containing known concentrations of target gene fragments. Quantitative PCR was performed using an ABI VIIA 7-cycle real-time PCR system (Applied Biosystems, Foster City, CA, USA) in a 10 μL reaction system with three repeats. PCR was performed at 95°C for 5 min, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. Phosphorus conversion-related functional genes gcd (Quinoprotein glucose dehydrogenase), phoC (Acid phosphatase), phoD (Alkaline phosphatase), and pqqC (pyrroloquinoline-quinone synthase) were determined; the aluminum tolerance functional gene queC (Queuosine biosynthesis protein) was determined. Primer sequences for phosphorus-solubilizing and aluminum-tolerant gene amplification are shown in Table 8. Results are detailed below. Figure 8-9 .

[0121] Table 8 Primer sequence information for phosphorus-solubilizing and aluminum-resistant gene amplification.

[0122]

[0123]

[0124] like Figure 8-9 As shown, except for phoD, the expression levels of the phosphorus-solubilizing gene and aluminum-tolerant gene in RB1+RF1 were significantly higher than those in other treatment groups, indicating that the synthetic microbial community of the present invention has a superior phosphorus-aluminum conversion function than phosphorus-solubilizing and aluminum-tolerant microorganisms, and can further promote the phosphorus-aluminum conversion in the soil to promote crop growth.

[0125] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A phosphorus-soluble, aluminum-tolerant microorganism, characterized in that, The microorganism is Ochrobactrum teleogrylli 05217 or Talaromyces fuscoviridis 20762; The microorganisms Ochrobactrum teleogrylli Accession number 05217 is deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 6, 2024, with accession number GDMCC No: 64715. The microorganisms Talaromyces fuscoviridis Accession number 20762 is deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 6, 2024, with accession number GDMCC No: 64713.

2. A synthetic microbial community, characterized in that, The synthetic microbial community includes strains Ochrobactrum teleogrylli 05217 and strain Talaromyces fuscoviridis 20762; The strain Ochrobactrum teleogrylli Accession number 05217 is deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 6, 2024, with accession number GDMCC No: 64715. The strain Talaromyces fuscoviridis Accession number 20762 is deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 6, 2024, with accession number GDMCC No: 64713; The Ochrobactrum teleogrylli 05217 and Talaromyces fuscoviridis The concentration ratio of 20762 is Ochrobactrum teleogrylli 05217: Talaromyces fuscoviridis 20762 = 1:

1.

3. A microbial inoculant, characterized in that, The microbial agent includes the phosphorus-solubilizing and aluminum-resistant microorganisms of claim 1 or the synthetic microbial community of claim 2.

4. The microbial agent as described in claim 3, characterized in that, The strains in the microbial agent Ochrobactrum teleogrylli The bacterial concentration of 05217 was 10. 7~8 CFU / mL; The strains in the microbial agent Talaromyces fuscoviridis The spore concentration of 20762 is 10. 6~7 CFU / mL.

5. The application of the phosphorus-solubilizing and aluminum-resistant microorganisms as described in claim 1, the synthetic microbial community as described in claim 2, or the microbial agent as described in claim 3 in promoting the growth of Chinese cabbage.

6. The application of the phosphorus-solubilizing and aluminum-resistant microorganisms as described in claim 1, the synthetic microbial community as described in claim 2, or the microbial agent as described in claim 3 in promoting the phosphorus-aluminum conversion in the rhizosphere of Chinese cabbage.

7. A method for promoting the growth of Chinese cabbage, characterized in that, The process includes the following steps: applying the phosphorus-solubilizing and aluminum-resistant microorganisms of claim 1, the synthetic microbial community of claim 2, or the microbial agent of claim 3 to the roots of crops, wherein the concentration of the phosphorus-solubilizing and aluminum-resistant microorganisms, the synthetic microbial community, or the compound microbial agent is 10. 6 ~10 8 CFU / mL.

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