Preparation and application of fungus sticks against sunflower sclerotinia

Abstract

The invention discloses the preparation and application of a fungus stick antagonizing sunflower sclerotinia disease, and the preparation includes the following steps: step S1, crushing naturally dried corn stalks to obtain corn stalk particles; step S2, preparing an endophytic bacterial agent; step S3, uniformly mixing the corn stalk particles and the bacterial agent to obtain a granulated material; step S4, granulating the granulated material using a granulating device to obtain a granular biocontrol agent; step S5, drying the granular biocontrol agent to obtain a fungus stick antagonizing sunflower sclerotinia disease. The fungus stick is used for the prevention and control of sunflower sclerotinia disease, which can effectively enhance the resistance of sunflower to sclerotinia disease, improve the prevention and control effect of sunflower sclerotinia disease, and meet the prevention and control demand of sclerotinia disease in sunflower production.

Description

Technical Field

[0001] The invention relates to the technical field of sunflower sclerotinia pathogen prevention and control, and specifically relates to the preparation and application of fungus sticks for antagonizing sunflower sclerotinia pathogen. Background Art

[0002] Sunflower (Helianthus annuus L.) is an important high-quality oil crop widely planted around the world. my country is a major producer and consumer of sunflowers, and sunflower oil occupies an important position in the consumption structure of edible oils. The degree of damage caused by sunflower sclerotinia to sunflower production is increasing and spreading year by year, which has an adverse impact on sunflower production. Sunflower sclerotinia is a serious fungal soil-borne disease caused by Sclerotinia sclerotiorum. The pathogen [Sclerotiniasclerotiorum (Lib.) de Bary = Sclerotinia libertiana Fuckel.] is a facultative parasite. When sunflower is infected with sclerotinia, mild cases will lead to an increase in the husk rate, a decrease in protein content and oil content, and the oil will taste bitter, which will reduce the edible value and commercial value of sunflower seeds; severe cases will lead to a total crop failure, causing huge economic losses and obstacles to sunflower production.

[0003] In recent years, due to the lack of sunflower varieties with outstanding resistance, with the continuous increase in the planting area of ​​sunflowers, the large-scale occurrence of sclerotinia disease from the flowering period to the withering period has become increasingly serious. At present, the prevention and control measures for sunflower sclerotinia disease are still mainly based on crop rotation, but due to the difficulty of crop rotation with gramineous crops, and the fact that the pathogen can survive in the soil in the form of sclerotia for 10 years, crop rotation to prevent and control sunflower sclerotinia disease can only be a stopgap measure. In addition, although certain disease prevention effects have been achieved in the prevention and control of sclerotinia disease by chemical agents, the subsequent problems of environmental pollution, pesticide residues in soil and rivers caused by chemical control are becoming increasingly prominent. In addition, the plants of sunflowers are too tall during the flowering period, and it is very inconvenient to spray agents in the field, which increases the difficulty of chemical prevention and control, making it difficult to implement chemical agent technology on a large scale. Biological control refers to reducing the number of pathogens or weakening the pathogenicity of sclerotinia by secreting products of live microorganisms, thereby reducing the occurrence of diseases caused by pathogens, and this prevention and control measure has gradually shown its importance in production. Therefore, the use of biological control in the prevention and control of sclerotinia disease is an effective measure for environmentally friendly green production of sunflower.

[0004] Plant endophytes refer to fungi or bacteria that live in healthy plant tissues or organs at some or all stages of their life cycle. Endophytes will not cause obvious symptoms in plants and can form a harmonious and stable symbiotic relationship with plants. Plant endophytic Bacillus is widely present in plants and can co-evolve with plants. On the one hand, endophytes can obtain the energy and material nutrition necessary for growth through the plant body, and on the other hand, they can affect the plant body through their own metabolites or signal transduction, thereby effectively promoting plant growth and the performance of biocontrol functions. They have broad-spectrum antibacterial activity and extremely strong stress resistance; because the secondary metabolites they produce are rich in variety, stable, and have broad-spectrum antibacterial activity, they can promote plant growth, so in recent years it has been widely used in the biological control of plant diseases. In the prior art, when using endophytes to control sunflower sclerotinia, sunflower seeds are usually directly soaked in bacterial solution or sunflower plants are directly sprayed with bacterial solution. The control effect of this application method is not ideal. Summary of the invention

[0005] To this end, the technical problem to be solved by the present invention is to provide a method for preparing and applying a fungus stick capable of antagonizing sunflower sclerotinia disease, so as to solve the technical problem that the existing use of endophytic bacteria to control sunflower sclerotinia disease has a poor control effect.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] The preparation of the fungus stick for antagonizing sunflower sclerotinia disease comprises the following steps:

[0008] Step S1, crushing naturally dried corn stalks to obtain corn stalk particles;

[0009] Step S2, preparing an endophytic bacterial agent;

[0010] Step S3, mixing the corn stalk particles and the bacterial agent evenly to obtain a granulated material;

[0011] Step S4, granulating the granulated material using a granulating device to obtain a granular biocontrol agent;

[0012] Step S5, drying the granular biocontrol agent to obtain fungus sticks antagonizing sunflower sclerotinia pathogen.

[0013] In the preparation of the above-mentioned antagonistic sunflower sclerotinia bacteria sticks, in step S1, the particle size of the corn straw particles is 5 to 10 mm (a particle size less than 5 mm or greater than 10 mm will affect the density of the prepared bacteria sticks, thereby affecting the storage and sustained release of Bacillus subtilis Y116 in the bacteria sticks), and the moisture content in the corn straw is 10 to 20 wt%; if the moisture content of the straw is too high or too low, it will affect the extrusion forming effect during the production of the bacteria sticks.

[0014] In the preparation of the above-mentioned antagonistic sunflower sclerotinia bacteria stick, in step S2, the preparation method of the endophytic bacterial agent is:

[0015] Step S2-1, under sterile conditions, a single colony of the endophytic bacterial strain is picked up with an inoculation loop, and inoculated into 30 mL of seed culture medium for constant temperature shaking culture, wherein the constant temperature shaking culture conditions are: constant temperature shaking culture at 180 rpm and 28° C. for 2 days; after the culture is completed, a seed culture solution is obtained; the endophytic bacteria is Bacillus subtilis Y116, and the deposit number is CGMCC 27043;

[0016] Step S2-2, inoculating the seed culture solution into the fermentation medium for fermentation, the inoculation amount of the seed culture solution is 5% of the fermentation medium, the fermentation conditions are: culture at 200rpm, 28°C for 4 days with ventilation, and the dissolved oxygen content in the fermentation medium is maintained at 10-16%; after the fermentation is completed, filter to obtain the bacterial agent of endophytic bacteria.

[0017] In the preparation of the above-mentioned antagonistic sunflower sclerotinia fungus stick, in step S2-1, the seed culture medium is a Luria-Bertani liquid culture medium, which is composed of the following components: 5g sodium chloride, 5g yeast extract, 10g peptone, and 1000mL distilled water; the pH of the seed culture medium is 7.2-7.5;

[0018] The fermentation medium consists of the following components: 30 g of peptone, 30 g of yeast extract, 30 g of soluble starch, and 60 g of sodium chloride.

[0019] In the preparation of the above-mentioned antagonistic sunflower sclerotinia fungus sticks, in step S3, the mass volume ratio of corn stalk particles to the bacterial agent is 4-5 kg / L. A small amount of corn stalk particles will waste the bacterial agent; a large amount of corn stalk particles will affect the fermentation efficiency.

[0020] In the preparation of the above-mentioned antagonistic sunflower sclerotinia disease fungus sticks, urea is also added to the granulated material, and the mass of the added urea is 0.5-0.7% of the mass of the corn straw particles; the role of adding urea is to optimize the carbon-nitrogen ratio in the fungus sticks, promote the abundance of bacteria, and is beneficial to the reproduction of bacteria. Compared with the fungus sticks without urea, the addition of 0.5-0.7% of the mass of the corn straw particles of urea can enable the fungus sticks to obtain better and longer-lasting sclerotinia disease prevention and control effects after application.

[0021] In the preparation of the above-mentioned antagonistic sunflower sclerotinia fungus sticks, in step S4, the granulation equipment is the mobile biochar granulator in patent CN209138573U, and the obtained granular biocontrol agent has a diameter of 4-5 mm, a length of 10-15 mm, and a density of 0.4-0.5 g / cm 3 .

[0022] In the preparation of the above-mentioned antagonistic sunflower sclerotinia pathogen bacteria sticks, in step S5, the drying method is air-drying in the sun or air-drying at room temperature; air-drying in the sun or air-drying at room temperature until the water content of the granular biocontrol agent is 20-30wt%, thereby obtaining antagonistic sunflower sclerotinia pathogen bacteria sticks; controlling the water content of the bacteria sticks to 20-30wt% is beneficial to the survival of the bacteria and the storage of the bacteria sticks.

[0023] The application of the antagonistic sunflower sclerotinia disease fungus stick is to prepare the antagonistic sunflower sclerotinia disease fungus stick using the preparation method of the antagonistic sunflower sclerotinia disease fungus stick and use it for the prevention and treatment of sunflower sclerotinia disease.

[0024] The application of the above-mentioned antagonistic sunflower sclerotinia pathogen fungus sticks is as follows: during the budding period of sunflowers, the fungus sticks are applied jointly by broadcasting them between the rows of sunflowers and spot-seeding them in the rhizosphere; the total application amount of the fungus sticks is calculated based on 15.0 g per sunflower plant, and the ratio of the application amount of the fungus sticks broadcast between the rows and the spot-seeding fungus sticks is 1:1.

[0025] The technical solution of the present invention achieves the following beneficial technical effects:

[0026] 1. The fermentation method, granulation method and field application method of endophytic Bacillus subtilis of sunflower antagonistic to Sclerotinia sclerotiorum of the present invention can solve the problem of difficulty in preventing and controlling sclerotinia disease in sunflower production. The present invention processes the biocontrol agent Bacillus subtilis Y116 antagonistic to Sclerotinia sclerotiorum into a granular form by granulation, which is not only more convenient for mechanized application, but also the endophytic bacteria in the prepared bacterial sticks can be slowly released into the sunflower plant under the action of soil moisture after application, thereby effectively enhancing the resistance of sunflower to sclerotinia disease, improving the prevention and control effect of sunflower sclerotinia disease, and meeting the prevention and control demand of sclerotinia disease in sunflower production; this can not only reduce the use of chemical reagents, but also has a positive effect on environmentally friendly agricultural production.

[0027] 2. When preparing the microbial agent, the present invention controls the types and ratios of the components in the culture medium, as well as the fermentation conditions, so that the prepared microbial agent is mixed with corn straw particles and urea and then granulated to obtain a microbial stick with good sustained release and microbial agent activity; by controlling the size and density of the microbial sticks obtained by granulation by a granulator within a specific range, the Bacillus subtilis Y116 in the microbial sticks can be placed in an oxygen-deficient and water-deficient dormant state under a room temperature and dry storage environment, and when applied to the soil, under the action of soil moisture, the microbial sticks slowly absorb water and disperse, so that the Bacillus subtilis Y116 in the microbial sticks are reactivated and enter the sunflower plant, thereby enhancing the resistance of the sunflower to sclerotinia disease.

[0028] 3. When applying, the combined application of mushroom sticks and spot-seeding mushroom sticks between rows of sunflowers is used. Compared with the combined application of mushroom sticks and root irrigation with bacterial agents between rows of sunflowers and root irrigation with single bacterial agents, the control effect of sunflower sclerotinia disease is better. This is because the combined application of mushroom sticks and spot-seeding mushroom sticks can simultaneously antagonize the sclerotinia in the deep rhizosphere and on the surface. At the same time, the Bacillus subtilis Y116 in the mushroom sticks is slowly activated and released under the action of moisture in the soil, and has a long-term control effect; compared with application in the early and late flowering stages, application in the budding stage has a better control effect on sunflower sclerotinia disease. This is because application in the budding stage can effectively inhibit the initial infection of sclerotinia hyphae on the root system and stem base of sunflowers, thereby achieving better control effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Actual photos of sunflower materials used in the embodiments of the present invention;

[0030] Figure 2 The Bacillus subtilis strain Y116 used in the embodiments of the present invention;

[0031] Figure 3 Cluster analysis diagram of 16SrDNA of Bacillus subtilis strains used in the examples of the present invention;

[0032] Figure 4 Actual photos of the granulator used in the embodiments of the present invention and the produced mushroom sticks;

[0033] Figure 5 A mechanical device for crushing corn stalks in an embodiment of the present invention. DETAILED DESCRIPTION

[0034] Example 1

[0035] The preparation process of the fungus sticks for antagonizing sunflower sclerotinia in this embodiment is as follows:

[0036] Step S1, crushing the naturally dried corn stalks to a particle size of 5 to 10 mm to obtain corn stalk particles; the moisture content of the naturally dried corn stalks is 20 wt%; the crusher used in this embodiment is Figure 5 .

[0037] Step S2: preparing an endophytic bacterial agent, the specific preparation method is as follows:

[0038] Step S2-1, under sterile conditions, pick a single colony of the endophytic bacterial strain with an inoculation loop, inoculate it into 30 mL of seed culture medium and culture it at constant temperature and shaking. The conditions of constant temperature and shaking culture are: constant temperature and shaking culture at 180 rpm and 28° C. for 2 days; after the culture is completed, obtain the seed culture solution;

[0039] The endophyte used in this embodiment was isolated from resistant sunflower seeds by the Pathology Laboratory of the College of Horticulture and Plant Protection of Inner Mongolia Agricultural University. The endophyte is Bacillus subtilis Y116, and was deposited in the General Microbiology Center of the China Culture Collection Administration on April 7, 2023. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The classification name is Bacillus subtilis, and the deposit number is CGMCCNO.27043. The antagonistic effect of the bacterium was published online by the members of the invention team of the present invention in the journal "Crops Journal", and the paper title is "Isolation, Screening and Identification of Endophytes in Sunflower Seeds".

[0040] The method for preserving the bacterial agent is as follows: sterilize 40% glycerol with high temperature and high pressure, activate the purified endophytic Bacillus subtilis Y116 strain in LB liquid medium for 24 hours, and culture it to the logarithmic phase for bacterial preservation. That is, add 1mL of 40% glycerol and 1mL of bacterial solution to the sterilized bacterial storage tube, mix well, and store in a -20℃ refrigerator for long-term storage;

[0041] The seed culture medium used in this example is Luria-Bertani liquid culture medium, which is composed of the following components: 5 g sodium chloride, 5 g yeast extract, 10 g peptone, and 1000 mL distilled water; the pH of the seed culture medium is 7.2-7.5;

[0042] Step S2-2, inoculating the seed culture solution into 250 mL of fermentation medium for fermentation, the inoculation amount of the seed culture solution is 5% of the fermentation medium, the fermentation conditions are: culture at 200 rpm, 28 ° C for 4 days, and the dissolved oxygen content in the fermentation medium is maintained at 10-16%; after the fermentation is completed, filter to obtain the bacterial agent of endophyte; the fermenter used in this embodiment is produced by Shanghai Baoxing Biological Equipment Engineering Co., Ltd.

[0043] The fermentation medium used in this example is composed of the following components: 30 g of peptone, 30 g of yeast extract, 30 g of soluble starch, and 60 g of sodium chloride.

[0044] Step S3, 100 kg of corn stalk particles, 20 L of bacterial agent and 0.5 kg of urea (N: 46%) are mixed evenly to obtain a granulated material;

[0045] Step S4: Granulate the granulated material using a granulating device to obtain a granular biocontrol bacteria stick with a diameter of 4 to 5 mm and a length of 10 to 15 mm, and a density of 0.4 to 0.5 g / cm 3 ;

[0046] The granulation equipment in this embodiment is a mobile biochar granulator in patent CN209138573U, including a chassis, a motor, a coupling, a reduction box, an extrusion bin, a roller bearing, a discharge hopper, a feed hopper, a feed hopper adjustment handle, and an extrusion die; the specific structure is shown in patent CN209138573U, which will not be repeated here, and the actual figure is shown in Figure 4 During granulation, the granulated materials mixed in proportion are put into the hopper of the granulator, the machine is started, and the feed amount is controlled by adjusting the feed adjustment handle of the hopper to obtain a granular biocontrol agent rich in Bacillus subtilis Y116 that antagonizes Sclerotinia sclerotiorum.

[0047] Step S5, drying the granular biocontrol agent and then bagging it to obtain antagonistic sunflower sclerotinia pathogen bacteria sticks; the drying method is air-drying in the sun or air-drying at room temperature; air-drying in the sun or air-drying at room temperature until the water content of the granular biocontrol agent is within 20-30wt%, thus obtaining antagonistic sunflower sclerotinia pathogen bacteria sticks, and storing them at room temperature after packaging to prevent them from being wetted by rain, snow and water.

[0048] Example 2

[0049] The application method of the fungus sticks against sunflower sclerotinia in this example is: the fungus agent and fungus sticks against sunflower sclerotinia prepared in Example 1 are used for the prevention and treatment of sunflower sclerotinia. Three different application methods are set: treatment 1, treatment 2 and treatment 3, and a blank control treatment CK is set.

[0050] The application method of treatment 1 was: combined application of inter-row scattering of mushroom sticks and rhizosphere spot seeding of mushroom sticks;

[0051] The application method of treatment 2 is: inter-row spreading of fungus sticks and root irrigation of fungus agent;

[0052] The application method of treatment 3 was: root irrigation with a single bacterial agent.

[0053] After the mushroom sticks are spread in the field, under the action of water, the antagonistic endophytes are slowly released during the water-soluble dispersion of the mushroom sticks, so as to achieve the long-term prevention and control of the infection of Sclerotinia sclerotiorum during the growth process of sunflowers; and the endophytes in the bacterial agent are directly released into the sunflower plants.

[0054] The application method of CK treatment is: no application of bacterial sticks and bacterial agents.

[0055] Time for applying mushroom sticks: Apply mushroom sticks in the root zone during the budding stage, early flowering stage and late flowering stage of sunflower to compare the control effect; Application amount of mushroom sticks: 15.0g of mushroom sticks are applied per sunflower plant, and the ratio of application of mushroom sticks between rows and root-spotted mushroom sticks is 1:1; The application amount of the mushroom agent for root irrigation is 0.03L / plant.

[0056] In this example, an experiment was conducted in an edible sunflower field with a seedling density of 1200 plants / acre. The mushroom sticks prepared in Example 1 were used at different developmental stages of sunflowers and the control effect of sunflower sclerotinia was evaluated. The results are shown in Tables 1 to 3.

[0057] Table 1 Application effect at bud stage

[0058]

[0059] Table 2 Application effect at the early stage of flowering

[0060]

[0061] Table 3 Application effect in late flowering period

[0062]

[0063] From the experimental results in Tables 1 to 3, it can be seen that the application of bacterial sticks and bacterial agents in the budding stage, early flowering stage and late flowering stage can significantly improve the disease resistance of sunflower. As can be seen from Table 1, in the budding stage, the incidence of sunflower sclerotinia in each treatment group was CK>treatment 3>treatment 2>treatment 1, and the control effects of the three treatments in the budding stage were significantly better than those of the control group CK, especially treatments 1 and 2, with a relative control effect of more than 83%.

[0064] As can be seen from Table 2, when intervention was carried out in the early stage of sunflower flowering, the number of diseased plants with sunflower sclerotinia increased, and the incidence rate was CK>treatment 1>treatment 3>treatment 2, among which treatment 2 was the best, with a relative control effect of 75.27%.

[0065] As can be seen from Table 3, the incidence of sunflower sclerotinia disease in each treatment group in the late flowering period was CK>treatment 3>treatment 1>treatment 2. The control effects of treatments 1 and 2 were both good, especially the control effect of treatment 2. In summary, the disease resistance effects of the bacterial agent and bacterial stick treatment were significantly better than those of the blank control.

[0066] The control effect of treatment 1 was better than that of treatment 2 when the intervention was carried out in the budding stage, while the control effect of treatment 2 was better than that of treatment 1 when the intervention was carried out in the early and late flowering stages. This may be because during the budding stage, Sclerotinia sclerotiorum has basically not infected the sunflower. At this time, the application of mushroom sticks can slowly release Bacillus subtilis Y116 into the soil, which has a long-term antagonistic effect on Sclerotinia sclerotiorum; when intervening in the early and late flowering stages, Sclerotinia sclerotiorum has already infected a part of the sunflower plants. At this time, all the mushroom sticks cannot activate and release a large amount of Bacillus subtilis Y116 in a short time. At this time, the combined application of mushroom sticks between rows and root irrigation with fungal agents can quickly allow the moisture in the fungal agent to enter the mushroom sticks, so that the Bacillus subtilis Y116 in the mushroom sticks can be activated and reproduced in a short time, thereby intensively eliminating a part of the infected Sclerotinia sclerotiorum in a relatively short time, thus achieving a relatively good control effect.

[0067] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. A method for preparing a fungus stick for antagonizing sunflower sclerotinia disease, characterized in that: The steps include: Step S1, crushing naturally dried corn stalks to obtain corn stalk particles; Step S2, preparing an endophytic bacterial agent; The preparation method of the endophyte agent is as follows: Step S2-1, under sterile conditions, a single colony of the endophytic bacterial strain is picked up with an inoculation loop, and inoculated into 30 mL of seed culture medium for constant temperature shaking culture, wherein the constant temperature shaking culture conditions are: constant temperature shaking culture at 180 rpm and 28° C. for 2 days; after the culture is completed, a seed culture solution is obtained; the endophytic bacteria is Bacillus subtilis Y116, and the deposit number is CGMCC 27043; Step S2-2, inoculating the seed culture solution into the fermentation medium for fermentation, wherein the inoculation amount of the seed culture solution is 5% of the fermentation medium, and the fermentation conditions are: cultured at 200 rpm, 28° C. for 4 days with ventilation, and the dissolved oxygen content in the fermentation medium is maintained at 10-16%; after the fermentation is completed, filtering is performed to obtain an endophytic bacterial agent; Step S3, mixing the corn stalk particles and the bacterial agent evenly to obtain a granulated material; Step S4, granulating the granulated material using a granulating device to obtain a granular biocontrol agent; Step S5, drying the granular biocontrol agent to obtain fungus sticks antagonizing sunflower sclerotinia pathogen.

2. The method for making the antagonistic sunflower sclerotinia pathogen bacteria stick according to claim 1, characterized in that: In step S1, the particle size of the corn stalk particles is 5-10 mm, and the moisture content of the corn stalks is 10-20 wt %.

3. The method for making the antagonistic sunflower sclerotinia pathogen bacteria stick according to claim 1, characterized in that: The seed culture medium is composed of the following components: 5 g sodium chloride, 5 g yeast extract, 10 g peptone, and 1000 mL distilled water; the pH of the seed culture medium is 7.2 to 7.5; The fermentation medium consists of the following components: 30 g of peptone, 30 g of yeast extract, 30 g of soluble starch, and 60 g of sodium chloride.

4. The method for making the antagonistic sunflower sclerotinia pathogen bacteria stick according to claim 1, characterized in that: In step S3, the mass volume ratio of corn stalk particles to bacterial agent is 4-5 kg / L.

5. The method for making the antagonistic sunflower sclerotinia pathogen bacteria stick according to claim 4, characterized in that: Urea is also added to the granulated material, and the mass of the added urea is 0.5-0.7% of the mass of the corn straw particles.

6. The method for preparing the fungus sticks for antagonizing sunflower sclerotinia disease according to claim 1, characterized in that: In step S5, the drying method is air-drying in the sun or air-drying at room temperature; the drying in the sun or air-drying at room temperature is performed until the water content of the granular biocontrol agent is 20-30wt%, thereby obtaining fungus sticks antagonistic to sunflower sclerotinia disease.

7. The use of a fungus stick for antagonizing sunflower sclerotinia disease, characterized in that: The antagonistic sunflower sclerotinia disease fungus sticks prepared by the method for preparing the antagonistic sunflower sclerotinia disease fungus sticks as described in claim 1 are used for the prevention and treatment of sunflower sclerotinia disease.

8. The use of the fungus sticks for antagonizing sunflower sclerotinia disease according to claim 7, characterized in that: The application method is: during the budding period of sunflowers, the mushroom sticks are applied jointly by broadcasting mushroom sticks between the rows and spot-seeding mushroom sticks in the rhizosphere; the total application amount of the mushroom sticks is calculated based on 15.0g per sunflower plant, and the ratio of the application amount of mushroom sticks broadcasting between the rows and spot-seeding mushroom sticks in the rhizosphere is 1:1.

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