Microbial inoculant, method for its preparation and use

Abstract

This invention provides a microbial inoculant, its preparation method, and its application. The microbial inoculant includes yeast strain An-15, its culture medium, and an optional anti-adhesion agent; wherein yeast An-15 is classified as *Saccharomyces cerevisiae*, with accession number CCTCC NO: M2019643; wherein the culture medium includes a mixed sugar formed by mixing one or more sugars in equal proportions; the sugar includes pure sugar or a mixture of sugars; pure sugar includes sucrose, maltose, or glucose; the mixture of sugars includes raw sugar, brown sugar, red sugar, or black sugar. The fermentation broth obtained by fermenting this microbial inoculant, after dilution, can effectively prevent and control root rot in plants. The method of this invention for treating plant root rot has the advantages of being simple, convenient, low-cost, and environmentally friendly.

Description

Technical Field

[0001] This invention relates to the field of plant root rot control technology, and more specifically, to a microbial agent, its preparation method, and its application. Background Technology

[0002] Microbial fertilizers are live microbial preparations produced by industrializing target microorganisms (effective bacteria) through solid / liquid fermentation and other methods. They directly or indirectly improve soil fertility, maintain the balance of the rhizosphere microbiome, and degrade toxic and harmful substances. When applied to agricultural production, microbial fertilizers increase nutrient supply to plants or promote plant growth, improve agricultural product quality, and enhance the agricultural ecological environment through the life activities of the microorganisms they contain. Utilizing microorganisms for biological control is effective, sustainable, and environmentally friendly, significantly reducing the need for chemical fungicides. Currently, the control of plant diseases such as root rot mostly relies on chemical pesticide application, which not only causes pesticide residues and pollution in the soil but also seriously affects product quality and human health. For many years, yeast has been considered an organism with enormous application potential as a biological control agent for plant pathogens, possessing significant product development potential.

[0003] Plant root rot refers to a disease in which plant roots are infected by fungi, bacteria, or other pathogens, leading to the rotting and necrosis of root tissues. This disease affects the plant's ability to absorb nutrients and water, resulting in stunted growth, slowed growth, or even death. Root rot is one of the most common diseases affecting plant growth, severely impacting plant growth and yield.

[0004] Yeasts are tiny eukaryotic organisms currently widely used in food, animal husbandry, brewing, food flavoring, and biotechnology. In recent years, yeast metabolites have also been increasingly applied in plant nutrition and protection. However, yeasts themselves have received little development. In fact, yeasts can also be used as beneficial microorganisms in plant nutrition and protection, directly or indirectly having a positive impact on plant growth and protection. Some yeasts do not produce any biotoxins, can inhibit the growth of pathogens, stimulate plant growth, promote plant resistance to physiological stress, and increase plant nutrient utilization.

[0005] Therefore, it is crucial to develop a microbial agent for preventing and controlling plant root rot, with yeast as the main component. Summary of the Invention

[0006] The main objective of this invention is to provide a microbial agent, its preparation method, and its application, in order to solve the problem of poor control effect of existing microbial agents for preventing and controlling plant root rot.

[0007] To achieve the above objectives, according to a first aspect of the present invention, a microbial agent is provided, the microbial agent comprising yeast An-15, its culture medium, and an optional anti-adhesion agent;

[0008] Among them, the above-mentioned yeast strain An-15 is classified as Saccharomyces cerevisiae and was deposited at the China Center for Type Culture Collection on August 19, 2019, at Wuhan University, Wuhan, China, with accession number CCTCCNO: M2019643.

[0009] The culture medium mentioned above includes a mixed sugar formed by mixing one or more sugars in equal proportions;

[0010] The sugars mentioned above include sugars in pure form or sugar mixtures;

[0011] The above-mentioned sugars in pure form include sucrose, maltose, or glucose;

[0012] The sugars in the above-mentioned mixed forms include raw sugar, brown sugar, red sugar, or black sugar.

[0013] Furthermore, the content of the above-mentioned yeast AN-15 is 1.0*10^9 CFU / g~2.0*10^9 CFU / g;

[0014] Preferably, the culture medium is brown sugar;

[0015] Preferably, the anti-blocking agent is selected from any one or more of the following: corn starch, attapulgite, kaolin, diatomaceous earth, or bentonite; more preferably, it is corn starch;

[0016] Preferably, the microbial agent includes an anti-adhesion agent, and the weight ratio of the culture medium to the anti-adhesion agent is (4~9):1.

[0017] To achieve the above objective, according to a second aspect of the present invention, a method for preparing a microbial inoculant is provided, the method comprising:

[0018] The above-mentioned microbial inoculant was obtained by mixing yeast strain An-15 with the culture medium.

[0019] Among them, the above-mentioned yeast strain An-15 is classified as Saccharomyces cerevisiae and was deposited at the China Center for Type Culture Collection on August 19, 2019, at Wuhan University, Wuhan, China, with accession number CCTCCNO: M2019643.

[0020] The culture medium mentioned above includes a mixed sugar formed by mixing one or more sugars in equal proportions;

[0021] The sugars mentioned above include sugars in pure form or sugar mixtures;

[0022] The above-mentioned sugars in pure form include sucrose, maltose, or glucose;

[0023] The sugars in the above-mentioned mixed forms include raw sugar, brown sugar, red sugar, or black sugar.

[0024] Furthermore, the above preparation method includes:

[0025] The above culture medium and anti-adhesion agent were mixed to obtain mixture 1; and

[0026] The above-mentioned yeast strain An-15 and the above-mentioned mixture 1 were mixed to obtain the above-mentioned microbial inoculum;

[0027] Preferably, the anti-blocking agent is selected from any one or more of the following: corn starch, attapulgite, kaolin, diatomaceous earth, or bentonite; more preferably, it is corn starch;

[0028] Preferably, before obtaining the above mixture 1, the above culture medium and the above anti-adhesion agent are mixed and then pulverized;

[0029] More preferably, it is pulverized to 10-80 mesh;

[0030] Preferably, a mixer is used for the above mixing;

[0031] More preferably, the mixing time is 5 to 10 minutes.

[0032] To achieve the above objectives, according to a third aspect of the present invention, a method for preparing microbial fertilizer is provided, the method comprising:

[0033] The above-mentioned microbial inoculant or the microbial inoculant prepared by the above preparation method is mixed with water and fermented to obtain a fermentation broth;

[0034] The above fermentation broth was diluted to obtain the above microbial fertilizer.

[0035] Furthermore, the mass ratio of the above-mentioned microbial inoculant to the above-mentioned water is 1:(25~50).

[0036] Preferably, the fermentation time is 1 to 2 days and the fermentation temperature is 25 to 30°C.

[0037] To achieve the above objectives, according to a fourth aspect of the present invention, a microbial fertilizer is provided, wherein the microbial fertilizer is prepared by the above method.

[0038] Furthermore, the content of the above-mentioned yeast An-15 in the above-mentioned microbial fertilizer is 1.0*10^7 CFU / mL~1.0*10^8 CFU / mL.

[0039] To achieve the above objectives, according to a fifth aspect of the present invention, a method for preventing and controlling plant root rot is provided, the method comprising: applying the above-mentioned microbial fertilizer to the above-mentioned plants.

[0040] Furthermore, the above-mentioned microbial fertilizer is applied to the above-mentioned plants using any one or more of the following methods: fertigation, drip irrigation, or spraying;

[0041] Preferably, the plant is a tomato or a cucumber;

[0042] Preferably, the application rate of the above-mentioned microbial fertilizer is 2-3 kg / mu;

[0043] Preferably, the above-mentioned microbial fertilizer is applied before or at the early stage of disease onset.

[0044] Preferably, the above-mentioned microbial fertilizer is applied at least 3 to 4 times;

[0045] Preferably, the above-mentioned microbial fertilizer is applied at intervals of 5 to 7 days.

[0046] By applying the technical solution of this invention, yeast An-15 and its culture medium are mixed to prepare a microbial agent. The fermentation liquid obtained by fermenting this microbial agent is diluted and applied to plants (e.g., cucumbers and tomatoes) to effectively prevent root rot in plants (e.g., cucumbers and tomatoes), thereby promoting plant growth. This helps to minimize or eliminate the use of pesticides and improve plant quality, meeting the development needs of green agriculture and sustainable agriculture. Attached Figure Description

[0047] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0048] Figure 1 A schematic diagram of bacterial growth after co-culturing yeast An-15 and cucumber root rot pathogen Fusarium oxysporum according to Embodiment 1 of the present invention is shown.

[0049] Figure 2 The effects of PDA plates prepared with different nutrient concentrations according to Example 2 of the present invention on the growth of yeast An-15 and cucumber root rot pathogen Fusarium oxysporum are shown.

[0050] Figure 3 The effect of volatile organic compounds produced by yeast An-15 according to Example 3 of the present invention on the growth of Fusarium oxysporum, the pathogenic fungus of cucumber root rot, is shown.

[0051] Figure 4 A flowchart for preparing microbial fertilizer according to an embodiment of the present invention is shown.

[0052] Figure 5 The image shows the leaf condition of a tomato seedling with root rot on August 15, 2024, before drenching, according to Embodiment 4 of the present invention.

[0053] Figure 6 The image shows the root condition of a tomato seedling suffering from root rot on August 15, 2024, before drenching, according to Embodiment 4 of the present invention.

[0054] Figure 7 The illustration shows that the leaves of tomato seedlings suffering from root rot were inhibited after applying the microbial fertilizer of the present invention on August 17, 2024, according to Embodiment 4 of the present invention.

[0055] Figure 8 The image shows the leaf condition of cucumber seedlings with root rot on August 14, 2024, before drenching, according to Embodiment 5 of the present invention.

[0056] Figure 9 The image shows the root condition of cucumber seedlings suffering from root rot on August 14, 2024, before drenching, according to Embodiment 5 of the present invention.

[0057] Figure 10 The illustration shows the suppression of leaf growth (overall growth) in cucumber seedlings suffering from root rot after applying the microbial fertilizer of the present invention on August 18, 2024, according to Embodiment 5 of the present invention.

[0058] Figure 11 The illustration shows the suppression of leaf growth in cucumber seedlings suffering from root rot after application of the microbial fertilizer of the present invention on August 18, 2024, according to Embodiment 5 of the present invention (root diagram of a single cucumber plant).

[0059] Figure 12 The illustration shows the suppression of root rot in cucumber seedlings after application of the microbial fertilizer of the present invention on August 18, 2024, according to Embodiment 5 of the present invention. Detailed Implementation

[0060] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0061] Terminology Explanation:

[0062] First irrigation: An agricultural planting technique referring to the first application of water and fertilizer during the crop's growth process. This technique is usually carried out in the early stages of crop growth, i.e., the first growth period after sowing, with the aim of promoting crop growth, increasing yield, and improving crop resistance. Through the first irrigation, the crop can quickly absorb nutrients and grow rapidly, laying a good foundation for subsequent growth and development.

[0063] Raw sugar: Sugar extracted from sugarcane juice and obtained through preliminary processing (usually including evaporation crystallization and concentration crystallization). A centrifuge is used to separate the crystalline sugar from the remaining molasses, yielding relatively pure raw sugar that still retains some molasses. It typically contains a certain amount of molasses and small amounts of minerals such as calcium, magnesium, iron, and potassium.

[0064] Brown sugar: It is obtained by boiling and concentrating sugarcane juice without undergoing centrifugation. It is more refined than red sugar and black sugar, and retains fewer natural components (such as a large amount of natural pigments, minerals and flavor substances).

[0065] Brown sugar: Raw sugar formed by directly boiling and concentrating sugarcane juice. It usually does not undergo centrifugal separation. It is less refined than raw granulated sugar but more refined than black sugar, and retains more natural components (such as a large amount of natural pigments, minerals and flavor substances).

[0066] Brown sugar is made by concentrating sugarcane juice through long-term boiling (longer than that of raw sugar and brown sugar). It does not undergo centrifugation and is less refined than raw sugar and brown sugar, retaining more natural components (such as a large amount of natural pigments, minerals and flavor substances).

[0067] As mentioned in the background section, existing microbial agents for controlling root rot in plants (e.g., cucumbers and tomatoes) have poor efficacy. Some yeasts do not produce toxic or harmful substances and can directly or indirectly have a positive impact on plant growth and protection. Therefore, in this invention, the inventors attempted to develop a highly effective microbial agent for controlling root rot in cucumbers or tomatoes, using yeast as the main component, and thus proposed a series of protective solutions for this invention.

[0068] In a first typical embodiment of the present invention, a microbial agent is provided, which includes yeast AN-15, its culture medium, and an optional anti-adhesion agent.

[0069] The yeast strain An-15 was classified as Saccharomyces cerevisiae and deposited at the China Center for Type Culture Collection (CCTCC) on August 19, 2019, at Wuhan University, Wuhan, China, with accession number CCTCCNO: M2019643.

[0070] The culture medium includes a sugar mixture formed by mixing one or more sugars in equal proportions; the sugar includes sugar in pure form or sugar in mixture form; the sugar in pure form includes sucrose, maltose or glucose; the sugar in mixture form includes raw sugar, brown sugar or black sugar.

[0071] Microbial culture media contain various elements, trace elements, and vitamins required by microorganisms, which meet their needs for growth and reproduction. The aforementioned culture medium provides ample nutrients for yeast An-15, promoting its growth and reproduction and producing abundant metabolites. Only a small amount of the original yeast An-15 is needed to achieve highly effective control of root rot in plants such as cucumbers or tomatoes (see [link]). Figure 4 ).

[0072] The microbial inoculant of the present invention can effectively prevent and control root rot in plants (e.g., cucumbers and tomatoes). Furthermore, the microbial inoculant of the present invention has a simple composition, containing only one active ingredient, yeast AN-15, making it inexpensive and easy to prepare.

[0073] In a preferred embodiment of the present invention, the culture medium is brown sugar. Brown sugar is rich in nutrients, and using brown sugar as the culture medium for yeast An-15 is beneficial to the growth of yeast An-15. In a preferred embodiment of the present invention, the content of yeast An-15 is 1.0*10^9 CFU / g~2.0*10^9 CFU / g. Using this concentration of yeast has the beneficial effect of a high initial fermentation cell content.

[0074] To prevent the yeast strain An-15 in the microbial inoculant from adhering to the culture medium, thus adversely affecting the growth and reproduction of the yeast, in a preferred embodiment of the present invention, the microbial inoculant further includes an anti-adhesion agent. In a more preferred embodiment of the present invention, the anti-adhesion agent is selected from any one or more of the following: corn starch, attapulgite, kaolin, diatomaceous earth, or bentonite. Using the above-mentioned anti-adhesion agent has the beneficial effect of preventing the yeast and culture medium from adhering. Corn starch is more preferred. Compared with other anti-adhesion agents, corn starch is a fully water-soluble anti-adhesion agent. Fully water-soluble anti-adhesion agents have good solubility and will not clog agricultural drip irrigation and sprinkler equipment.

[0075] Optimizing the weight ratio of the culture medium and the anti-blocking agent further promotes yeast growth, reproduction, and metabolism. In a preferred embodiment of the present invention, the weight ratio of the culture medium to the anti-blocking agent is (4-9):1. This ratio provides the advantages of sufficient nutrition, lower raw material costs, better product appearance, and more stable shelf life.

[0076] In a second typical embodiment of the present invention, a method for preparing a microbial inoculant is provided. The method includes: mixing the aforementioned yeast strain An-15 with the aforementioned culture medium to obtain the aforementioned microbial inoculant; wherein the yeast strain An-15 is classified as *Saccharomyces cerevisiae*, and was deposited on August 19, 2019, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M2019643; wherein the culture medium comprises a mixed sugar consisting of one or more sugars in equal proportions; the sugar comprises sugar in pure form or a mixture of sugars; the pure sugar comprises sucrose, maltose, or glucose; the mixture of sugars comprises raw sugar, brown sugar, red sugar, or black sugar. This method for preparing the microbial inoculant has the advantage of being simple to operate.

[0077] In a preferred embodiment of the present invention, the preparation method includes: mixing the culture medium and the anti-adhesion agent to obtain mixture 1; and mixing the yeast strain An-15 with mixture 1 to obtain the microbial agent. Since the culture medium is viscous, mixing the culture medium and the anti-adhesion agent first can better disperse the culture medium, and adding yeast and mixing will make the yeast more evenly dispersed in the culture medium.

[0078] To ensure more uniform mixing, in a preferred embodiment of the invention, a mixer is used for mixing; more preferably, the mixing time is 5-10 minutes. Mixing under these conditions allows the yeast, culture medium, and anti-blocking agent to be mixed more evenly, which is beneficial for subsequent fermentation culture.

[0079] To ensure uniform mixing of the aforementioned yeast strain An-15, the aforementioned binder, and the aforementioned culture medium, and to facilitate the effective utilization of nutrients in the culture medium by yeast An-15 during fermentation, in a preferred embodiment of the present invention, the preparation method further includes: before obtaining the aforementioned mixture 1, mixing and pulverizing the aforementioned culture medium and the aforementioned anti-blocking agent. More preferably, pulverizing to 10-80 mesh. Pulverizing the culture medium and the anti-blocking agent to 10-80 mesh helps to mix the yeast more uniformly and helps the yeast utilize the nutrients in the culture medium.

[0080] In a third typical embodiment of the present invention, a method for preparing microbial fertilizer is provided. The method includes: mixing the aforementioned microbial agent or a microbial agent prepared using the aforementioned method with water and fermenting the mixture to obtain a fermentation broth; and diluting the fermentation broth to obtain microbial fertilizer. The microbial fertilizer of the present invention has the advantages of low cost and good effect in preventing and controlling root rot.

[0081] In a preferred embodiment of the present invention, the fermentation time is 2-3 days, and the fermentation temperature is 25-30°C. Fermentation under these conditions promotes the rapid growth and reproduction of yeast An-15, resulting in faster and more efficient production of microbial fertilizer. It is important to note that daily stirring during fermentation is recommended. Typically, fermentation is complete when a white film appears on the surface after 2 days, and white foam appears and an alcoholic aroma is emitted after 3 days.

[0082] The ratio of microbial inoculant to water may affect the proliferation rate of yeast. In a preferred embodiment of the present invention, the mass ratio of the microbial inoculant to the water is 1 kg: (25~50) kg. Fermentation under these conditions has the beneficial effect of promoting the yeast proliferation rate and increasing the number of yeast cells. To ensure the control effect, the fermentation broth needs to be diluted after fermentation to obtain the microbial fertilizer. The diluted fermentation broth has a beneficial effect of effectively inhibiting pathogens.

[0083] It should be noted that the fermentation broth can be diluted appropriately depending on the severity of the plant root rot. In a preferred embodiment of the present invention, the content of yeast An-15 in the diluted fermentation broth, i.e., the microbial fertilizer, is 1.0*10^7 CFU / mL to 1.0*10^8 CFU / mL. If the plant root rot is severe, the yeast content in the microbial fertilizer can be appropriately increased.

[0084] In a fourth typical embodiment of the present invention, a microbial fertilizer is provided. This microbial fertilizer is prepared using the method described above. Applying the microbial fertilizer of the present invention to plants can effectively prevent and control root rot. In a preferred embodiment of the present invention, the content of the aforementioned yeast strain An-15 in the microbial fertilizer is 1.0*10^7 CFU / mL to 1.0*10^8 CFU / mL. Using this concentration of microbial fertilizer can effectively prevent and control root rot in plants.

[0085] In a fifth typical embodiment of the present invention, a method for preventing and controlling plant root rot is provided, the method comprising: applying the above-mentioned microbial fertilizer to the above-mentioned plants.

[0086] The microbial fertilizer of this invention can effectively prevent and control root rot in plants (e.g., cucumbers and tomatoes). Furthermore, the method for preventing and controlling root rot in plants (e.g., cucumbers and tomatoes) according to this invention is simple, easy to operate, and low in cost. It only requires mixing the aforementioned microbial agent with water and then fermenting it at a low temperature for a short time to obtain the microbial fertilizer for preventing and controlling root rot in plants (e.g., cucumbers and tomatoes). It features simple operation and convenient use. Using the microbial fertilizer can improve the survival rate of plants such as tomatoes or cucumbers, promote root growth, and prevent and control root rot.

[0087] Furthermore, root rot is a disease caused by fungal or bacterial infection of plant roots. These pathogens invade the plant roots, destroying root cells and preventing the plant from absorbing nutrients and water, ultimately leading to stunted growth and even death. Different plants infected with the same pathogen exhibit similar pathogenic mechanisms; therefore, the same microbial agent can control root rot in various plants (caused by the same pathogen). The microbial fertilizer of this invention can effectively control root rot in various plants caused by Fusarium oxysporum, such as tomatoes, cucumbers, loofahs, peppers, and potatoes. In a preferred embodiment of this invention, the aforementioned plants are tomatoes or cucumbers. Applying the above-mentioned microbial agent to cucumbers and tomatoes can effectively control root rot in cucumbers and tomatoes.

[0088] Depending on the fertilization habits of different plants, the above-mentioned microbial fertilizer can be applied in different ways. In a preferred embodiment of the present invention, the above-mentioned microbial fertilizer is applied to the above-mentioned plants using any one or more of the following methods: fertigation, drip irrigation, or spraying.

[0089] When using the microbial fertilizer of the present invention, the applicable amount, number of applications, and application frequency vary depending on the type of plant applied and the disease being controlled. The appropriate amount, number of applications, and application frequency can be selected according to the type of plant and the severity of the disease. In a preferred embodiment of the present invention, the application amount of the above-mentioned microbial fertilizer is 2-3 kg / mu; the number of applications of the above-mentioned microbial fertilizer is 3-4 times; and the application frequency of the above-mentioned microbial fertilizer is every 5-7 days.

[0090] To ensure yeast gains dominance and better resists pathogen invasion, in a preferred embodiment of this invention, the above-mentioned microbial fertilizer is applied before or in the early stage of disease (when only a few (e.g., 3-5) lateral roots and fibrous roots show yellowing and gradually spread to the main root; initially, the plant does not show symptoms). In the late stage of disease, pathogens dominate the plant's population. Adding yeast at this time can help resist the pathogens, but its effect is limited and may not effectively save the plant. Therefore, to allow yeast to gain a dominant population and achieve better resistance, it is recommended to use the above-mentioned microbial fertilizer during or in the early stage of disease.

[0091] The present application will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed in the present application.

[0092] Example 1: Yeast has a significant inhibitory effect on the growth of cucumber root rot fungi.

[0093] The yeast strain selected in this embodiment is Saccharomyces cerevisiae, a yeast strain from the Angel Yeast Company's yeast strain bank. It was deposited at the China Center for Type Culture Collection (CCTCC) on August 19, 2019, with accession number CCTCC NO: M2019643. This yeast strain was inoculated into potato dextrose agar (PDA) medium and incubated at 28°C for 48 hours.

[0094] The root rot fungus selected in this embodiment is the cucumber root rot pathogen Fusarium oxysporum preserved in our laboratory. In order to obtain active fungi, PDA agar blocks containing root rot pathogen spores were transferred to fresh PDA medium and cultured at 28°C for 7-14 days.

[0095] The antagonistic activity of yeast against the root rot pathogen was evaluated by co-culturing yeast strain An-15 and the cucumber root rot pathogen on PDA plates. A 4 mm diameter block of actively growing fungal pathogen was inoculated in the center of the plate, followed by a ring of active yeast cells approximately 3 cm away. The plates were incubated at 28°C for 7 days. A control plate containing only the fungal pathogen was used. Each treatment was performed in triplicate. Antagonistic activity was indicated by the formation of an inhibition band on the plate.

[0096] The mycelial growth inhibition rate was calculated using the formula {(R1‐R2) / R1}×100, where R1 is the mycelial growth radius of the control treatment and R2 is the mycelial growth radius of the yeast fungus An-15 treatment.

[0097] Experimental results are as follows Figure 1 As shown in Table 1, the results of measuring the mycelial growth radius and calculating the inhibition rate are as follows: the inhibition rate of yeast An-15 against cucumber root rot pathogens is 43.33±0.05%, indicating that yeast An-15 has a good inhibitory effect on cucumber root rot pathogens.

[0098] Table 1

[0099]

[0100] Example 2: Yeast and cucumber root rot fungi compete for nutrients.

[0101] The competition for nutrients was estimated by observing the inhibitory effect of yeast on the growth of fungal pathogens on culture media with different nutrient concentrations using a dual culture method. PDA plates were prepared with four different nutrient concentrations: normal nutrient concentration (28g PDA powder + 20g agar), half normal nutrient concentration (14g / L PDA powder + 20g agar), one-quarter normal nutrient concentration (7g / L PDA powder + 20g agar), and one-tenth normal nutrient concentration (2.8g / L PDA powder + 20g agar).

[0102] The mycelial growth inhibition rate was calculated using the formula {(R1‐R2) / R1}×100, where R1 is the mycelial growth radius of the control treatment and R2 is the mycelial growth radius of the yeast fungus An-15 treatment.

[0103] Experimental results are as follows Figure 2 As shown in Table 2, the results of measuring the mycelial growth radius and calculating the inhibition rate are as follows: As the nutrient concentration in the PDA medium decreases, the inhibition rate of yeast An-15 against cucumber root rot pathogens also decreases, indicating that yeasts with different activities have a significant impact on the growth of root rot fungi, and there is a nutrient competition relationship between the two.

[0104] Table 2

[0105]

[0106] Example 3: Yeast can produce antifungal volatile organic compounds.

[0107] The antifungal volatile organic compounds produced by yeast strain An-15 were evaluated using the double-layer plate method. A cell suspension of yeast An-15, cultured in YPD liquid medium for 2 days, was prepared by suspending it in sterile water at a concentration of 10⁻⁶. 8 Cells / mL (adjust OD600 to 1.0). Aliquots (100 μL) of yeast cell suspension were evenly spread onto YM agar plates and incubated at 28°C for 2 days. After incubation, the caps of the plates were removed, and the plates were inverted and placed on the bottom of another PDA plate inoculated with a 4 mm diameter disc of actively growing fungal pathogen placed in the center. Both plates were sealed with sealing film and incubated at 28°C for 7 days. The control treatment consisted of PDA plates inoculated only with discs of actively growing cucumber root rot pathogens. Each treatment was performed in triplicate.

[0108] The percentage of inhibition of fungal growth by volatile organic compounds produced by yeast An-15 was calculated using the formula {(R1‐R2) / R1}×100, where R1 is the mycelial growth radius of the control treatment and R2 is the mycelial growth radius of the yeast An-15 treatment.

[0109] Experimental results are as follows Figure 3 As shown in Table 3, the results of measuring the mycelial inhibition radius and calculating the inhibition rate are as follows: the volatile organic compounds produced by yeast An-15 have an inhibition rate of 43.33±0.05% against cucumber root rot pathogens, indicating that the antifungal volatile organic compounds produced by yeast An-15 can significantly inhibit the growth of cucumber root rot pathogens.

[0110] Table 3

[0111]

[0112] Example 4

[0113] Preparation of microbial fertilizer:

[0114] (1) Weigh according to the following mass parts: 1.7 kg of brown sugar, 0.2 kg of corn starch, and the content of yeast AN-15 is 1.0*10^9 CFU / g. It should be noted that the yeast content refers to the yeast content in the microbial agent (including brown sugar, corn starch and yeast).

[0115] (2) Put the above brown sugar and corn starch into a mixer and mix evenly for 5 minutes. After mixing, grind the mixture into 10 mesh using a pulverizer.

[0116] (3) Add yeast An-15 to the mixture of brown sugar and corn starch, stir and mix for 5 minutes to make microbial fertilizer.

[0117] How to use:

[0118] Add tap water to the microbial fertilizer to dissolve it, with the amount of tap water being 25 times the mass of the microbial agent. Stir thoroughly and place in a warm indoor place (30℃) for natural fermentation for 2 days. It is ready when a white film appears on the surface, white foam appears, and an alcoholic aroma is emitted. Dilute before use; the diluted yeast content should be 1.0*10^7 CFU / ml. Apply directly to crops via drip irrigation. Note that the diluted yeast content refers to the yeast content in the fermentation liquid. The application rate of the above microbial fertilizer is 2 kg / acre; the application frequency is 4 times, with an interval of 7 days between applications.

[0119] Experimental Field 1:

[0120] Date: August 15, 2024; Location: Xihei Village, Yingli Town, Shouguang City, Shandong Province; Experimental crop: Tomato.

[0121] Before application, root rot was present in the greenhouse, causing seedlings to wilt and their roots to turn yellow and rot. The root rot was suppressed the day after the first irrigation with the microbial agent, and the seedlings' leaves became upright and normal, their roots developed normally, and their growth was excellent. Figure 5 The image shows the leaf condition of tomato seedlings with root rot before application of foliar spray on August 15, 2024. Figure 6 The image shows the root condition of tomato seedlings with root rot before application of foliar spray on August 15, 2024. Figure 7 The illustration shows that the leaves of tomato seedlings suffering from root rot were suppressed after applying the microbial fertilizer of the present invention on August 17, 2024.

[0122] Example 5

[0123] Preparation of microbial fertilizer:

[0124] (1) Weigh according to the following mass parts: 1.6 kg of brown sugar, 0.4 kg of corn starch, and the content of yeast AN-15 is 2.0*10^9 CFU / g. It should be noted that the yeast content refers to the yeast content in the microbial agent (including brown sugar, corn starch and yeast).

[0125] (2) Put brown sugar and corn starch into a mixer and mix evenly for 10 minutes. After mixing, grind them into 80 mesh using a grinder.

[0126] (3) Add the yeast to the mixture of brown sugar and corn starch, stir and mix for 10 minutes to make microbial fertilizer.

[0127] How to use:

[0128] Add tap water to the microbial fertilizer to dissolve it, with the amount of tap water being 50 times the mass of the microbial agent. After stirring thoroughly, place it in a warm indoor place (25℃) and allow it to ferment naturally for 2 days. It is ready when a white film appears on the surface, white foam appears, and an alcoholic aroma is emitted. Dilute before use; the concentration of the diluted yeast solution is approximately 2.0*10^7 CFU / ml. Apply directly to crops via drip irrigation. The application rate of the above microbial fertilizer is 2 kg / acre; the application frequency is 3 times, with an interval of 5 days between applications.

[0129] Experimental Field 2:

[0130] Date: August 16, 2024; Location: Zhangjiatun Village, Hualong Town, Shouguang City, Shandong Province; Experimental crop: cucumber.

[0131] Before use, the previous crop planted in the greenhouse did not grow for a month and suffered from severe root rot (see...). Figure 8 and Figure 9 Two days after applying the microbial fertilizer of this invention during the first irrigation of the next crop, rooting was significantly improved, survival rate was high, and growth was excellent (see...). Figure 10 , Figure 11 and Figure 12 ).

[0132] Example 6

[0133] Preparation of microbial fertilizer:

[0134] (1) Weigh according to the following mass parts: 900 kg of brown sugar, 100 kg of corn starch, and the yeast content is 1.1*10^9 CFU / g. It should be noted that the yeast content refers to the yeast content in the microbial agent (including brown sugar, corn starch and yeast).

[0135] (2) First, put brown sugar and corn starch into a mixer and mix evenly for 8 minutes. After mixing, grind them into 80 mesh using a grinder.

[0136] (3) Add the yeast to the mixture of brown sugar and corn starch, stir and mix for 8 minutes to make microbial fertilizer.

[0137] How to use:

[0138] Add tap water to the microbial fertilizer to dissolve it, with the amount of tap water being 49 times the mass of the microbial agent. After stirring thoroughly, place it in a warm indoor place (27℃) and allow it to ferment naturally for 1.5 days. Dilute it before use. The yeast content after dilution is shown in Table 4. Apply it directly to the drip irrigation of crops.

[0139] Experiments were conducted in greenhouses where cucumber and tomato root rot had occurred for many years. The planting area for cucumbers and tomatoes was approximately 2,500 seedlings per acre. One acre was selected for each treatment group, while the control group received no treatment and was fertilized and watered normally.

[0140] Control group 1 used Saccharomyces sp. (Ningbo Mingzhou Biotechnology Co., Ltd., model BMZ126481) to replace the yeast in this patented microbial fertilizer, at a dosage of 2 kg / mu.

[0141] Control group 2 used 2 kg / mu of Hanseniaspora vineae (provided by the Microbial Resource Classification Laboratory of the School of Food and Bioengineering, Zhengzhou University of Light Industry, No. WYCCW10371) to replace the yeast in this patented microbial fertilizer.

[0142] The experimental group used microbial fertilizer made from the yeast strain (CCTCC NO: M 2019643) in this patent, with a dosage of 2 kg / mu.

[0143] First, dissolve the fertilizer in 50 times its volume of tap water, stir thoroughly, and place it in a warm indoor place (27℃) for natural fermentation for 2 days. Then, test the total number of yeast cells in the fermentation liquid. Next, dilute the fertilizer to 1.0*10^7 CFU / ml in all three groups (control group 1, control group 2, and experimental group) and apply it with irrigation water. Apply the fertilizer with irrigation water once every 7 days. Observe and count the occurrence of root rot (leaf wilting, root necrosis, plant death, etc.) on days 7, 45, 90, and 120, and calculate the incidence of root rot.

[0144] Table 4. Total yeast count in fermentation broth of microbial fertilizers with different formulations

[0145]

[0146] Table 5. Incidence of root rot

[0147]

[0148] The above results indicate that the microbial fertilizer made from yeast An-15 for preventing root rot in plants has a significant effect on the prevention and control of root rot in cucumbers and tomatoes. After two days of fermentation, the total number of yeasts in the fermentation liquid was significantly higher than that in control groups 1 and 2, and the long-term incidence of root rot was only 5%, significantly lower than that in control groups 1 and 2. This shows that under the same addition amount, the yeast of this patent not only has a fast proliferation rate, but also has a significantly better effect on preventing and controlling root rot than the control group.

[0149] Example 7

[0150] In Example 6, the brown sugar in the experimental group was replaced with sucrose, maltose, glucose, raw sugar, granulated sugar, brown sugar, and black sugar, respectively. The rest of the preparation was the same as in Example 6. The yeast content in the diluted fermentation broth is shown in Table 6. The incidence of root rot is shown in Table 7.

[0151] Table 6

[0152]

[0153] Table 7

[0154]

[0155] Example 8

[0156] The difference between this embodiment and the experimental group in Embodiment 6 is the preparation of the microbial fertilizer; the rest is the same as the experimental group in Embodiment 6.

[0157] Preparation of microbial fertilizer in this embodiment:

[0158] (1) Weigh according to the following mass parts: 4 kg of brown sugar, 0.4 kg of corn starch, and 0.5*10^9 CFU / g of yeast AN-15. It should be noted that the yeast content refers to the yeast content in the microbial agent (including brown sugar, corn starch and yeast).

[0159] (2) Put brown sugar and corn starch into a mixer and mix evenly for 2 minutes. After mixing, grind the mixture into 90 mesh using a grinder.

[0160] (3) Add the yeast to the mixture of brown sugar and corn starch, stir and mix for 2 minutes to make microbial fertilizer.

[0161] How to use:

[0162] Add tap water to the microbial fertilizer to dissolve it; the amount of tap water should be 20 times the mass of the microbial agent. After stirring thoroughly, place it in a warm indoor place (20℃) and allow it to ferment naturally for 5 days. It is ready when a white film appears on the surface, white foam appears, and an alcoholic aroma is emitted. Dilute before use. The yeast content in the diluted fermentation liquid is shown in Table 8. The incidence of root rot is shown in Table 9.

[0163] Table 8

[0164]

[0165] Table 9

[0166]

[0167] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: the microbial agent has excellent control effects on diseases such as root rot in plants (e.g., cucumbers and tomatoes). The microbial agent is used via tap water fermentation, which is simple to operate, convenient to use, and novel in approach. This microbial agent can increase microbial abundance, activate soil structure, and promote plant growth.

[0168] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A microbial inoculant, characterized in that, The microbial agent includes yeast An-15, its culture medium, and an optional anti-adhesion agent; The yeast strain An-15 is classified as Saccharomyces cerevisiae and was deposited at the China Center for Type Culture Collection (CCTCC) on August 19, 2019, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M2019643. The culture medium includes a sugar mixture formed by mixing one or more sugars in equal proportions; The sugar includes sugar in pure form or sugar in mixture form; The sugar in its pure form includes sucrose, maltose, or glucose; The sugar in the form of the mixture includes raw sugar, brown sugar, red sugar, or black sugar.

2. The microbial agent according to claim 1, characterized in that, The content of yeast AN-15 is 1.0*10^9 CFU / g~2.0*10^9 CFU / g; Preferably, the culture medium is brown sugar; Preferably, the anti-blocking agent is selected from any one or more of the following: corn starch, attapulgite, kaolin, diatomaceous earth, or bentonite; more preferably, it is corn starch; Preferably, the microbial agent includes an anti-adhesion agent, and the weight ratio of the culture medium to the anti-adhesion agent is (4~9):

1.

3. A method for preparing a microbial inoculant, characterized in that, The preparation method includes: The yeast strain An-15 was mixed with the culture medium to obtain the microbial inoculum. The yeast strain An-15 is classified as Saccharomyces cerevisiae and was deposited at the China Center for Type Culture Collection (CCTCC) on August 19, 2019, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M2019643. The culture medium includes a sugar mixture formed by mixing one or more sugars in equal proportions; The sugar includes sugar in pure form or sugar in mixture form; The sugar in its pure form includes sucrose, maltose, or glucose; The sugar in the form of the mixture includes raw sugar, brown sugar, red sugar, or black sugar.

4. The preparation method according to claim 3, characterized in that, The preparation method includes: The culture medium and the anti-adhesion agent were mixed to obtain mixture 1; and The yeast strain An-15 and the mixture 1 are mixed to obtain the microbial inoculant; Preferably, the anti-blocking agent is selected from any one or more of the following: corn starch, attapulgite, kaolin, diatomaceous earth, or bentonite; more preferably, it is corn starch; Preferably, before obtaining the mixture 1, the culture medium and the anti-adhesion agent are mixed and then pulverized; more preferably, they are pulverized to 10-80 mesh. Preferably, a mixer is used for the mixing; More preferably, the mixing time is 5 to 10 minutes.

5. A method for preparing microbial fertilizer, characterized in that, The method includes: The microbial agent according to claim 1 or 2 or the microbial agent prepared by the preparation method according to claim 3 or 4 is mixed with water and fermented to obtain a fermentation broth; The fermentation broth was diluted to obtain the microbial fertilizer.

6. The method according to claim 5, characterized in that, The mass ratio of the microbial agent to the water is 1:(25~50); Preferably, the fermentation time is 1 to 2 days, and the fermentation temperature is 25 to 30°C.

7. A microbial fertilizer, characterized in that, The microbial fertilizer is the microbial fertilizer prepared by the method described in claim 5 or 6.

8. The microbial fertilizer according to claim 7, characterized in that, The content of yeast AN-15 in the microbial fertilizer is 1.0*10^7 CFU / mL~1.0*10^8 CFU / mL.

9. A method for preventing and controlling plant root rot, characterized in that, The method includes: The microbial fertilizer of claim 7 or 8 is applied to the plant.

10. The method according to claim 9, characterized in that, The microbial fertilizer is applied to the plants using any one or more of the following methods: fertigation, drip irrigation, or spraying; Preferably, the plant is a tomato or a cucumber; Preferably, the application rate of the microbial fertilizer is 2-3 kg / mu; Preferably, the microbial fertilizer is applied before or at the early stage of disease onset. Preferably, the microbial fertilizer is applied at least 3 to 4 times; Preferably, the microbial fertilizer is applied at intervals of 5 to 7 days.

Images