Crop probiotics as well as preparation and application thereof in agricultural production

Abstract

The invention discloses crop probiotics as well as preparation and application thereof in agricultural production, and belongs to the technical field of microorganisms. The strain is Saitozyma off.MH006 separated from soil, original materials and culture base materials are easy to obtain, and the culture mode is simple and easy to implement; and after being highly diluted, the bacterial strain can be directly added into crop planting soil and near the root system of a plant, so that the colonization of the bacterial strain on the crop rhizosphere soil is realized, and the relative abundance of dominant populations is changed by increasing the diversity of rhizosphere microbial populations and community structures and functions, thereby promoting the development of the root system of the plant and improving the yield of the plant. The root system of the crop is more developed and deeper, so that the crop can absorb more nutrients, the physiological traits of the whole plant are further influenced, and the growth and development of the crop are promoted.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a crop probiotic and preparation and application thereof in agricultural production. BACKGROUND

[0002] With the development of economic society and the upgrading of consumption structure, the demand for grain in China is rising, and the super large-scale demand for grain formed by more than 1.4 billion people brings huge pressure on land resources and water resources. In the face of the uncertainty risk of global grain industry supply chain, the complex international situation and the development trend of domestic grain supply and demand tension, the state attaches great importance to food security issues. However, the excessive use of pesticides and fertilizers disturbs the inherent structure of the soil, causes soil compaction, leads to soil fertility decline, and brings a series of ecological environmental pollution and agricultural product residue problems, which seriously hinder the carbon sequestration and sustainable development of China's agriculture. Even cause plant growth and development to be abnormal, reduce the quality of agricultural products. With the improvement of people's living standards, higher requirements are put forward for the quality of agricultural products. Therefore, the research on the planting technology and plant physiology of agricultural products is emerging, and the application of microbial inoculants in green plant breeding has become a new research hotspot. Beneficial microorganisms not only can improve soil quality, increase soil fertility, and improve crop stress resistance, but also can increase crop yield and improve crop quality. Studies have shown that plant growth-promoting bacteria (PGPB) can produce plant growth hormones, have nitrogen fixation effect, can expand the relative abundance of plant rhizosphere bacterial community to promote plant growth, strengthen the absorption and utilization of plant nutrients (phosphorus dissolution, potassium dissolution and iron carrier synthesis), alleviate biological and non-biological stress, and regulate plant root configuration and rhizosphere microbial community structure and other growth-promoting and stress-resistant mechanisms.

[0003] Microbial fertilizer is a special product gathered by one or more microbial components, taking the life activities of microorganisms as the center, and providing specific nutrient fertilizer products for crops. The biological activity in microbial fertilizer can effectively regulate and improve the biology of plant-microorganism-soil ecosystem, and some microorganisms can colonize in the rhizosphere of crops to promote the growth and development of plants. Microbial resources are rich and diverse, and their action mechanism is relatively complex, so the development and application prospect of microbial resources developed into biological bacterial fertilizer is very extensive, and has become a research hotspot in the global related field. In the agricultural industry, microbial fertilizer can not only improve the soil ecological environment and increase soil fertility, but also promote the absorption of nutrients by plants, effectively reduce the content of harmful substances, improve the quality of crop products, further achieve the quality standard of green and non-pollution, and promote the development of green ecological agriculture. SUMMARY

[0004] Saitozyma aff.MH006 involved in the technical scheme of the present application is a kind of fungi widely distributed in nature, with some characteristics of yeast, it can grow in a variety of environments, including soil, water and plant surface, etc., play an important role in the ecosystem, including decomposing organic matter and participating in certain biochemical cycles, therefore they have important value in biotechnology, ecology and environmental science. But so far, Saitozyma aff.MH006 as agricultural probiotics has rarely been reported.

[0005] The present application aims to provide a kind of crop probiotics: Saitozyma aff.MH006, preparation method, and as crop probiotics in the application case of agricultural production. Saitozyma aff.MH006 can change soil structure, increase soil moisture content, while it can also change the structure and function of plant root microbial community, improve the nutrient cycle of rhizosphere soil, and then promote the growth and development of plants. By developing related products, soil nutrients and nutrient elements can be efficiently utilized, crop yield can be improved, and the use of chemical fertilizers can be reduced or replaced, the soil ecological environment can be protected, and its sustainable utilization can be increased.

[0006] The present application is realized by the following technical schemes:

[0007] A kind of crop probiotics, the probiotics are fungi, and the fungi are Saitozyma aff.MH006;

[0008] The Saitozyma aff.MH006 has growth and reproduction functions in the pH range of 5.0-10.0.

[0009] Application of a kind of crop probiotics in preparing products for promoting plant root development and improving physiological properties of plant ground surface.

[0010] Application of a kind of crop probiotics in preparing soil improvement preparation.

[0011] Application of a kind of crop probiotics in preparing crop growth-promoting preparation.

[0012] Application of a kind of crop probiotics in preparing degradable agricultural mulching film preparation.

[0013] Application of a kind of crop probiotics in preparing soil water-retaining preparation.

[0014] A preparation method of a kind of crop probiotics, comprising the following steps:

[0015] S1, select the soil of the grassland around Baotou City, Inner Mongolia Autonomous Region, dissolve the soil in water, stir uniformly, take the upper layer of the supernatant after 20-30 min of precipitation, and add it to the purified culture medium for the first time of strain purification culture, and obtain a single strain;

[0016] In the first strain purification culture process, a rare earth solution is added to the purified culture medium as a selective agent;

[0017] S2, the single strain in S1 is coated and cultured on the propagation culture medium, and is put into the incubator for propagation culture, and is collected after 4-5 days to obtain Saitozyma aff.MH006 strain.

[0018] As preferred, the first strain purification culture lasts for 110-120 h; the culture temperature is 10-30 DEG C, and the pH is 2.0-12.0;

[0019] The propagation culture temperature is 10-30 DEG C, and the pH is 5.0-8.0;

[0020] The purified culture medium is prepared from 10 parts of corn flour, 10 parts of sucrose, 20 parts of agar powder and 1000 parts of distilled water;

[0021] The propagation culture medium is prepared from 5 parts of corn flour, 20 parts of agar powder and 1000 parts of distilled water.

[0022] A method for using crop probiotics, wherein the Saitozyma aff.MH006 strain is diluted in distilled water to obtain a strain diluent for use;

[0023] The mass concentration of the strain in the strain diluent is 0.05-0.4%.

[0024] The crops suitable for the above-mentioned strain include wheat, fast-growing cabbage and white radish.

[0025] Compared with the prior art, the present application has at least the following technical effects:

[0026] (I) The present application provides a Saitozyma aff.MH006 and a preparation method thereof, and an application case of the Saitozyma aff.MH006 as a crop probiotic in agricultural production. The Saitozyma aff.MH006 can change the soil structure and increase the soil moisture content, and it can also change the structure and function of the plant root microbial community, improve the nutrient cycling of the rhizosphere soil, and further promote the growth and development of the plant. By developing related products, the nutrients and nutrient elements in the soil can be efficiently utilized, the crop yield can be improved, the use of chemical fertilizers can be reduced or replaced, the soil ecological environment can be protected, and the sustainable utilization can be increased.

[0027] (II) The strain is Saitozyma aff. MH006 isolated from soil. The original material and culture medium are easy to obtain, the culture method is simple, and it is easy to realize. After high dilution, it can be directly added to the soil of planted crops and the vicinity of plant roots to realize the colonization of the strain in the rhizosphere soil of crops. By increasing the diversity of microbial population and community structure and function in the rhizosphere, and changing the relative abundance of dominant population, the development of plant roots is promoted, the roots of crops are more developed, and the roots are deeper, which is beneficial to the crops to absorb more nutrients, thereby affecting the physiological characteristics of the whole plant and promoting the growth and development of crops.

[0028] (III) The strain has the following beneficial effects:

[0029] (1) High efficiency of promoting growth: Saitozyma aff. MH006 has high activity of promoting growth, which can effectively promote the root development of plants and improve the physiological characteristics of the ground part of plants, such as chlorophyll content and protein content.

[0030] (2) Wide acid-base adaptability: Saitozyma aff. MH006 has wide acid-base adaptability and can grow and reproduce in the pH range of 5.0-10.0, which improves its applicability in agricultural production.

[0031] (3) Environmental friendliness: Saitozyma aff. MH006 is found to colonize in various environments, including soil, water and plant surface. Therefore, its application in soil improvement and crop growth promotion is more environmentally friendly and more in line with the requirements of sustainable development of modern agriculture.

[0032] (4) Easy to produce and apply: Saitozyma aff. MH006 has high production efficiency in the production process, and the product is easy to store, transport and apply.

[0033] (5) Saitozyma aff. MH006 has certain agricultural mulching degradation ability, which has a positive effect on eliminating microplastic pollution in farmland.

[0034] (6) The soil added with Saitozyma aff. MH006 has significantly improved water retention capacity, so it has a certain positive effect on water-saving irrigation. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 The growth curve of Saitozyma aff. MH006 in different environments in Example 1 is shown in the figure;

[0036] Figure 2Figure of comparison of root system of wheat after inoculation with Saitozyma aff. MH006 at different concentrations for 20 days in Example 1;

[0037] Figure 3 Figure of comparison of average length of root system of wheat after inoculation with Saitozyma aff. MH006 at different concentrations for 20 days in Example 1;

[0038] Figure 4 Figure of comparison of average diameter of root system of wheat after inoculation with Saitozyma aff. MH006 at different concentrations for 20 days in Example 1;

[0039] Figure 5 Figure of comparison of average projected area of root system of wheat after inoculation with Saitozyma aff. MH006 at different concentrations for 20 days in Example 1;

[0040] Figure 6 Figure of comparison of average volume of root system of wheat after inoculation with Saitozyma aff. MH006 at different concentrations for 20 days in Example 1;

[0041] Figure 7 Figure of comparison of root system of fast-growing Chinese cabbage after inoculation with Saitozyma aff. MH006 at different concentrations for 20 days;

[0042] Figure 8 Figure of comparison of root system of white radish after inoculation with Saitozyma aff. MH006 at different concentrations for 20 days. DETAILED DESCRIPTION

[0043] The embodiments of the present application will be described in detail below with examples, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application, and the specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer, and the reagents or instruments not mentioned by the manufacturer are all conventional products that can be purchased on the market.

[0044] Example 1:

[0045] 1.1 Purification of Saitozyma aff. MH006

[0046] (1) 10.0 g of corn flour, 10.0 g of sucrose, and 20.0 g of agar powder were added to 1000 ml of distilled water to obtain a purification medium, and the purification medium was placed in an autoclave for high-pressure sterilization at a temperature of 121°C for 25 min;

[0047] After autoclaving, rare earth solution (200 μg ~ 2 mg / L medium) was added to the medium as a selective agent to make solid culture plates.

[0048] Grassland around Baotou City, Inner Mongolia Autonomous Region, Bayan Obo mining area, 100 grams of soil for grass growth was dissolved in 500 ml water, fully stirred, precipitated for 30 min, then inoculated with a loop, dipped in the upper suspension on the purified medium, and streaked for culture to obtain single colony strains;

[0049] (2) Strain propagation: 5.0 g of corn meal and 20.0 g of agar powder were added to 1000 ml of distilled water to obtain a propagation medium. The propagation medium was placed in an autoclave and autoclaved (temperature 121℃, time 25 min). After autoclaving, solid culture plates were prepared. After the propagation medium was dried, single colony strains were selected and inoculated on the propagation medium using the smear culture method. All the above operations were completed in a clean bench;

[0050] (3) Culture: The smeared solid culture plates were placed in an incubator for culture. The culture temperature was set to 10-30℃.

[0051] (4) The strains were collected with a cell scraper after five days.

[0052] The collected strains were quantitatively diluted and used for soil improvement and crop growth promotion ability determination.

[0053] The above strains were identified and preserved, and the strain was named Saitozyma aff. MH006.

[0054] As shown in Figure 1 , it is the growth curve of Saitozyma aff. MH006 in different environments. Figure A is the growth curve of Saitozyma aff. MH006 at different pH values; Figure B is the growth curve of Saitozyma aff. MH006 at different temperatures.

[0055] (5) The application of Saitozyma aff. MH006 strain in preparing soil improvement and plant growth promotion products.

[0056] When Saitozyma aff. MH006 is applied to promote plant growth, it is identified by the following steps:

[0057] The Saitozyma aff. MH006 strain was diluted in distilled water to obtain a strain diluent, and the mass concentration of the strain diluent was 0.05%-0.4%;

[0058] Take 2.5 kg of soil into a potting tray, add 500 ml of bacterial diluent, and plant seeds in the potting soil the next day. Plant cultivation is carried out in an artificial climate chamber. After 20 days, take samples, take photos, and record the physiological characteristics of the plants. Measure the physiological and biochemical indicators of the soil and plants.

[0059] The bacterial strain obtained in Example 1 is identified as follows.

[0060] Verification Example 1: Verification of Wheat Growth

[0061] 1.1 Prepare 40 pots of equal amounts (2.5 kg / pot) of sandy soil, divide them into five groups, each with 8 pots, and label them as CK, T1, T2, T3, and T4.

[0062] 1.2 The control group (CK group) adds 500 ml of distilled water to each pot of sandy soil.

[0063] Prepare the T1 group bacterial diluent (bacterial stock solution: distilled water 1:4000), shake thoroughly, mix evenly, and then add 500 ml of bacterial diluent to each pot of sandy soil in the T1 group.

[0064] Prepare the T2 group bacterial diluent (bacterial stock solution: distilled water 1:2000), shake thoroughly, mix evenly, and then add 500 ml of bacterial diluent to each pot of sandy soil in the T2 group.

[0065] Prepare the T3 group bacterial diluent (bacterial stock solution: distilled water 1:1000), shake thoroughly, mix evenly, and then add 500 ml of bacterial diluent to each pot of sandy soil in the T3 group.

[0066] Prepare the T4 group bacterial diluent (bacterial stock solution: distilled water 1:500), shake thoroughly, mix evenly, and then add 500 ml of bacterial diluent to each pot of sandy soil in the T4 group.

[0067] Plant 25 wheat seeds in each pot of sandy soil the next day. After sowing, add 100 ml of distilled water to each group to remove air from the soil and facilitate seed germination.

[0068] 1.3 Cultivate wheat in an artificial climate chamber (25°C, 16h light, 8h darkness) for 20 days. Take photos and collect samples on the twentieth day. Measure the stems and leaves of the wheat using a thousandth vernier caliper. Scan the roots of the wheat using a root scanning instrument. After scanning, calculate the average root length, average diameter, average projected area, and average volume of the wheat using a LA-S plant image analysis system to obtain experimental data.

[0069] 1.4 The experimental data were standardized using Excel, and one-way ANOVA was performed using SPSS 26.0 software to determine the differences in wheat growth indicators. The results were analyzed in chart form.

[0070] Table 1 Characterization of wheat roots, stems, and leaves

[0071]

[0072] As shown in Table 1, the traits of crops (i.e., groups T1, T2, T3, and T4) with the addition of Saitozyma aff.MH006 from Example 1 changed significantly. Among them, the average diameter of wheat roots added with Saitozyma aff.MH001 (i.e., the control group) decreased significantly compared with the control group. The finer the roots, the more conducive it is for the crop to take root in the soil, absorb more nutrients, and provide sufficient nutrients for crop growth and development.

[0073] like Figure 2 The image shows a comparison of wheat root systems 20 days after inoculation with different concentrations of Saitozyma aff.MH006.

[0074] like Figure 3 The figure shown is a comparative analysis of the average root length of wheat 20 days after inoculation with different concentrations of Saitozyma aff.MH006.

[0075] like Figure 4 The figure shown is a comparative analysis of the average diameter of wheat roots 20 days after inoculation with different concentrations of Saitozyma aff.MH006.

[0076] like Figure 5 The figure shown is a comparative analysis of the average projected area of ​​wheat roots 20 days after inoculation with different concentrations of Saitozyma aff.MH006.

[0077] like Figure 6 The figure shown is a comparative analysis of the average root volume of wheat 20 days after inoculation with different concentrations of Saitozyma aff.MH006.

[0078] Results combined Figures 2-6 It can be seen that Saitozyma aff.MH006 has a significant effect on crop roots, and the effect is most significant at a concentration of 0.2%.

[0079] Because the root system is more developed, it also promotes the growth of leaves to a certain extent, but it has no effect on the stem length and will not cause excessive growth.

[0080] Therefore, Saitozyma aff.MH006 has a direct promoting effect on crop growth and development.

[0081] Table 2 Wheat root, stem, leaf dry weight and wet weight characterization

[0082]

[0083] From the analysis of Table 2, it can be seen that the addition of Saitozyma aff. MH006 bacterial solution not only improves the root system characteristics, but also increases the weight (dry and wet weight) of the plant, thus having a significant promoting effect on the growth of the plant.

[0084] Verification Example 2: Verification of Fast-growing Chinese Cabbage Growth

[0085] 2.1 Prepare 40 pots of equal amount (2.5 kg / pot) of wind-sand soil, divide into five groups, 8 pots each;

[0086] 2.2 The control group (CK group) adds 500 ml of distilled water to each pot of wind-sand soil;

[0087] Dilute the bacterial solution according to the method of Verification Example 1, and add it to the culture pots containing wind-sand soil after dilution, respectively marked as T1, T2, T3 and T4 treatment groups;

[0088] Replace the wheat seeds with fast-growing Chinese cabbage seeds, and the other operations are the same as Verification Example 1.

[0089] As shown in Figure 7 , the root system of fast-growing Chinese cabbage after 20 days of inoculation with different concentrations of Saitozyma aff. MH006.

[0090] Table 3 Root, stem and leaf characterization of fast-growing Chinese cabbage

[0091]

[0092] According to Table 3 combined with Figure 7 , it can be seen that the characteristics of fast-growing Chinese cabbage added with Saitozyma aff. MH006 in Example 1 have changed, thus it can be known that the application of Saitozyma aff. MH006 bacterial solution can significantly increase the weight (dry and wet weight) of the plant, therefore Saitozyma aff. MH006 has a direct promoting effect on the growth and development of crops.

[0093] Verification Example 3: Verification of White Radish Growth

[0094] 3.1 Prepare 40 pots of equal amount (2.5 kg / pot) of wind-sand soil, divide into five groups, 8 pots each;

[0095] 3.2 The control group (CK group) adds 500 ml of distilled water to each pot of wind-sand soil;

[0096] The bacterial solution was diluted in the same manner as in Verification Example 1, and after dilution, it was added to culture pots containing wind sand soil, and was recorded as T1, T2, T3 and T4 treatment groups, respectively.

[0097] The wheat seeds were replaced with white radish seeds, and the other operations were the same as in Verification Example 1.

[0098] As shown in Figure 8 , the white radish root system comparison chart after 20 days of inoculation with different concentrations of Saitozyma aff. MH006.

[0099] Table 4 Root and leaf characteristics of white radish

[0100]

[0101]

[0102] According to Table 4 in combination with Figure 8 , it can be seen that the addition of Saitozyma aff. MH006 in Example 1 has a significant effect on the growth traits of white radish, which is beneficial to the rooting of white radish and the growth and development of stem and leaf parts. Therefore, Saitozyma aff. MH006 can significantly increase the weight (dry and wet weight) of plant roots, stems and leaves, thus having an obvious promoting effect on the growth of the aboveground part of the plant. Saitozyma aff. MH006 has a direct promoting effect on the growth and development of crops.

[0103] Verification Example 4: Effect of bacterial strain on soil

[0104] In order to further verify the principle of Saitozyma aff. MH006 promoting plant growth and development, the following analyzes the changes in the rhizosphere microbial community of the wheat roots in Test Example 1 in detail.

[0105] Analysis of soil bacteria:

[0106] Table 5 Difference significance test and variance test of rhizosphere bacterial community structure

[0107] Group R-value P-value CK-T1 0.904 0.009 CK-T2 0.964 0.01 CK-T3 0.856 0.013 CK-T4 0.832 0.008 T1-T2 0.364 0.013 T1-T3 0.712 0.006 T1-T4 0.72 0.006 T2-T3 0.716 0.008 T2-T3 0.352 0.021 T3-T4 0.068 0.228

[0108] Note: P < 0.05, significant difference; P < 0.01, extremely significant difference.

[0109] As shown in Table 5, compared with the control group, the bacterial community structure of T1, T3 and T4 changed extremely significantly, and the bacterial community structure of T2 changed significantly compared with the control group.

[0110] The comparison between each Saitozyma aff.MH006 addition group showed that, except for the comparison between T3 and T4, the differences between the other groups were significant.

[0111] Verification example 5: analysis of soil fungi:

[0112] Table 6: Rhizosphere fungal community structure difference significance test and variance test

[0113] Group R-value P-value CK-T1 0.492 0.015 CK-T2 0.524 0.01 CK-T3 0.984 0.009 CK-T4 0.656 0.006 T1-T2 0.4 0.011 T1-T3 0.72 0.009 T1-T4 0.496 0.005 T2-T3 0.712 0.013 T2-T4 0.552 0.011 T3-T4 0.124 0.068

[0114] Note: P<0.05, significant difference; P<0.01, extremely significant difference.

[0115] As shown in Table 6, the differences in fungal community structure between T1, T3 and T4 and the control group were extremely significant, and the difference between the T2 treatment group and the control group also reached a significant level. The comparison between each Saitozyma aff.MH006 addition group showed that, except for the comparison between T3 and T4, the differences between the other groups were significant.

[0116] Therefore, Saitozyma aff.MH006 can change the plant root microbial community structure, increase the diversity of microbial communities (including bacteria and fungi), improve the species evenness of microbial communities, and increase the relative abundance of plant probiotics; by affecting the rhizosphere microbial population and community diversity, Saitozyma aff.MH006 can promote plant root development, and thus affect the physiological characteristics of the whole plant.

[0117] Finally, it should be noted that the above description is only a preferred embodiment of the present application and is not intended to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A crop probiotic, characterized in that, The probiotic is a fungus, and the fungus is Saitozyma aff. MH006. The Saitozyma aff. MH006 has a growth and reproduction function in a pH range of 5.0-10.

0.

2. Application of the crop probiotic in claim 1 in preparation of a product for promoting root development of plants and improving physiological properties of ground parts of plants.

3. Application of the crop probiotic in claim 1 in preparation of a soil improvement preparation.

4. Application of the crop probiotic in claim 1 in preparation of a crop growth promoting preparation.

5. Application of the crop probiotic in claim 1 in preparation of a preparation for degrading agricultural mulch.

6. Application of the crop probiotic in claim 1 in preparation of a soil water retention preparation.

7. A method of preparing a crop probiotic according to claim 1, wherein, The method comprises the following steps: S1. Selecting soil in grassland around Baotou White Cloud and Bo mining area in Inner Mongolia Autonomous Region, dissolving the soil in water, stirring uniformly, taking the upper suspension after 20-30 min of sedimentation, and adding the suspension to a pure culture medium for first strain purification culture to obtain a single strain; A rare earth solution is added to the pure culture medium as a selection agent during the first strain purification culture; S2. The single strain in S1 is coated on a propagation culture medium and placed in a culture box for propagation culture, and the Saitozyma aff. MH006 strain is obtained after 4-5 days of collection.

8. The method of claim 7, wherein the crop probiotic is prepared by, The first strain purification culture lasts for 110-120 h, the culture temperature is 10-30℃, and the pH is 2.0-12.0; The propagation culture temperature is 10-30℃, and the pH is 5.0-8.0; The pure culture medium is prepared from 10 parts of corn powder, 10 parts of sucrose, 20 parts of agar powder, and 1000 parts of distilled water; The propagation culture medium is prepared from 5 parts of corn powder, 20 parts of agar powder, and 1000 parts of distilled water.

9. A method of using the crop probiotic of claim 1, wherein, The Saitozyma aff. MH006 strain is diluted in distilled water to obtain a strain diluent for use; The mass concentration of the strain in the strain diluent is 0.05-0.4%.

Images