Actinomucor elegans EM-27, microbial agent and application

Abstract

The invention discloses actinomucor elegans EM-27, a microbial agent and application, and belongs to the technical field of microbial application. The strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on April 24, 2025, the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO.41924. According to the invention, an actinomucor elegans strain is separated from wheat rhizosphere, and antagonism experiments find that 8.7% of rhizosphere bacteria have antagonism on the actinomucor elegans, and 91.3% of rhizosphere bacteria and actinomucor elegans can coexist. And by applying actinomucor elegans, the growth and development of wheat can be obviously promoted. Therefore, the actinomucor elegans has a good application prospect in the aspect of improving plant growth and development.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial application, in particular to a strain of Actinobacillus elegans EM-27, a microbial agent and application thereof. Background Art

[0002] The application of chemical fertilizers effectively guarantees grain production, but biased or excessive fertilization causes the physical, chemical, and biological indicators of the soil to develop in an unfavorable direction, mainly resulting in a decrease in soil organic matter content, deterioration of physical and chemical properties, and changes in soil microbial activity and community structure, which in turn affects the soil's ability to supply and retain fertilizers and to mineralize and decompose soil nutrients.

[0003] Microbial agents not only transform insoluble phosphorus and potassium compounds in the soil but also enhance crop resistance to pests and diseases, significantly promoting crop growth. Increased microbial metabolism and enzyme activity promote the mineralization and decomposition of soil organic matter and the release of nutrients, providing a favorable root zone environment for crop growth and improving crop quality.

[0004] Actinomucor elegans is a filamentous fungus belonging to the phylum Mucoromycota, class Mucoromycetes, orders Mucorales, family Mucoraceae, and genus Actinomucor. Its main characteristic is its cottony, mucor-like colony, no more than 1 cm in height, initially white or light orange-yellow, later turning dark bluish-brown. It has creeping hyphae and rhizodactyls. The sporangium stalk is erect, with branches concentrated at the apex. A large sporangium is located at the apex of the main branch, and below it are three to eight whorled branches, each bearing a sporangium. The main branch of the sporangium stalk is approximately 30 microns in diameter, and each branch typically has a transverse septum. The sporangium is spherical, generally no larger than 80 microns in diameter on the main branch, but sometimes reaching 120 microns, and 20 to 50 microns in diameter on the branches. After aging, the cyst wall dissolves or cracks, leaving behind a cyst collar. The cyst axis is inconsistent, and in larger spore cysts, it is oval to pear-shaped, 50-60 × 30-40 microns. Actinobacillus elegans is widely present in nature, distributed in soil and on plant roots. Currently, Actinobacillus elegans is mainly used in cheese fermentation and is one of the main Mucor species used in fermented bean curd production in my country. There are currently no reports on the use of Actinobacillus elegans to promote plant growth. Summary of the Invention

[0005] The purpose of the present invention is to provide a strain of Actinomucor elegans EM-27, a microbial agent and an application thereof, so as to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is a strain of Actinomucor elegans EM-27, which was deposited in the General Microbiology Center of the China Culture Collection Administration on April 24, 2025. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.41924.

[0008] The second technical solution of the present invention is a microbial agent, including the Actinomucor elegans EM-27.

[0009] The third technical solution of the present invention is the use of the Actinomucor elegans EM-27 or the microbial agent in the preparation of products that promote plant growth.

[0010] A fourth technical solution of the present invention is a product for promoting plant growth, comprising the Actinomucor elegans EM-27 or the microbial agent; the plant comprises wheat.

[0011] The fifth technical solution of the present invention is the use of the Actinomucor elegans EM-27 or the microbial agent in the preparation of products antagonizing bacteria.

[0012] The sixth technical solution of the present invention is a product for antagonizing bacteria, comprising the Actinomucor elegantissima EM-27 or the microbial agent; the bacteria include Ferrovibrio, Ectopseudomonas, Pseudomonas, Kribbella, Ochrobactrum, Bosea, Bradyrhizobium, Niabella, Agromyces, Rheinheimera, Variovorax, Microbacterium, Protaetiibacter, Arthrobacter, Agrobacterium, Cellulosimicrobium, Ramlibacter, Streptomyces, Brevibacillus, Polaromonas, Actinotalea, and Nocardioides.

[0013] Based on the above technical solution, the present invention has the following technical effects:

[0014] The present invention isolated a strain of Actinobacillus elegans from the wheat rhizosphere. Antagonistic experiments revealed that 8.7% of rhizosphere bacteria were antagonistic to it, while 91.3% of rhizosphere bacteria could coexist with Actinobacillus elegans. Furthermore, application of Actinobacillus elegans significantly promoted wheat growth and development. Therefore, Actinobacillus elegans has promising application prospects in improving plant growth and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 This is the colony morphology of Actinomucor elegans EM-27.

[0017] Figure 2 To construct a phylogenetic tree based on ITS gene sequences.

[0018] Figure 3 This is the antagonistic graph between strain EM-27 and rhizospheric bacteria.

[0019] Figure 4 Strain EM-27 promotes wheat plant height.

[0020] Figure 5 Strain EM-27 promotes wheat dry weight. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0024] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0027] An embodiment of the present invention provides an Actinomucor elegans EM-27 strain, which was deposited on April 24, 2025 at the General Microbiology Center of the China Culture Collection Administration, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC NO.41924.

[0028] The embodiment of the present invention also provides a microbial agent, including the Actinomucor elegans EM-27.

[0029] The embodiments of the present invention also provide the use of the Actinomucor elegans EM-27 or the microbial agent in preparing a product that promotes plant growth.

[0030] In some specific embodiments, the plant comprises wheat.

[0031] An embodiment of the present invention further provides a product for promoting plant growth, comprising the Actinomucor elegans EM-27 or the microbial agent; the plant comprises wheat.

[0032] The embodiments of the present invention also provide the use of the Actinomucor elegans EM-27 or the microbial agent in preparing products for antagonizing bacteria.

[0033] In some specific embodiments, the bacteria include Ferrovibrio, Ectopseudomonas, Pseudomonas, Kribbella, Ochrobactrum, Bosea, Bradyrhizobium, Niabella, Agromyces, Rheinheimera, Variovorax, Microbacterium, Protaetiibacter, Arthrobacter, Agrobacterium, Cellulosimicrobium, Ramlibacter, Streptomyces, Brevibacillus, Polaromonas, Actinotalea, and Nocardioides.

[0034] An embodiment of the present invention also provides an antagonistic bacterial product, comprising the Actinomucor elegans EM-27 or the microbial agent; the bacteria include Ferrovibrio, Ectopseudomonas, Pseudomonas, Kribbella, Ochrobactrum, Bosea, Bradyrhizobium, Niabella, Agromyces, Rheinheimera, Variovorax, Microbacterium, Protaetiibacter, Arthrobacter, Agrobacterium, Cellulosimicrobium, Ramlibacter, Streptomyces, Brevibacillus, Polaromonas, Actinotalea, and Nocardioides.

[0035] Example 1

[0036] Isolation, identification and preservation of strain EM-27

[0037] The Actinomucor elegans EM-27 described in the present invention was isolated from the rhizosphere of potted wheat. The soil was taken from Quzhou County, Handan City, Hebei Province, and the wheat variety was Zhongmai 578. The wheat root system was completely removed, the soil attached to the roots was shaken off, and the rhizosphere soil attached to the root surface was washed with PBS buffer. A 10-fold gradient dilution method was used. 1 g of soil was weighed and poured into a triangular flask containing 90 mL of sterile water. The mixture was thoroughly shaken and mixed. The supernatant was diluted to a concentration of 10 -3 , 10 -4 , 10 -5 , 10 -6Soil suspension. Fungal isolation was performed using the dilution plate method. 100 μL of the dilution was spread onto a PDA medium (containing 1% Rose Bengal and 1% streptomycin) plate. After incubation at 28°C for 3-5 days, colonies with distinct morphological characteristics were selected from the plate and purified repeatedly on PDA plates.

[0038] The fungal genome was extracted using a fungal genome extraction kit, and genomic DNA was used as a template for PCR amplification using universal primers for fungal ITS sequences, upstream primer ITS1 (SEQ ID NO. 1: 5'-TCC GTAGGT GAA CCT GCG G-3') and downstream primer ITS4 (SEQ ID NO. 2: 5'-TCC TCC GCT TAT TGATAT GC-3').

[0039] A 50 μL PCR reaction system was used, consisting of 2 μL template DNA, 2 μL upstream primer (0.25 μM), 2 μL downstream primer (0.25 μM), 25 μL PCR Master-Mix (2×), and 19 μL sterile water. PCR reaction conditions were as follows: 95°C for 5 min; 30 cycles of 95°C for 30 sec, 53°C for 30 sec, and 72°C for 30 sec; and 72°C for 10 min.

[0040] The amplification results were sequenced, and the nucleotide sequence of the ITS of the EM-27 strain was shown as SEQ ID NO.3.

[0041] SEQ ID NO. 3: GATTATATAACTTGAGGGGAACTGGGCTTACGGGCTTGGTTTT.

[0042] The sequencing results were compared with the GenBank nucleic acid database by BLAST. The results showed that the homology between strain EM-27 and Actinomucor elegans was 98.81% ( Figure 2 ).

[0043] The biological characteristics of the isolated strain EM-27 were observed. The bacterial colonies were cultured on PDA medium at 28°C for 5 days. The bacterial colonies on PDA medium were similar in morphology to those of Mucor, cotton-like, initially white or light orange-yellow, then dark green-brown-yellow, with creeping hyphae and rhizoids. Figure 1 ).

[0044] Through the above identification results, strain EM-27 was confirmed to be Actinomucor elegans. The strain was deposited in the General Microbiology Center of China Culture Collection Administration on April 24, 2025. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.41924.

[0045] Example 2

[0046] Antagonism test between strain EM-27 and bacteria

[0047] The activated Actinomucor elegans EM-27 bacterial cake was inoculated into the center of a PDA solid plate and cultured at 28°C for 5 days to allow the colony to fill the plate. The spores on the culture medium were washed with sterile water and the spore suspension was collected. The spores were counted under a microscope using a hemocytometer. The total concentration of the spore suspension was finally calculated and diluted to 1×10 7 Spore suspension was inoculated at a 1% (v:v) inoculum into melted PDA medium at approximately 50°C, mixed thoroughly, and plated. After the plate solidified, 448 bacterial colonies isolated from the wheat rhizosphere were picked using a sterile toothpick and inoculated onto plates containing Actinomucor elegans. Three replicates were performed for each treatment, and the cells were incubated in a 28°C incubator for 3 days to observe the antagonistic effects of the bacteria against EM-27.

[0048] The results showed that all 448 bacterial strains isolated from wheat rhizosphere could grow on the plates containing EM-27 strain, and significant inhibition zones were observed for 39 of them ( Figure 3 ), 8.7% of the bacteria were antagonistic to the EM-27 strain, and the analysis found that 39 strains belonged to 22 genera.

[0049] Table 1 Antagonistic effect of rhizospheric bacteria and strain EM-27

[0050]

[0051] Example 3

[0052] Experiment on the Growth-Promoting Effect of Actinomucor elegantissima EM-27 in Potted Plants

[0053] Preparation of spore suspension of Actinomucor elegans EM-27: EM-27 strain was inoculated on PDA solid medium and cultured in a 28°C light incubator for 5 days. After removal, about 5 ml of sterile water was added to the medium and spores were scraped off with a spreader to obtain a spore suspension. The number of spores was counted using a hemocytometer and diluted to 1 × 10 7 CFU / mL spore suspension.

[0054] Soil was collected from Quzhou, Hebei Province, and 2 kg of sterilized soil was placed in each pot. Zhongmai 578 wheat was used for the experiment. Wheat seeds were soaked in a 2% (m / v, g / mL) sodium hypochlorite solution for 10 min, disinfected, then washed several times with sterile water, and spread on sterile filter paper for germination. The main embryonic axis of the wheat was exposed, and 2 wheat plants with the same growth potential were sown in each pot. Two treatments (3 replicates for each treatment) were set up, as follows: CK (not inoculated with bacteria) and EM-27 (inoculated with Actinomucor elegantissima EM-27). When the wheat was grown to the seedling stage, 20 mL of spore suspension was watered at the root, and the growth index of the plant was measured after two weeks of growth.

[0055] Table 2 Effects of strain EM-27 on wheat plant height and dry weight

[0056]

[0057] The results showed that from Figure 4 、 Figure 5 It can be seen that inoculation with Actinomucor elegans EM-27 can significantly increase the plant height and dry weight of wheat. The plant height increased by 18.2%, and the dry weight of the plant increased by 14.6%.

[0058] In summary, the Actinomucor elegans EM-27 provided by the present invention can significantly promote the growth and development of wheat and has good application prospects.

[0059] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the implementation methods of the present invention. A person skilled in the art will be able to make other variations or modifications based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A strain of Actinomucor elegans EM-27, characterized in that: The strain was deposited in the General Microbiology Center of China Culture Collection Administration on April 24, 2025. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.41924.

2. A microbial agent, characterized in that: The invention comprises the Actinomucor elegans EM-27 described in claim 1.

3. Use of the Actinomucor elegans EM-27 according to claim 1 or the microbial agent according to claim 2 in the preparation of a product that promotes plant growth.

4. The use according to claim 3, characterized in that The plants include wheat.

5. A product for promoting plant growth, characterized in that: The method comprises the Actinomucor elegans EM-27 according to claim 1 or the microbial agent according to claim 2; and the plant comprises wheat.

6. Use of the Actinomucor elegans EM-27 according to claim 1 or the microbial agent according to claim 2 in the preparation of products antagonizing bacteria.

7. The use according to claim 6, characterized in that The bacteria include Ferrovibrio, Ectopseudomonas, Pseudomonas, Kribbella, Ochrobactrum, Bosea, Bradyrhizobium, Niabella, Agromyces, Rheinheimera, Variovorax, Microbacte rium, Protaetiibacter, Arthrobacter, Agrobacterium, Cellulosimicrobium, Ramlibacter, Streptomyces, Brevibacillus, Polaromonas, Actinotalea and Nocardioides.

8. A product for antagonizing bacteria, characterized in that The invention comprises the Actinomucor elegantissima EM-27 according to claim 1 or the microbial agent according to claim 2; the bacteria include Ferrovibrio, Ectopseudomonas, Pseudomonas, Kribbella, Ochrobactrum, Bosea, Bradyrhizobium, Niabella, Agromyces, Rheinheimera, Variovorax, Microbacterium, Protaetiibacter, Arthrobacter, Agrobacterium, Cellulosimicrobium, Ramlibacter, Streptomyces, Brevibacillus, Polaromonas, Actinotalea, and Nocardioides.

Images