Aspergillus heteromorphus AJF-Z1 and application thereof

Abstract

The application belongs to the technical field of microbial inoculants, and particularly relates to a heteromorphic aspergillus AJF-Z1 and application thereof. The heteromorphic aspergillus is preserved in the China General Microbiological Culture Collection Center (CGMCC) on April 24, 2025, and the preservation number is CGMCC No. 41922. The heteromorphic aspergillus AJF-Z1 strain provided in the application has an obvious promoting effect on the growth and nodulation conditions of soybeans under the potting test conditions, thereby constructing an efficient and stable technology for regulating the symbiotic nodulation of host soybeans and rhizobium by an exogenous fungus, and regulating the root development, nodulation nitrogen fixation and stress resistance of soybeans from multiple dimensions.

Description

Technical Field

[0001] This invention belongs to the field of microbial inoculant technology, specifically relating to a strain of Aspergillus heterotrophus AJF-Z1 and its applications. Background Technology

[0002] Soybeans are an important food and feed crop, providing humans with high-quality oil and protein resources. With changes in living standards and dietary structure in my country, the demand for soybean feed from the livestock and poultry industry has been increasing year by year. However, my country's arable land resources are limited, and soybean yields are lower than those of staple crops. To ensure that total grain output does not decline, increasing yield per unit area is the fundamental way to develop my country's soybean industry, given the limited arable land area. However, if the nutrient supply problem in soybean production cannot be effectively solved, it will seriously hinder the development of my country's soybean industry.

[0003] Beneficial microorganisms have many functions, including helping plants acquire nutrients and improving their tolerance to various biotic and abiotic stresses. Beneficial microbial communities have become a key component in promoting soil health and sustainable agricultural development. Certain functional fungi can indirectly promote root development and nutrient absorption in host plants by secreting secondary metabolites (such as plant hormones and chitinases), activating plant immune responses, or improving the rhizosphere microenvironment. Soybean, a typical legume, can form a symbiotic nitrogen-fixing system with rhizobia in the soil. Soybean provides a carbon source for rhizobia, while the ammonia reduced by the rhizobia meets the nitrogen requirements for soybean growth and development.

[0004] However, the synergistic mechanism between fungi and rhizobia in existing technologies has not been systematically elucidated, especially in terms of optimizing the ratio of compound microbial agents, targeted screening of functional strains, and stability in field application. Most studies only focus on the effects of single microbial species, failing to fully utilize the interaction network among fungi, rhizobia, and plants. This results in the incomplete release of the symbiotic system's potential for promoting nodulation, stress resistance, and yield increase. Therefore, inoculating beneficial microorganisms into a symbiotic relationship with soybean rhizobia, and improving soybean nodulation through changes in rhizosphere microorganisms, will become one of the effective ways to increase soybean yield. Summary of the Invention

[0005] Based on this, the present invention isolated a strain of Aspergillus heterocaryoticus AJF-Z1, which can significantly improve the efficiency of synergistic nodulation between soybean and rhizobium and promote the growth of the host soybean.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions:

[0007] The present invention provides a strain of Aspergillus heterocaryoticus AJF-Z1, which was deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 24, 2025, with the accession number CGMCCNo.41922.

[0008] In another aspect, the present invention provides a microbial agent comprising the aforementioned Aspergillus heterotrophus AJF-Z1.

[0009] Preferably, the above-mentioned microbial agents include freeze-dried agents, solid agents, suspensions, or fermentation broths.

[0010] In another aspect, the present invention provides a bio-fertilizer comprising the above-mentioned Aspergillus heterotrophus AJF-Z1 or the above-mentioned microbial agent.

[0011] In another aspect, the present invention provides the application of the above-mentioned Aspergillus heteromorphus AJF-Z1 or the above-mentioned microbial agent or the above-mentioned bio-fertilizer in promoting soybean growth.

[0012] Preferably, in the above applications, the soybean variety is Zhonghuang 37 or Dongsheng 1.

[0013] Preferably, in the above applications, Aspergillus heterotrophus AJF-Z1, or the microbial inoculant, or the bio-fertilizer has one or more of the following functions:

[0014] (a) It has the function of increasing soybean plant height;

[0015] (b) It has the function of increasing the number of soybean pods;

[0016] (c) It has the function of increasing the dry weight of soybeans;

[0017] (d) It has the function of increasing the fresh weight of soybeans;

[0018] (e) It has the function of increasing the number of soybean root nodules.

[0019] In another aspect, the present invention provides a method for promoting soybean growth, the method comprising: applying the above-mentioned Aspergillus heterophylla AJF-Z1 or the above-mentioned microbial agent or the above-mentioned bio-fertilizer to the soybean roots by irrigation.

[0020] Preferably, in the above method, the soybean variety is Zhonghuang 37 or Dongsheng 1.

[0021] Preferably, in the above method, the method includes: applying the microbial agent to the soybean roots by irrigation, wherein the microbial agent is a suspension, and the amount of bacterial strain in the suspension is (0.8-1.2)×10⁻⁶. 7 CFU / mL, the volume-to-mass ratio of suspension to soil is 20mL:(1.5-2.5)kg.

[0022] The preservation information of Aspergillus sheterocaryoticus AJF-Z1 in this invention is as follows: Preservation institution: China General Microbiological Culture Collection Center (CGMCC); Preservation address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing; Preservation date: April 24, 2025; Preservation number: CGMCC No. 41922; Classification and name: Aspergillus sheterocaryoticus.

[0023] The beneficial effects of this invention include: the Aspergillus heterotrophus AJF-Z1 strain provided by this invention has a significant promoting effect on soybean growth and nodulation under pot experiment conditions, and regulates soybean root development, nodulation nitrogen fixation and stress resistance in multiple dimensions. Attached Figure Description

[0024] Figure 1 Phylogenetic analysis of ITS rRNA in Aspergillus heteromorphus AJF-Z1

[0025] Figure 2 Here is a colony morphology diagram of Aspergillus heteromorphus AJF-Z1:

[0026] Figure 3 The results of the assay of Aspergillus heteromorphus AJF-Z1 on the nodulation function of Dongsheng No. 1;

[0027] Figure 4 This study describes the effect of Aspergillus heteromorphus AJF-Z1 on the nodulation function of Zhonghuang 37. Detailed Implementation

[0028] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0029] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0030] In a first aspect, the present invention provides a strain of Aspergillus heterocaryoticus AJF-Z1, which was deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 24, 2025, with the accession number CGMCC No. 41922.

[0031] It should be noted that the Aspergillus heterophylla AJF-Z1 strain provided by this invention has a significant promoting effect on soybean growth and nodulation under pot experiment conditions, by regulating soybean root development, nodulation nitrogen fixation, and stress resistance in multiple dimensions. Furthermore, sequencing revealed that the nucleotide sequence of the ITS of this Aspergillus heterophylla AJF-Z1 strain is shown in SEQ NO: 1.

[0032] Secondly, embodiments of the present invention provide a microbial agent, which includes the aforementioned Aspergillus heterotrophus AJF-Z1.

[0033] It should be noted that the Aspergillus heterotrophus AJF-Z1 in this invention can be processed to obtain different microbial inoculants, such as freeze-dried inoculants, solid inoculants, suspensions, or fermentation broths. The preparation methods for different microbial inoculants are well known in the art, and those skilled in the art can choose according to different application requirements.

[0034] In some specific examples, the aforementioned microbial agents can be solid agents. Solid agents are prepared by loading Aspergillus heterotrophus AJF-Z1 onto a carrier. The carrier is a key medium for immobilizing the microorganisms and maintaining their activity; it must possess high adsorption capacity, air permeability, and chemical stability, which are well-known in the art. Examples include natural organic carriers (corn cob, wheat bran, sawdust, peat moss, vermiculite, etc., providing the carbon source and porous structure required for microbial growth), inorganic or composite carriers (ultra-polymer materials (used for slow-release antimicrobial agents), corn flour and soybean mixtures, enhancing mechanical strength or extending the lifespan of the microbial agent), or functionalized carriers (adding wheat koji (containing natural enzymes), marigold extract (for synergistic degradation of pollutants), improving the overall efficacy of the microbial agent), etc.

[0035] In some specific examples, the aforementioned microbial agents can be suspensions. A bacterial suspension refers to a homogeneous suspension formed by dispersing microbial cells (live or inactivated bacteria) in a sterile liquid (such as sterile water, physiological saline, buffer solution, or culture medium) through physical or chemical methods.

[0036] In some specific examples, the aforementioned microbial inoculant can be a fermentation broth. Fermentation broth refers to a mixed liquid containing microbial cells, metabolites, and culture medium components, produced after a specific microbial strain has multiplied in a liquid culture medium. Its core value lies in the active substances (such as enzymes, antibiotics, hormones, etc.) secreted by the microorganisms during fermentation. Those skilled in the art can obtain it by culturing using conventional liquid culture media.

[0037] Thirdly, embodiments of the present invention provide a bio-fertilizer, which includes the above-mentioned Aspergillus heterotrophus AJF-Z1 or the above-mentioned microbial agent.

[0038] It should be noted that the above-mentioned Aspergillus heterophylla AJF-Z1 or the above-mentioned microbial agent can be added with auxiliary materials suitable for use in soil to prepare bio-fertilizer.

[0039] Fourthly, embodiments of the present invention provide the application of the above-mentioned Aspergillus heteromorphus AJF-Z1 or the above-mentioned microbial agent or the above-mentioned bio-fertilizer in promoting soybean growth.

[0040] It should be noted that, as mentioned above, Aspergillus heterophylla AJF-Z1 in this invention can significantly promote soybean growth. Similarly, microbial agents or bio-fertilizers containing Aspergillus heterophylla AJF-Z1 can also significantly promote soybean growth.

[0041] In some specific examples, the soybean varieties used in the above applications are Zhonghuang 37 or Dongsheng 1.

[0042] It should be noted that the Aspergillus heterophylla AJF-Z1 strain, microbial inoculant, or bio-fertilizer in this invention can promote the growth of any soybean variety, especially Zhonghuang 37 or Dongsheng 1 soybeans. Inoculation with the Aspergillus heterophylla AJF-Z1 strain of this invention can significantly increase the plant height, number of pods, dry weight, fresh weight, and number of root nodules in soybeans.

[0043] In some specific examples, in the above applications, Aspergillus heterotrophus AJF-Z1 or microbial inoculants or biofertilizers have one or more of the following functions:

[0044] (a) It has the function of increasing soybean plant height;

[0045] (b) It has the function of increasing the number of soybean pods;

[0046] (c) It has the function of increasing the dry weight of soybeans;

[0047] (d) It has the function of increasing the fresh weight of soybeans;

[0048] (e) It has the function of increasing the number of soybean root nodules.

[0049] Fifthly, embodiments of the present invention provide a method for promoting soybean growth, the method comprising: applying the above-mentioned Aspergillus heterophylla AJF-Z1 or the above-mentioned microbial agent or the above-mentioned bio-fertilizer to the soybean roots by irrigation.

[0050] In some specific examples, the soybean variety used in the above method is Zhonghuang 37 or Dongsheng 1.

[0051] In some specific examples, the above method includes: applying the above-mentioned microbial agent to the roots of soybeans by irrigation; the microbial agent is a suspension, and the amount of bacterial strain in the suspension is (0.8-1.2)×10⁻⁶. 7 CFU / mL, the volume-to-mass ratio of suspension to soil is 20mL:(1.5-2.5)kg.

[0052] It should be noted that the volume-to-mass ratio of the suspension to the soil is 20 mL : (1.5-2.5) kg, for example, 20 mL : 1.7 kg, 20 mL : 2 kg, or 20 mL : 2.3 kg, etc. Furthermore, it should be understood that this ratio is not limited to mL: kg and can be varied according to specific international standards.

[0053] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0054] Example 1

[0055] This invention provides the source, isolation, purification, and identification of Aspergillus heterocaryoticus AJF-Z1.

[0056] (1) Source of AJF-Z1 strain

[0057] The Aspergillus heterocaryoticus AJF-Z1 strain used in this invention is a strain isolated from tobacco in Erhai Lake.

[0058] (2) Identification of AJF-Z1 strain

[0059] Using the genomic DNA of strain AJF-Z1 as a template, ITS rDNA (ITS rDNA sequence as shown in SEQ ID NO: 1) was amplified and then sequenced. The specific amplification method was as follows: the strain was scraped from the purified single-colony plate with a pipette and transferred to a sterile mortar. Liquid nitrogen was added repeatedly for rapid grinding. The mixture was then transferred to a 1.5 mL centrifuge tube containing 100 μL of ultrapure water and mixed thoroughly. The tube was heated in a 95°C metal bath for 15 min, immediately placed in a -20°C freezer for 2 min, and then centrifuged at 14000 rpm for 3 min. The supernatant obtained was used as the template for PCR amplification. The amplification primers were universal fungal primers: primer ITS1 (the sequence of primer ITS1 is shown in SEQ ID NO: 2: TCCGTAGGTGAACCTGCGG) and primer ITS4 (the sequence of primer ITS4 is shown in SEQ ID NO: 3: TCTCCGCTTATTGATATGC). The amplification system consisted of 20 μL of PCR reaction mixture and 10 μL of Taq Master. Mixtures included 0.5 μL of forward and reverse primers, 8 μL of H2O, and 1 μL of DNA template. PCR reaction conditions: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 1 min, 55℃ annealing for 1 min, 72℃ extension for 1 min, 30 cycles; 72℃ final extension for 10 min. PCR products were analyzed using 1.5% agarose gel electrophoresis to determine successful amplification. Sequence alignment using the Ezbiocloud database showed that strain MR-Z1 had a 99.41% similarity to *Aspergillus heterocaryoticus*, a closely related species. A phylogenetic tree was constructed using MEGA11 software. Figure 1 It was found that it clustered with Aspergillus sheterocaryoticus, and was named Aspergillus sheterocaryoticus AJF-Z1 accordingly.

[0060] In addition, the biological characteristics of the AJF-Z1 strain were observed, specifically including: the AJF-Z1 strain was cultured on PDA solid medium (potato: 200g, glucose: 20g, agar powder: 16g, the same below) at 28℃ for 3 days, and its colony morphology was as follows. Figure 2 As shown, the initial growth stage consists of dense, round, white mycelia. During growth, brownish-green spores are produced from the center of the colony, which then turns brown. Aspergillus heteromorphus colonies grow slowly, taking 1-2 weeks to produce spores, and the colonies are relatively small. Eventually, the entire colony appears brown on PDA medium.

[0061] Example 2

[0062] In the following examples, the preparation of Aspergillus heterophylla AJF-Z1 spore suspension was as follows: AJF-Z1 strain was inoculated onto PDA solid medium and cultured in a 25°C light incubator for 7 days until spores appeared. After incubation, approximately 5 mL of sterile water was added to the medium, and the spores were scraped off with a spreader and filtered into a sterile Erlenmeyer flask lined with two layers of sterile gauze to obtain the Aspergillus heterophylla AJF-Z1 spore suspension. The number of spores was counted using a hemocytometer, ensuring that the spore count was 1 × 10⁻⁶ spores. 7 cfu mL -1 .

[0063] This invention provides an embodiment of the actual growth-promoting effect of AJF-Z1 on soybeans, using Zhonghuang 37 and Dongsheng 1 as the selected soybean varieties. Details are as follows:

[0064] (1) Select a batch of plump and intact soybean seeds from Zhonghuang 37 and Dongsheng 1 respectively, and soak them in a solution of 3% sodium hypochlorite solution and sterile water at a volume ratio of 1:1 for 3 minutes in tissue culture bottles.

[0065] (2) Then rinse 10 times with sterile water to achieve disinfection.

[0066] (3) After disinfection, the seeds are soaked in a saturated calcium sulfate solution for 6 hours under ventilation conditions, then placed on a sterilized tray lined with moist gauze and germinated in a shaded environment for 2 days. Soybeans with roots about 2cm long are selected for sowing.

[0067] (4) Fill each pot with 2 kg of soil and apply 1 × 10⁻⁶ spores. 7 cfu mL -1 20 mL of Aspergillus heterophylla AJF-Z1 spore suspension (hereinafter referred to as suspension) was diluted 10 times and applied to each potted plant.

[0068] (5) Two different types of germinated soybeans were sown in soybean pots, with 4 soybeans planted in each pot. Each treatment was replicated 3 times. In addition, a blank control CK without bacterial solution was set up and placed in the Shangzhuang Experimental Station of China Agricultural University.

[0069] (6) Thin the seedlings 5 ​​days after emergence, leaving 2 seedlings with similar growth in each pot; after the thinning, pour 20 mL of diluted bacterial solution onto the roots of the soybeans.

[0070] (7) After soybeans have grown for 50 days, samples were collected and the above-ground and underground parts of the plants were collected separately to measure the corresponding indicators. The average value of each indicator was taken from two plants in each pot.

[0071] The indicators of Dongsheng No. 1 soybean are shown in Table 1, including the number of root nodules. Figure 3 As shown.

[0072] Table 1. Indicators of growth-promoting and yield-increasing effects of strain AJF-Z1 on Dongsheng No. 1 soybean.

[0073] Plant height (cm) Number of pods (g) Dry weight of the plant (g) Fresh weight of the plant (g) CK 31.3±0.94 2.36±0.27 3.37±0.16 5.81±0.45 AJF-Z1 36,6±4.4 2.88±0.12 3.64±0.27 11.5±0.94

[0074] In addition, the indicators of Zhonghuang 37 soybean are shown in Table 2, including its root nodule count. Figure 4 As shown.

[0075] Table 2. Indicators of AJF-Z1 strain on promoting growth and increasing yield of Zhonghuang 37 soybean.

[0076] Plant height (cm) Number of pods (g) Dry weight of the plant (g) Fresh weight of the plant (g) CK 31.6±1.69 5.66±1.24 4.88±0.14 7.86±0.95 AJF-Z1 44.67±2.05 5±0.81 5.57±0.31 9.89±2.07

[0077] As shown in Tables 1 and 2, *Aspergillus heterocaryoticus* AJF-Z1 increased plant height, fresh weight, dry weight, and pod number in two different soybean varieties. Furthermore, from... Figure 3 and Figure 4 The promoting effect of MR-Z1 strain on nodulation of two different soybean varieties can be seen directly. Compared with CK, MR-Z1 can increase nodulation of Zhonghuang 37 by 40.47% and Dongsheng 1 by 46.4%.

[0078] In summary, the strains provided by this invention promote growth, increase fresh weight, dry weight, and number of root nodules in different soybean varieties. Through a symbiotic system, the strains regulate the nitrogen-fixing ability of soybean nodules and promote their growth and development, breaking through the technical bottlenecks of traditional bio-fertilizers and providing innovative solutions for green agriculture.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. 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 spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A strain of Aspergillus heterocaryoticus AJF-Z1, characterized in that, This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 24, 2025, with accession number CGMCCNo.41922.

2. A microbial inoculant, characterized in that, The microbial agent includes Aspergillus heterotrophus AJF-Z1 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, Microbial inoculants include freeze-dried inoculants, solid inoculants, suspensions, or fermentation broths.

4. A bio-fertilizer, characterized in that, Includes Aspergillus heteromorphus AJF-Z1 as described in claim 1 or the microbial agent as described in claim 2 or 3.

5. The application of Aspergillus heterophylla AJF-Z1 as described in claim 1, or the microbial agent as described in claim 2 or 3, or the bio-fertilizer as described in claim 4, in promoting soybean growth.

6. The application according to claim 5, characterized in that, The soybean varieties are Zhonghuang 37 or Dongsheng 1.

7. The application according to claim 5 or 6, characterized in that, Promoting soybean growth includes: (a) Increase soybean plant height; (b) Increase the number of soybean pods; (c) Increase the dry weight of soybeans; (d) Increase the fresh weight of soybeans; (e) Increase the number of soybean root nodules.

8. A method for promoting soybean growth, characterized in that, The method includes applying the Aspergillus heterophylla AJF-Z1 of claim 1, or the microbial agent of claim 2 or 3, or the bio-fertilizer of claim 4 to the soybean roots by irrigation.

9. The method according to claim 8, characterized in that, The soybean varieties are Zhonghuang 37 or Dongsheng 1.

10. The method according to claim 8 or 9, characterized in that, The method includes: applying the microbial agent according to claim 2 or 3 to the roots of soybeans by irrigation; the microbial agent is a suspension, and the amount of bacterial strain in the suspension is (0.8-1.2)×10⁻⁶. 7 CFU / mL, the volume-to-mass ratio of suspension to soil is 20mL:(1.5-2.5)kg.

Images