Root fungi having efficient phosphorus solubilizing and siderophore producing capabilities and uses thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a suaeda salsa rhizosphere fungus, namely Aspergillus tubingensis RS-31, and a function of the Aspergillus tubingensis RS-31. The invention discloses an Aspergillus tubingensis RS-31, and the preservation number of the Aspergillus tubingensis RS-31 is CGMCC (China General Microbiological Culture Collection Center) No. 41724. The invention also provides application of the Aspergillus tubingensis RS-31. The Aspergillus tubingensis RS-31 has the capability of efficiently dissolving insoluble phosphorus and efficiently generating siderophores, so that the Aspergillus tubingensis RS-31 can be used for improving soil and promoting plant growth.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a Suaeda salsa rhizosphere fungus Aspergillus tubingensis RS-31 strain and its function. Background Art

[0002] The long evolutionary process has established an inseparable connection between plants and microorganisms. In order to adapt to the complex living environment, plants and microorganisms form a symbiotic system, such as the mutualistic symbiosis between rhizobia and legumes. In the symbiotic relationship, plants and microorganisms are interdependent and influence each other, forming a highly coordinated and balanced system. Plants provide matrix and signal molecules for microbial growth, and can regulate microbial communities through secretions to promote their own adaptation to different environments; microorganisms can promote plant nutrient absorption and growth and development, and improve the ability of plants to resist external stress. As the transitional area between plants and external soil, the root system is an important place for microbial colonization and activity, and is also the most active area for plant-microorganism interactions. Due to its relatively high species diversity and functional diversity, root microorganisms, as potential biocontrol resources and microbial agent resources, have important application potential in agricultural and forestry production and other fields.

[0003] Iron is an important element required for the life activities of organisms. It plays an important role in life processes such as biochemical metabolic reactions and electron transfer. Although the iron content in soil is rich, it is mainly in the form of trivalent iron ions (Fe 3+ ) exists in the form of iron, and the solubility of its oxides or hydroxides is extremely low, which greatly reduces the biological effectiveness of iron. Previous studies have found that soil microorganisms can chelate iron in nature or host cells by synthesizing and secreting small molecule compounds with high affinity for trivalent iron ions. Iron carrier microorganisms are closely related to the sustainable development of agriculture as a special and valuable resource. The use of iron carriers secreted by microorganisms is expected to become an important measure to develop new iron supplements or additives and improve the utilization rate of iron. However, the existing microbial resources that secrete iron carriers are still very limited, and a large amount of resources need to be further explored. In addition, existing studies have focused on bacteria that secrete iron carriers, such as Acinetobacter baumannii and Bacillus. There is still a lack of sufficient research on fungi that secrete iron carriers.

[0004] Phosphorus is also one of the indispensable components of plant growth. In recent years, in pursuit of higher economic benefits, humans have applied excessive amounts of phosphorus fertilizer to the soil, resulting in a serious imbalance of soil nutrients. However, after soluble phosphorus fertilizer is applied to the soil, only part of it can be absorbed and utilized by plants, and the rest reacts with soil components and is removed from the soil phase, fixed in the soil in the form of insoluble phosphates (such as calcium phosphate or iron phosphate) that are difficult for plants to use. Therefore, although the phosphorus content in the soil is high, the effective phosphorus content available to plants is very low. Existing studies have shown that there are abundant phosphate-dissolving bacteria in the soil, which can not only improve plant phosphorus nutrition, but also promote the metabolic activities of beneficial microorganisms in the soil and regulate soil phosphorus utilization. Similarly, the phosphate-dissolving bacteria reported are mostly bacteria, and fungi are less. However, phosphate-dissolving bacteria are prone to lose their ability to dissolve phosphorus after multiple subcultures, and they cannot be restored after loss. In contrast, phosphate-dissolving fungi are relatively genetically stable and generally not easy to lose their ability to dissolve phosphorus. Therefore, it is extremely important to explore more fungal strain resources with phosphate-dissolving functions.

[0005] Since soil is an extremely complex ecological system, many soils may be deficient in both iron and phosphorus. Most of the fungal strains involved in existing research or inventions only have the function of dissolving phosphorus or producing siderophores. Therefore, it is more worthy of attention to explore and utilize fungal resources that have the functions of secreting siderophores and dissolving phosphorus.

[0006] The invention of CN119120209A, "Aspergillus tubingensis strain and its application", discloses Aspergillus tubingensis TL-B31f, with a deposit number of CCTCC M 20241814. The Aspergillus tubingensis strain has the ability to efficiently dissolve phosphate, produce siderophores and 3-indoleacetic acid (IAA), and can be used as a growth promoter in non-grain cultivated land, improve soil matrix components, make non-grain cultivated land suitable for rice planting, promote rice growth, and thus help increase grain production.

[0007] The invention of CN117603886B "A salt-alkali tolerant rhizosphere growth-promoting agent, its preparation method and application" informs: the Aspergillus tubingensis RGPF-1 is classified and named as Aspergillus tubingensis, with a preservation number of CGMCC No. 40731. The Aspergillus tubingensis RGPF-1 has the characteristics of nitrogen fixation, phosphorus solubilization and pH lowering. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a root fungus---Aspergillus tubingensis RS-31 and its application.

[0009] In order to solve the above technical problems, the present invention provides Aspergillus tubingensis RS-31, whose deposit number is CGMCC No 41724.

[0010] The present invention also provides the use of the Aspergillus tubingensis RS-31: it has the ability to efficiently dissolve insoluble phosphorus and efficiently produce iron carriers, so it can be used for soil improvement and promoting plant growth.

[0011] The Aspergillus tubingensis RS-31 of the present invention is derived from the root soil fungi of Suaeda salsa, and has the functions of dissolving phosphate and secreting siderophores at the same time, and it is found that its siderophore production ability is very strong, and its siderophore activity index is as high as 89.2. Therefore, the strain has extremely high application potential.

[0012] Note: Soil improvement is manifested in that it can promote the dissolution of insoluble phosphorus, thereby increasing the nutrient content of the soil; promoting plant growth is manifested in that it can assist plants in absorbing nutrients through the secretion of iron carriers and improve the plants' resistance to stress, thereby achieving the effect of promoting plant growth. This strain has a promoting effect on the growth and nutrient absorption of trees. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0014] Figure 1 The results show the culture characteristics of strain Aspergillus tubingensis RS-31 in conventional PDA medium.

[0015] Figure 2 The phosphate solubilization characteristics of strain Aspergillus tubingensis RS-31; when the strain is cultured on the detection medium, a transparent phosphate solubilization hydrolysis zone will be produced, where (A) is the front side of the plate culture, and (B) is the back side of the plate culture.

[0016] Figure 3 Evidence for the siderophore-producing trait of Aspergillus tubingensis RS-31 and nine other strains. The siderophores secreted by the strains are released and take away iron from the culture medium, thus producing a purple-red halo around the colonies. DETAILED DESCRIPTION

[0017] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0018] As a general rule, the culture media mentioned below need to be sterilized with high pressure steam at 121°C for 20 minutes before use.

[0019] Example 1. Isolation and culture of strain RS-31

[0020] 1. Preparation of isolation and culture medium

[0021] Czapek screening medium: 20 g glucose, 20 g sea salt, 3 g NaNO3, 1 g K2HPO4, 0.5 g MgSO4·H2O, 0.5 g KCl, 0.01 g FeSO4·7H2O, dissolved in appropriate amount of double distilled water, add double distilled water to 1 L, adjust the pH to 5.6, add 15 g agar; sterilize at 121°C high pressure steam for 20 min; cool the culture medium to about 45°C, add streptomycin sulfate and tetracycline hydrochloride with final concentrations of 0.05 g / L and 0.02 g / L to inhibit bacterial contamination.

[0022] PDA culture medium: Take 200g of peeled potatoes, cut into small pieces, add double-distilled water to boil, filter with four layers of gauze, add 20g of glucose, add double-distilled water to make the volume 1L, and finally add 15g of agar; sterilize with high-pressure steam at 121℃ for 20min; this is PDA culture medium.

[0023] 2. The present invention relates to the isolation and cultivation of RS-31 strain

[0024] The material Suaeda salsa used in the present invention was collected from the coastal tidal flats of Dongying City, Shandong Province. The soil outside the roots of the Suaeda salsa root system was carefully shaken off, and the old roots were removed with sterile scissors. About 3g of the tenderer roots were picked and placed in a 50ml centrifuge tube filled with 40ml 1xPBS buffer, and the root soil was fully shaken to suspend the rhizosphere soil. The root sample was taken out, and the root soil suspension was centrifuged at 6000g for 15min at 10°C, the supernatant was poured out, and the root soil was collected. After sufficient collection, 1g of the root soil was taken in 99ml 1xPBS buffer, and resuspended and mixed. Diluted in sequence according to the volume ratio of 1:10 to obtain 10 respectively. -3 , 10 -4 , 10 -5 Three concentrations. Select 10 -4 and 10 -5 Mix the two concentrations thoroughly and take 100 μl of each, then apply it on the surface of Czapek screening medium. Cultivate at 25℃ in the dark for 7 days, observe in time, and when the culture medium shows mycelium growth, transfer to PDA medium for enrichment and purification until pure colony RS-31 is obtained. When RS-31 is cultured on PDA medium, the colony is as follows Figure 1 shown.

[0025] Example 2, Molecular Biological Identification of Strain RS-31

[0026] 1. DNA extraction, PCR amplification and Sanger sequencing

[0027] Pick about 100 mg of RS-31 mycelium into a 1.5 ml sterile centrifuge tube, add an appropriate amount of steel beads to the centrifuge tube, freeze it in liquid nitrogen, and use a sample grinder to fully break the mycelium. Then use the modified CTAB method to extract the total fungal DNA, and measure the concentration and purity of the DNA. Using DNA as a template, ITS, LUS, SSU, TEF1 and TUB2 fragments were amplified respectively. The primer information is shown in Table 1. The amplification system is 50 μl: template DNA, 2 μl; forward and reverse primers, 0.5 μl each; Taq enzyme, 0.3 μl; dNTP, 4 μl; 10xPCR buffer, 5 μl; sterile ultrapure water, 37.7 μl. The PCR amplification program includes: 94℃ pre-denaturation, 4 min; 35 cycles, including 94℃ pre-denaturation for 40 s, 55℃ annealing for 50 s, 72℃ extension for 1 min; final extension for 10 min. The obtained PCR products were sequenced by Sanger sequencing.

[0028] Table 1. Genes and primers used in molecular biological identification of the present invention.

[0029]

[0030] 2. Sequence determination and species identification

[0031] After amplification and sequencing, the ITS sequence was obtained as shown in SEQ ID NO: 1; the LSU sequence was obtained as shown in SEQ ID NO: 2; the SSU sequence was obtained as shown in SEQ ID NO: 3; the TEF1 sequence was obtained as shown in SEQ ID NO: 4; and the TUB2 sequence was obtained as shown in SEQ ID NO: 5.

[0032] The sequences obtained by sequencing were compared with the existing sequences in the NCBI database, and it was found that the sequences obtained from the five genes were extremely similar to the corresponding sequences of Aspergillus tubingensis - the similarity reached 99% or 100%. Based on this, it can be confirmed that the isolated RS-31 strain is Aspergillus tubingensis. The deposit information of the strain RS-31 is as follows:

[0033] The deposited name is Aspergillus tubingensis, the depository unit is the General Microbiology Center of China Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCCNO.41724, and the deposit date is December 19, 2024.

[0034] Example 3: Phosphate-dissolving function of strain RS-31

[0035] 1. Preparation of strain RS-31

[0036] The strain RS-31 was cultured on PDA solid medium (temperature was 25°C) for 7 days, and a hole was punched from the edge of the colony with a borer for later use.

[0037] 2. Verification of the phosphate-dissolving function of strain RS-31

[0038] Prepare the Montgena inorganic phosphorus solid culture medium (the inorganic phosphorus component is calcium phosphate).

[0039] The formula of the Montgina inorganic phosphorus solid culture medium is as follows:

[0040] Dissolve 10g of glucose, 0.5g of (NH4)2SO4, 0.3g of NaCl, 0.3g of KCl, 0.03g of FeSO4·7H2O, 0.03g of MnSO4, and 0.3g of MgSO4·7H2O in an appropriate amount of double distilled water, make up to 1L with double distilled water, and finally add 10g of agar and 5g of Ca3(PO4)2. Sterilize with high-pressure steam at 121℃ for 20min.

[0041] Use a sterile puncher to take a 5mm diameter bacterial block and inoculate it on the Montkina inorganic phosphate plate medium, and culture it in a 25℃ incubator for 5 days. If the strain can secrete substances that dissolve calcium phosphate, the substances secreted by the strain will diffuse around the colony during the growth of the strain, thereby dissolving calcium phosphate and forming a transparent phosphate-dissolving hydrolysis zone. Figure 2 As shown, there is a clear transparent zone around the colony of RS-31 strain, indicating that the strain has the ability to dissolve insoluble phosphorus (inorganic phosphorus). That is, the diameter of the phosphorus dissolving hydrolysis zone after 5 days of cultivation is 2.39±0.34cm.

[0042] Example 4: Verification of the siderophore secretion function of strain RS-31

[0043] 1. Preparation of strain RS-31

[0044] The strain was cultured on PDA solid medium for 7 days (temperature was 25°C), and a hole was punched from the edge of the pure culture fungal colony plate with a borer (diameter 5 mm) for later use.

[0045] 2. Verification of the functional characteristics of siderophore production by strain RS-31

[0046] Siderophore refers to a kind of Fe-binding substance secreted by bacteria or fungi into the surrounding environment under iron-deficient conditions. 3+ A substance with extremely strong specific chelating effect.

[0047] The CAS plate method can be used to detect whether bacteria or fungi have the ability to secrete iron carriers. The detection principle is as follows: Chrome Azure S (CAS), trivalent iron ions and hexadecyltrimethylammonium bromide (HTDMA) can combine to form a sky-blue complex. When the culture plate is dyed with CAS dye, when the strain releases iron carriers into the culture plate and seizes iron, the aforementioned complex is destroyed and the color changes. That is, an orange-yellow or orange-red halo appears around the colony. By recording the presence or absence of the orange halo on the plate culture medium and measuring the diameter of the halo, the presence or absence of the strain's ability to secrete iron carriers and its strength can be determined.

[0048] The formula of the special solid culture medium (i.e., CAS staining medium) for detecting the ability of strains to secrete siderophore is as follows:

[0049] CAS powder 60.5mg, glucose 10g, MgSO4·7H2O 0.1g, acid hydrolyzed casein 10g, peptone 10g, add double distilled water to 1L, adjust pH to 6.8, and finally add 10g agar. Sterilize at 121℃ with high pressure steam for 20min.

[0050] After strain RS-31 was cultured on a CAS dye-stained plate for 3 days (culture temperature was 25°C), Figure 3 As shown in FIG. 1 , an obvious purple-red halo (diameter of the halo is 4.65±0.15 cm) appears around the RS-31 colony, indicating that the strain has a strong ability to produce siderophores. The present invention also measures the siderophore production function of similar strains screened in the invention process according to step 2 above, and the results are as follows: Figure 3 and Table 2 below. The results showed that the RS-31 strain had the strongest ability to produce siderophores.

[0051] Table 2. Siderophore diameter 3 days after inoculation

[0052] strain Iron carrier capacity / cm RS-31 4.65±0.15 RS-11 3.90±0.21 RS-12 3.58±0.22 RS-13 4.15±0.15 RS-17 3.86±0.16 RS-18 4.35±0.25 RS-42 4.00±0.19 RS-47 4.55±0.18 RS-53 4.39±0.14 RS-B17 3.63±0.19

[0053] Note: The above data are the average values ​​obtained from 4 parallel experiments.

[0054] The siderophore diameter of TL-B31f cultured at 25°C for 3 days reported in the background art is only about 2.44 cm, far lower than the 4.65 cm of RS-31 of the present invention. The siderophore halo around the colony of TL-B31f is light yellow in color.

[0055] 3. Quantitative detection of the ability of RS-31 strain to produce siderophore

[0056] The CAS method was also used to determine the iron carrier production. Five RS-31 bacterial blocks (5 mm in diameter) were placed in 100 mL of iron-limited MKB culture medium and cultured at 25°C and 150 r / min for 3 days. An equal volume of bacterial suspension was mixed with CAS detection solution and the absorbance was measured at 630 nm as the experimental group; the bacterial suspension was replaced with an equal volume of iron-limited MKB culture medium as the bacterial suspension as the blank control.

[0057] The size of the siderophore unit (SU) can characterize the ability of the strain to produce siderophores: SU (%) = (1-A s / A r )*100. r is the OD value of the uninoculated culture medium mixed with an equal volume of CAS detection solution, A s It is the OD value of the mixture of the strain culture solution and the equal volume of CAS detection solution. Based on the above method, it is calculated that the SU (%) of the strain RS-31 involved in the present invention is 89.2.

[0058] The formula of the iron-limited MKB culture medium for quantitative detection of the ability of strains to secrete iron carriers is as follows:

[0059] 5 g of casamino acid, 15 mL of glycerol, 2.5 g of K2HPO4, 0.2 g of MgSO4·7H2O, add double distilled water to make up to 1 L, and adjust the pH to 7.2.

[0060] The CAS detection solution was purchased from Beijing Coolbo Technology Co., Ltd., which is "10xCAS detection solution / siderophore detection solution (sterilized)" (product number: PM0821-2-100mL).

[0061] Finally, it should be noted that the above examples 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 a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. Aspergillus tubingensis RS-31, characterized by: The deposit number is CGMCC No.41724.

2. The use of Aspergillus tubingensis RS-31 as claimed in claim 1, characterized in that: It has the ability to dissolve insoluble phosphorus and produce iron carriers.

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