Application of Rhizocystis ulcerans in peanut cultivation

Abstract

This invention provides the application of *Rhizonhagus intraradices* in peanut cultivation, relating to the field of microbial technology. This invention demonstrates for the first time that *Rhizonhagus intraradices* BGSC-107 can promote peanut growth, including increasing germination rate, promoting root and stem development, and increasing fresh weight. Furthermore, this application also demonstrates that *Rhizonhagus intraradices* BGSC-107 has an inhibitory effect on *Cercospora personata*, the pathogen of peanut black spot disease, and can effectively prevent the occurrence of peanut black spot disease. This invention also provides a method for preparing a microbial agent containing *Rhizonhagus intraradices* BGSC-107, providing a theoretical basis for the industrial production of products containing this microbial agent.

Description

Technical Field

[0001] This application relates to the field of microbial technology, and in particular to the application of Rhizocystis ulcerans in peanut cultivation. Background Technology

[0002] *Rhizophagus intraradices* is an arbuscular mycorrhizal fungus whose hyphae penetrate the epidermal cells of plant roots, enabling it to form symbiotic relationships with 80% of higher plants on land. This symbiotic relationship provides the plant with essential mineral nutrients, particularly phosphorus and nitrogen, while the plant provides photosynthetic products as a carbon source for the fungus. *Rhizophagus intraradices* can form symbiotic relationships with a variety of plants, such as soybeans and peppers. However, interactions between *Rhizophagus intraradices* and peanuts have not yet been reported.

[0003] Peanuts, also known as groundnuts, are annual herbaceous plants belonging to the genus Arachis in the family Fabaceae. Originally from South America, peanuts are cultivated in over 100 countries worldwide, most widely in Asia, followed by Africa. According to Chinese literature, peanut cultivation predates that of Europe by approximately 100 years. Peanuts are hailed as "plant meat" due to their high oil content (up to 50%), excellent quality, and fragrant aroma. Besides being used for food, they are also used in printing and dyeing, papermaking, and other industries. Peanuts are also a traditional Chinese medicine, used to treat malnutrition, spleen and stomach disorders, coughs with phlegm, and insufficient lactation. However, peanut cultivation is currently plagued by various diseases, leading to reduced yields. Therefore, there is an urgent need to develop effective pesticides and fertilizers to control peanut diseases. Summary of the Invention

[0004] This invention is the first to discover that the root endophyte BGSC-107 can effectively promote peanut growth and inhibit the growth of peanut pathogens, thereby playing a role in preventing and controlling peanut diseases and providing a new type of bio-fertilizer for peanuts.

[0005] On the one hand, this application provides the application of Rhizonhagus intraradices in promoting peanut growth and controlling peanut diseases, wherein the Rhizonhagus intraradices is Rhizonhagus BGSC-107.

[0006] Preferably, promoting peanut growth includes increasing peanut germination rate, promoting peanut root and stem development, and increasing peanut fresh weight.

[0007] Furthermore, the peanut diseases include peanut white mold and / or peanut black spot.

[0008] Preferably, the peanut disease is peanut black spot disease.

[0009] The control measures are achieved by suppressing Cercospora personata, the pathogen of peanut black spot, and Sclerotium rolfsii, the pathogen of peanut white rot.

[0010] Among them, Rhizocystis ulcerans BGSC-107 can be cultured using general fungal culture methods.

[0011] On the other hand, this application also provides a method for preparing a fungal agent, wherein the fungal agent includes Rhizocystis jirovecii BGSC-107.

[0012] Furthermore, the method includes the following steps:

[0013] Step 1: Sterilize the mixed substrate, wherein the substrate comprises peat moss and vermiculite in a mass ratio of (4-6):1;

[0014] Step 2: Inoculate the substrate with 1%-5% of Rhizocystis jirovecii BGSC-107 to obtain a substrate containing the fungus.

[0015] Step 3: Sow alfalfa on the bacterial substrate. After germination, cultivate for 0.5-1.5 months, then dry, crush and granulate the alfalfa roots to obtain the bacterial agent.

[0016] Preferably, the matrix comprises peat moss and vermiculite in a mass ratio of 5:1.

[0017] Preferably, the inoculation amount is 3%.

[0018] Preferably, the germination is carried out for 1 month.

[0019] In a preferred embodiment, the method for preparing the microbial agent includes the following steps:

[0020] Step 1: Mix peat moss and vermiculite in a mass ratio of (4-6):1, sterilize at 121℃ for 1-2 hours to obtain the substrate;

[0021] Step 2: Inoculate the substrate with 1%-5% of Rhizocystis jirovecii BGSC-107 to obtain a substrate containing the fungus.

[0022] Step 3: Sow alfalfa on the substrate containing the bacteria, and cultivate the alfalfa using conventional methods. After germination, cultivate for 0.5-1.5 months, then take the alfalfa roots and dry them at 35℃-45℃, crush and granulate them to obtain the inoculum.

[0023] Those skilled in the art can select specific drying methods and crushing and granulation sizes according to actual needs, and no further limitations are made here.

[0024] On the other hand, this application also provides the bacterial agent prepared by the method described above.

[0025] Preferably, the microbial agent may include:

[0026] A1) Rhizocystis ulcerans BGSC-107;

[0027] A2) Dead fungal suspension of Rhizocystis jirovecii BGSC-107;

[0028] A3) Metabolites of Rhizocystis BGSC-107;

[0029] A4) Extract of Rhizocystis ulmoides BGSC-107;

[0030] A5) Mycelium of Rhizocystis Roots BGSC-107;

[0031] A6) Rhizocystis BGSC-107 spores.

[0032] Preferably, the microbial agent can promote peanut growth and / or prevent peanut diseases.

[0033] On the other hand, this application also provides a fertilizer containing Rhizocystis BGSC-107 or the microbial agent as described.

[0034] Preferably, the fertilizer may include excipients, which may be suitable solvents, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, thickeners, encapsulating agents, humectants, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc.

[0035] Preferably, the fertilizer is a peanut fertilizer, which can promote peanut growth and / or prevent peanut diseases.

[0036] On the other hand, this application also provides the application of the aforementioned microbial agent or fertilizer in promoting peanut growth and preventing peanut diseases.

[0037] Furthermore, the peanut diseases include peanut white mold and / or peanut black spot.

[0038] Preferably, the peanut disease is peanut black spot disease.

[0039] Those skilled in the art will understand that the dosage of the microbial agent or fertilizer can be adjusted according to actual conditions. In a preferred embodiment, the dosage of the microbial agent or fertilizer is 2g / plant.

[0040] The present invention has the following beneficial effects:

[0041] 1. This application demonstrates for the first time that *Rhizocystis ulmoides* BGSC-107 can promote peanut growth, including increasing germination rate, promoting root and stem development, and increasing fresh weight. Furthermore, this application also demonstrates that *Rhizocystis ulmoides* BGSC-107 has an inhibitory effect on *Cercospora coccidioides*, the pathogen of peanut black spot disease, and can effectively prevent the occurrence of peanut black spot disease.

[0042] 2. This application also provides a method for preparing a fungal agent containing Rhizocystis jirovecii BGSC-107, which provides a theoretical basis for the industrial production of products containing this fungal agent. Attached Figure Description

[0043] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, are used to interpret this application and do not constitute an undue limitation thereof. In the drawings:

[0044] Figure 1 This is a schematic diagram of peanut cultivation in Example 2. From left to right, pots 1-3 are the blank control group, and pots 4-6 are the BGSC-107 treatment group. Detailed Implementation

[0045] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0046] Unless otherwise specified in the examples, the conditions shall be performed according to the standard conditions or the conditions recommended by the manufacturer.

[0047] Among them, Rhizonhagus intraradices BGSC-107 was purchased from the Subtropical Arbuscular Mycorrhizal (AM) Fungal Resource Preservation Center; the peanut variety was Qihua No. 1.

[0048] Unless otherwise specified, in the following embodiments, reagents or instruments whose manufacturers are not indicated are all conventional products that can be purchased commercially.

[0049] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in the fields of microbiology, biochemistry, analytical chemistry, cell culture, and related areas.

[0050] In addition, the "water" mentioned in this invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.

[0051] In the following examples, unless otherwise specified, % means wt%, i.e., weight percentage.

[0052] Example 1: Culture of Rhizocystis jirovecii

[0053] In this embodiment, alfalfa is used to ferment Rhizocystis jirovecii BGSC-107, specifically including the following steps: The substrate is sterilized at 121℃ for 1 hour and filled into flower pots (10cm x 10cm x 10cm). Rhizocystis jirovecii BGSC-107 is added to 2 / 3 of the substrate in the flower pot, and alfalfa is sown about 2cm above the substrate (alfalfa seeds are sterilized by soaking in 10% sodium hypochlorite solution for 5 minutes). 30 seeds are sown per pot. After one week of emergence, the seedlings are thinned to 15 plants per pot. Hoagland nutrient solution is applied twice a week, and water is balanced once a week. Finally, one month after germination, the stems and leaves of the plants are removed. The alfalfa roots, Rhizocystis jirovecii BGSC-107, and substrate are dried at a low temperature of 35℃-45℃, pulverized, and granulated to obtain the Rhizocystis jirovecii BGSC-107 inoculum. The content of (live) spores in the fungal agent was detected by hemocytocyte counting method. The test results and specific conditions are shown in Table 1.

[0054] Table 1

[0055]

[0056]

[0057] Note: The proportions of the matrix components are by mass.

[0058] As shown in Table 1, when peat moss and vermiculite with a mass ratio of 5:1 are used as substrate materials and the inoculum amount is 3%, the Root Rhizocystis BGSC-107 inoculum agent with high spore content can be efficiently cultured within 1 month.

[0059] Example 2: Investigation on the growth-promoting effect of Rhizocystis jirovecii on peanuts

[0060] In this embodiment, the fungal agent prepared by the optimized method in Example 1 was applied to the rhizosphere of peanut to investigate the promoting effect of Rhizocystis jirovecii on peanut growth.

[0061] Specifically, the following steps were taken: Nutrient soil was used as the peanut culture medium. 2g / plant of the *Rhizopus oryzae* BGSC-107 inoculant obtained in Example 1 was added to 2 / 3 of the substrate in 10cm x 10cm x 10cm pots. An equal amount of water was applied to the blank control group. Peanut seeds were sterilized by soaking in 75% alcohol for 1 minute followed by soaking in 10% sodium hypochlorite solution for 15 minutes. One seed was then sown in each pot, for a total of 60 pots. Germination rate was recorded after emergence. Hoagland's nutrient solution was applied twice a week, with the same amount applied to each pot. The pots were randomly arranged in a block design, with the order changed weekly. After 21 days of cultivation, the peanut plants were removed, and root length, plant height, and fresh weight were measured using trypan blue staining. The results and specific conditions are shown in Table 2. Figure 1 As shown, all data are averages.

[0062] Table 2

[0063] Group Germination rate (%) Root length (cm) Plant height (cm) Fresh weight (g) Mycorrhizal infection rate (%) BGSC-107 processing group 94.25 11.59 15.41 5.60 75.26 control group 73.76 7.23 6.57 2.59 -

[0064] From Table 2 and Figure 1 The results showed that root endophytic cysts could effectively infect the peanut rhizosphere and promote peanut germination and growth.

[0065] Example 3: Investigation into the preventive and therapeutic effects of Rhizocystis jirovecii on common peanut diseases.

[0066] To further explore the interaction between Rhizocystis ulmiformis and peanut, this example tests the preventive and control effects of Rhizocystis ulmiformis on common peanut diseases.

[0067] The plate confrontation method was used to simultaneously inoculate equal amounts of *Rhizocystis ulmoides* and *Sclerotium rolfsii* (purchased from Taisto Biotechnology, catalog number: TS330731), the pathogen of peanut white rot, onto PDA solid plates. After incubation at 22℃-28℃ for one week, the presence of an inhibition zone between the two was observed. The same method was used to test the antagonism between *Rhizocystis ulmoides* and *Fusarium sp.* (purchased from Taisto Biotechnology, catalog number: TS332341), the pathogen of peanut root rot, and *Cercospora personata* (purchased from Taisto Biotechnology, catalog number: TS332354), the pathogen of peanut black spot. The specific results are shown in Table 3.

[0068] Table 3

[0069] Antagonistic situation Neat small sclerotium + Fusarium root rot of peanut - Cercospora globosum ++

[0070] Note: "++" indicates a clear inhibition band, "+" indicates the presence of an inhibition band, and "-" indicates the absence of an inhibition band.

[0071] As shown in Table 3, there is a clear inhibition zone between Rhizocystis ulmoides and Cercospora globosum, the pathogen of peanut black spot disease, which proves that Rhizocystis ulmoides is antagonistic to the pathogen of peanut black spot disease and has the potential to control peanut black spot disease.

[0072] Furthermore, a pot experiment for controlling peanut black spot disease was conducted in this embodiment. Specifically, nutrient soil was used as the peanut culture medium. 2g / plant of the optimized rhizocystis jirovecii agent obtained in Example 1 was added to 2 / 3 of the substrate in a 10cm x 10cm x 10cm pot. An equal amount of water was applied to the blank control group. Peanut seeds were sterilized by soaking in 75% alcohol for 1 minute and then in 10% sodium hypochlorite solution for 15 minutes before being inoculated into the pots, one seed per pot. Hoagland's nutrient solution was applied twice a week, with the same amount applied to each pot. The pots were arranged in a randomized block design, with the order changed weekly. After 14 days of culture, the pathogenic fungus *Cercospora glomerata* of peanut black spot disease was inoculated, with 250μL inoculated per peanut plant. After 21 days of culture, the control status was statistically analyzed. The disease index and control effect were calculated according to Table 4. The control effect is shown in Table 5. The calculation methods for the disease index and control effect are as follows.

[0073]

[0074] Table 4

[0075] Disease level Grading Standards Level 0 No lesions Level 1 The lesion diameter is 0-0.5 mm. Level 2 The lesion diameter is 0.5-2.0 mm. Level 3 The lesion diameter is 2.0-5.0 mm. Level 4 Lesions with a diameter greater than 5.0 mm

[0076] Table 5

[0077] Processing group Prevention and control efficacy / % BGSC-107 processing group 88.60 Blank control /

[0078] As shown in Table 5, the Rhizocystis BGSC-107 inoculant can effectively control peanut black spot disease.

[0079] The above description is merely an embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of this application should be included within the scope of the claims of this application.

Claims

1. Root endophytic rhizocysts ( Rhizonhagus intraradices Its application in the prevention and control of flowering plant diseases is characterized by, The root endophytic fungus is root endophytic fungus BGSC-107, and the peanut disease is peanut black spot disease.

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