A method for recycling waste strawberry substrate for antagonizing pests and diseases

Abstract

The invention discloses a method for recycling waste strawberry substrates, comprising the steps of: exposing cultivated waste strawberry substrates to the sun for 6-30 days between June and July each year, taking the waste strawberry substrates after exposure to the sun and mixing them with organic fertilizers in different proportions to form fresh strawberry substrates, planting, and obtaining waste strawberry substrates after planting and harvesting, and then exposing them to the sun. The invention optimizes the inoculation time and method of composting strains according to the characteristics of the composting stage, invents a step-by-step inoculation method, inoculates different strains at different stages, can reduce the loss of nutrients in the high-temperature stage of composting, and improve the quality of compost; and can reduce the antagonism between different strains, improve the activity of biocontrol bacteria, thereby facilitating the recycling of elevated strawberry substrates.

Description

Technical Field

[0001] The invention relates to the technical field of nutrient substrates, and in particular to a method for recycling discarded strawberry substrates. Background Art

[0002] Elevated substrate cultivation is currently the main cultivation method for strawberries in my country's intelligent greenhouses. With the continuous development and expansion of my country's planting industry, the traditional substrate that uses a simple mixture of raw materials such as peat, perlite, vermiculite, and coconut bran can no longer meet the high-quality and high-demand planting needs due to its poor water absorption, weak fertilizer storage capacity, easy collapse, poor resistance to pests and diseases, and low added value. After the elevated cultivation substrate is used for many crops, many rotten roots, pathogens or insect eggs, salt, and even nematodes will remain in it. In addition, the physical and chemical properties of the cultivation substrate itself will also change, which will greatly affect the cultivation effect of strawberries. If the substrate is replaced every year, the cost of strawberry cultivation will increase. How to extend the service life of the substrate and reduce its input cost has become a difficult problem that must be solved in elevated strawberry substrate cultivation.

[0003] In addition, strawberries are susceptible to diseases and insect pests such as gray mold, powdery mildew, anthracnose, red spiders, and thrips during their growth. With the widespread use of antibiotics and agriculture, the drug resistance of strawberry pathogens is also increasing. Therefore, green and biological control are increasingly valued, and more and more beneficial biocontrol bacteria have become important resources for reducing or replacing pesticides due to their environmental friendliness. However, due to the complexity and variability of the field environment, the biocontrol effect of beneficial bacteria is poor. This is not conducive to the promotion and application of strawberry matrix cultivation. Xiong Mingguo found in his research on the control effects of various microbial agents on strawberry diseases that composite microbial agents have the best control effect on anthracnose, with a control effect of >65%; the control effect on gray mold is relatively good, with a control effect of 55%; Patent CN114015617A mixes various biocontrol bacteria into a composite biological agent and adds it to the strawberry planting area in one step, which plays a role in antagonizing strawberry diseases (root rot, gray mold, wilt, anthracnose), but does not take into account the antagonism between biocontrol bacteria and the competition for nutrition and space, which will greatly reduce the efficacy of the biocontrol bacteria. Summary of the invention

[0004] In order to solve the above problems in the prior art, the present invention optimizes the inoculation time and method of composting bacteria according to the physical and chemical properties of the composting stage, and invents a step-by-step inoculation method to inoculate different bacteria at different stages. Compared with the one-step mixed inoculation method, this method can reduce the inactivation of biocontrol bacteria in the high-temperature stage of composting and improve the quality of organic fertilizer; it can also reduce the antagonism between different bacteria species and improve the activity of biocontrol bacteria, thereby facilitating the recycling of strawberry substrate.

[0005] The method for recycling waste strawberry substrates of the present invention comprises the following steps:

[0006] (1) Every year between June and July, the discarded strawberry substrate is exposed to the sun for 6-30 days.

[0007] (2) taking the sun-dried discarded strawberry substrate and mixing it with the composted organic fertilizer in different proportions to form a fresh strawberry substrate, and planting it after it is decomposed.

[0008] (3) After planting and harvesting, the discarded strawberry substrate is recovered.

[0009] (4) Repeat steps (1) and (2).

[0010] In the above-mentioned method for recycling waste strawberry substrate, preferably, the fresh strawberry substrate also includes other commonly used substrate raw materials required for strawberry cultivation.

[0011] In the above-mentioned method for recycling waste strawberry substrate, preferably, the other commonly used substrate raw materials include but are not limited to at least one of peat soil, perlite, vermiculite, coconut bran, earthworm castings, biochar, sheep manure, rapeseed cake, and rice husks.

[0012] In the above-mentioned method for recycling waste strawberry substrate, preferably, the other commonly used substrate raw materials are at least one of peat soil, earthworm castings, and biochar.

[0013] In the above-mentioned method for recycling waste strawberry substrate, preferably, the mass composition of the fresh strawberry substrate is: waste strawberry substrate: composted organic fertilizer = 6-7: 1-2; more preferably, the composition is: waste strawberry substrate: earthworm castings: biochar: composted organic fertilizer = 6-7: 1-2: 1-2: 1-2.

[0014] In the above-mentioned method for recycling waste strawberry substrate, preferably, the organic fertilizer is prepared by the following method: four mixed bacteria, namely, Bacillus stearothermophilus, Bacillus ferrugineus, Bacillus megaterium and Bacillus subtilis, are added to the organic fertilizer substrate for fermentation.

[0015] In the above-mentioned method for recycling waste strawberry substrate, preferably, the mass ratios of Bacillus stearothermophilus:Azotobacter rotundifolia:Bacillus megaterium:Bacillus subtilis are 1-2:1-2:1-2:1-2 respectively.

[0016] In the above-mentioned method for recycling waste strawberry substrate, preferably, the organic fertilizer is prepared by the following method: the order of adding four kinds of bacteria to the organic fertilizer substrate is as follows:

[0017] Bacillus stearothermophilus was added on days 0-5, Azotobacter rotundus was added on days 6-10, Bacillus megaterium was added on days 11-30, and Bacillus subtilis was added on days 31-60.

[0018] In the above-mentioned method for recycling waste strawberry substrate, preferably, 0.1-0.2% of Bacillus stearothermophilus by mass ratio of the organic fertilizer substrate raw material is added, 0.1-0.2% of Bacillus megaterium by mass ratio of the organic fertilizer substrate raw material is added, and 0.1-0.2% of Bacillus subtilis by mass ratio of the organic fertilizer substrate raw material is added.

[0019] In the above-mentioned method for recycling waste strawberry substrate, preferably, the organic fertilizer substrate includes but is not limited to at least one of sheep manure, rapeseed cake and rice husk.

[0020] In the above-mentioned method for recycling waste strawberry substrate, preferably, the round brown nitrogen-fixing bacteria is round brown nitrogen-fixing bacteria supported on multilayer graphene oxide, which is prepared by the following method:

[0021] (1) Activating the brown nitrogen-fixing bacteria;

[0022] (2) The activated round brown nitrogen-fixing bacteria are inoculated into an Ashby nitrogen-free liquid culture medium containing 0.5-1% by mass of multilayer graphene oxide; and the culture is carried out in a shaking incubator.

[0023] In the above-mentioned method for recycling waste strawberry substrate, preferably, the Bacillus megaterium is Bacillus megaterium supported on multilayer graphene oxide, which is prepared by the following method:

[0024] (1) Activating Bacillus megaterium strains;

[0025] (2) The activated Bacillus megaterium is inoculated into a liquid culture medium containing 0.5-1% by mass of multilayer graphene oxide and cultured in a shaking table.

[0026] In the above-mentioned method for recycling waste strawberry substrate, preferably, the Bacillus subtilis is Bacillus subtilis rich in nano-organic selenium, which is prepared by the following method:

[0027] (1) Activating Bacillus subtilis;

[0028] (2) inoculating the activated Bacillus subtilis into LB liquid medium containing sodium selenite for cultivation;

[0029] (3) adding 0.5-1% of the mass of the culture medium of multilayer graphene oxide to the culture solution obtained in step (2), and culturing on a shaking platform.

[0030] The present invention optimizes the inoculation time and method of composting bacteria according to the characteristics of the composting stage, invents a step-by-step inoculation method, and inoculates different bacteria at different stages. The method can reduce the inactivation of biocontrol bacteria in the high-temperature stage of composting and improve the quality of organic bacterial fertilizer; it can also reduce the antagonism between different bacteria and improve the activity of biocontrol bacteria. At present, the production of selenium-rich organic fertilizer mostly uses sodium selenite (Na2SeO3) solution as an inorganic selenium fertilizer to be added in the form of foliar spraying and root irrigation. Inorganic ionic selenium fertilizer is easily lost with water, which easily causes waste of selenium elements, while the added value of strawberries can be increased through reasonable organic selenium enrichment. The present invention uses Bacillus subtilis to produce nano-organic selenium fertilizer, solves the problem of easy loss of inorganic selenium fertilizer, and improves the utilization rate of selenium fertilizer. And graphene oxide has good thermal stability and adsorption effect, which can enhance the environmental adaptability of biocontrol bacteria and the slow release of selenium fertilizer. Using graphene oxide as a microbial carrier can isolate the high temperature stage of composting and the changeable climate changes, provide microorganisms with a local suitable microenvironment, and improve their adsorption capacity and ability to antagonize pests and diseases. DETAILED DESCRIPTION

[0031] The following embodiments are further descriptions of the content of the present invention as an explanation of the technical content of the present invention, but the essential content of the present invention is not limited to the following embodiments. Ordinary technicians in this field can and should know that any simple changes or replacements based on the essential spirit of the present invention should fall within the scope of protection required by the present invention.

[0032] Example 1

[0033] Preparation of Bacillus subtilis rich in nano-organic selenium

[0034] (1) Preparation of multilayer graphene oxide: Use a mixed solution of concentrated sulfuric acid: concentrated phosphoric acid (or concentrated sulfuric acid: concentrated nitric acid) (volume ratio of 7-8) and graphite powder, ultrasonically oscillate for 10 minutes, and fully react for 1-2 hours. Then, use potassium permanganate (the mass ratio of potassium permanganate: graphite powder is 6:1) and hydrogen peroxide (0.5-0.8%) to oxidize the graphite powder, stir well, and obtain multilayer graphene oxide, which is washed with deionized water for 3 times and set aside.

[0035] (2) Preparation of selenium-enriched Bacillus subtilis: After the freeze-dried powder of Bacillus subtilis was activated, it was cultured in a liquid LB medium at 30°C and 180 rpm for about 24 hours to obtain a seed solution, which was then inoculated into a fermentation medium at an inoculum concentration of 1-2% (volume concentration) and fermented at 30°C and 180 rpm for 2 days to obtain a fermentation broth with a live bacterial concentration of 5×10 7 ~1×10 8cfc / ml. The formula of the fermentation medium is prepared according to the following ratio: 10g peptone, 2.5g ammonium sulfate, 3g beef extract powder, 5g sodium chloride, 2g potassium hydrogen phosphate, 5g polyvinyl alcohol, 1000ml distilled water, pH7.25. The activated Bacillus subtilis is inoculated in LB liquid culture medium containing 20-30mg / L sodium selenite and cultured for 48-36h. Bacillus subtilis can convert inorganic selenium into organic selenium wrapped around Bacillus subtilis; Nano-selenium particle size verification method: Selenium-enriched Bacillus subtilis can further extract nano-selenium by ultrasonic crushing, including the following steps: placing the fermentation liquid in an ultrasonic crusher, 100W, 60HZ, 5s ON / 5s OFF, and crushing on ice for 20 minutes. The nano-particle size determination method is determined using the Malvern Zetasizer Nano ZS90 nano-particle size analyzer and its application program. The results show that the particle size range of nano-selenium produced by Bacillus subtilis is 63.07-65.89nm.

[0036] (3) Preparation of nano-organic selenium fertilizer: Multilayer graphene oxide is added to the selenium-rich LB expansion medium of Bacillus subtilis, and cultured aerobically on a shaker at 30°C and 180 rpm for 48-72 hours. Bacillus subtilis acts on the graphene oxide by adsorption, and the culture solution is added to the waste materials for aerobic composting.

[0037] Example 2

[0038] Activation culture of Bacillus stearothermophilus: The purchased lyophilized powder of Bacillus stearothermophilus was activated in the culture medium. The activation culture medium formula was: 0.8% peptone, 0.3% yeast powder, 0.3% NaCl, MnSO4 0.9mmol / L, CaCl210mmol / L; the best fermentation conditions were: pH7.0, inoculation amount 0.1%, culture time 24h, temperature 65℃. After obtaining the seed liquid, the inoculation amount of 1-2% (volume concentration) was inoculated into the expansion fermentation medium. The fermentation was carried out for 2 days at 65℃ and 180rpm to obtain the fermentation liquid. The concentration of live bacteria in the fermentation liquid was 5×10 7 ~1×10 8 cfc / ml.

[0039] Example 3

[0040] Cultivation of round brown nitrogen-fixing bacteria: The purchased round brown nitrogen-fixing bacteria freeze-dried powder was activated, shaken in a shaker at 25°C and 180rpm for 20 minutes, and 100pL was drawn on a sterile operating table and evenly spread on the Ashby nitrogen-free medium with a glass applicator. Ashby nitrogen-free medium formula: KH2PO4 0.2g / L; CaCO3 0.25g / L; MgSO4·7H2O 0.2g / L; mannitol 10g / L; NaCl 0.2g / L; agar 18g / L; CaSO4·2H2O 0.1g / L, pH adjusted to 7.0. The above culture medium without agar is a liquid culture medium. 0.5-1% multilayer graphene oxide is added to the liquid culture medium, and then the activated bacteria are inoculated and expanded: inoculation amount 1-5%, rotation speed 180rpm, constant temperature culture at 30°C for 3d, and the concentration of live bacteria in the fermentation broth is 5×10 7 ~1×10 8 cfc / ml.

[0041] Example 4

[0042] Bacillus megaterium culture: The purchased Bacillus megaterium freeze-dried powder was activated in the culture medium. The activation culture medium formula was: 1% bran, 0.5% soybean meal, 1% sodium chloride, 0.05% MnSO4, 18g / L agar; pH 7.0, temperature 30°C. The above culture medium minus the agar is a liquid culture medium. 0.5-1% multilayer graphene oxide is added to the liquid culture medium, and then the activated strain is inoculated and expanded. The inoculation amount is 1-5%, the rotation speed is 200r / min, and the number of effective live bacteria reaches 1×10 after 24h of fermentation. 8 cfu / mL.

[0043] Example 5

[0044] Through the analysis of the changes in the physical and chemical properties and nutrient elements of aerobic composting, it can be divided into a temperature rise fermentation stage (0-5d), a nitrogen fixation stage (5-10d), a phosphate solubilization stage (11-30d), and a degradation stage (31-60d), and a step-by-step inoculation method is carried out. In the high-temperature fermentation stage of composting, 0.1-0.2% of thermophilic stearic acid Bacillus is added to the mass ratio of the compost raw material; in the nitrogen fixation stage, 0.1-0.2% of round brown nitrogen-fixing bacteria is added to the mass ratio of the compost raw material; in the phosphate solubilization stage, 0.1-0.2% of selenium-enriched Bacillus subtilis is added to the mass ratio of the compost raw material.

[0045] From June to August 2022, it will be carried out in the planting greenhouse and composting fermentation greenhouse of the Anhui Provincial Collaborative Innovation Institute of Smart Agriculture in Hefei, Chinese Academy of Sciences. First, the waste strawberry substrate after planting and harvesting will be spread on the cement floor of the strawberry greenhouse with a thickness of 5-10cm, and exposed to the sun in the greenhouse for 20 days. The maximum temperature in the greenhouse can reach 50-60℃. The cultivated waste strawberry substrate will make full use of the vacant intermittent period of the greenhouse after the strawberry seedlings are removed (June-July), and will be exposed to the sun in the glass greenhouse (or film greenhouse) for 10-20 days, which can achieve the effect of high-temperature disinfection and sterilization.

[0046] Then sheep manure, rapeseed cake and rice husk were used as compost (organic fertilizer) matrix, mixed at a mass ratio of 2:2:1, and three composting schemes were set up:

[0047] 1# In the initial stage, mixed bacteria are added, with thermophilic Bacillus: brown nitrogen-fixing bacteria: Bacillus megaterium: selenium-enriched Bacillus subtilis = 1-2: 1-2: 1-2: 1-2;

[0048] 2#: Add 0.1-0.2% of Bacillus stearothermophilus by mass of the compost raw materials on the 0th day, add 0.1-0.2% of Azotobacter rotundifolia by mass of the compost raw materials on the 6th day, add 0.1-0.2% of Bacillus megaterium by mass of the compost raw materials on the 11th day, add 0.1-0.2% of Bacillus subtilis by mass of the compost raw materials on the 31st day, and adjust the moisture content to about 60% in time;

[0049] The CK group did not add any bacterial agents;

[0050] The compost raw materials were piled into a cone with a diameter of 1.5m and a height of 1.2m. The pile was turned over once a day when the temperature reached 50℃. If the temperature exceeded 65℃, the pile was turned over again. Water was added in time. 500g of samples were taken from multiple points of each group, mixed well, and brought back to the laboratory and stored in a refrigerator at room temperature or -40℃ for later use.

[0051] According to the water retention, air permeability, nutrients, biological activity and other characteristics of the substrate, earthworm castings and biochar are preferred as supplementary ingredients for strawberry substrates. They are mixed with composted organic fertilizer in different proportions during the high temperature stage of aerobic composting (waste strawberry substrate: earthworm castings: biochar: composted organic fertilizer = 6-7:1-2:1-2:1-2). After decomposition, the strawberries are planted and the growth characteristics, fruit quality and disease and pest resistance of the strawberries are observed. The results are shown in Tables 1-4 below.

[0052] Table 1 Matrix conditions

[0053] Grouping 1# 2# ck Matrix parameters One-step addition of bacteria Add bacteria step by step No bacteria added Matrix pH 6.89 6.91 6.94 EC(us / cm) 1.297 1.608 1.052 Organic matter (g / kg) 35.5 39.9 32.4 Nitrate nitrogen (mg / kg) 382.5 548.3 227.4 Hydrolyzed nitrogen (mg / kg) 489.2 539.6 477.3 Available potassium (mg / kg) 228.8 272.1 209.5 Available phosphorus (mg / kg) 93.1 101.1 86.2 Total selenium (mg / kg) 21.5 22.43 - Inorganic selenium (mg / kg) 0.117 0.128 -

[0054] Table 2 Growth characteristics of strawberry

[0055] Grouping 1# 2# ck parameter One-step addition of bacteria Add bacteria step by step No bacteria added Plant height (cm) 15.8 16.4 13.6 Number of stems 12 15 11 Stem diameter (mm) 3 4 2 Number of blades 36 40 32 Leaf width(cm) 4.3 4.6 3.9 Leaf length(cm) 4.9 5.2 4.3 <![CDATA[Leaf area (cm 2 )]]> 21.93 23.92 16.77

[0056] Table 3 Strawberry fruit quality

[0057] Grouping 1# 2# ck parameter One-step addition of bacteria Add bacteria step by step No bacteria added Average fresh weight of single fruit (g) 12.96 13.26 9.15 Total soluble sugar% 11.90％ 13.90％ 10.90％ Selenium content in fresh fruit (ug / kg) 77.44 98.72 - P (mg / kg) 260.45 183.55 214.01 K(mg / kg) 1367 1339 1464 Mg(mg / kg) 113.42 116.08 125.58 Fe(mg / kg) 12.28 8.64 31.24 Ca(mg / kg) 21.95 18.57 32.54

[0058] Table 4 Disease and insect pest resistance

[0059] Grouping 1# 2# ck parameter One-step addition of bacteria Add bacteria step by step No bacteria added Anthrax incidence % 10.7 0.89 13.89 Root rot incidence% 7.14 1.79 10.71 Powdery mildew incidence 5.56 2.68 14.81

[0060] As shown in Table 1, the technical data of the substrate with one-step bacteria addition are better than those without bacteria addition, and the technical data of the substrate with step-by-step bacteria addition are better than those of the substrate with one-step bacteria addition; as shown in Tables 2-4, if the discarded strawberry substrate after planting in previous years is directly used for the next crop of strawberry planting without the fermentation treatment of bacteria addition, it is obviously not conducive to the growth of strawberry fruit, and after the discarded strawberry substrate is treated with one-step bacteria addition, the strawberry growth condition is improved, but the discarded strawberry substrate treated with step-by-step bacteria addition is more conducive to the growth of strawberry after strawberry planting, and can better improve the soluble total sugar and fresh fruit selenium content of strawberry fruit, and also make strawberry have better disease and insect resistance. The present invention can realize the recycling and multi-crop use of the discarded strawberry substrate by effectively treating the discarded strawberry substrate, thereby creating greater value for elevated strawberry cultivation.

[0061] It should be noted that the above-mentioned technical contents of the present invention are only for explanation and clarification to enable those skilled in the art to understand the technical essence of the present invention, so the technical contents are not used to limit the substantial protection scope of the present invention. The substantial protection scope of the present invention shall be subject to the claims. Those skilled in the art should know that any modification, equivalent substitution and improvement based on the substantial spirit of the present invention shall be within the substantial protection scope of the present invention.

Claims

1. A method for recycling discarded strawberry substrates, comprising the steps of: (1) From June to July each year, the discarded strawberry substrate is exposed to the sun for 6 to 30 days. (2) taking the waste strawberry matrix after exposure to the sun, mixing it with the composted organic fertilizer in different proportions to form a fresh strawberry matrix, and planting it after it is decomposed, wherein the composted organic fertilizer is prepared by the following method: adding four mixed bacteria, namely, thermophilic Bacillus, round brown nitrogen-fixing bacteria, Bacillus megaterium, and Bacillus subtilis, to the organic fertilizer matrix and fermenting them step by step; the order of adding the four bacteria to the organic fertilizer matrix is ​​as follows: thermophilic Bacillus is added on the 0th to 5th day, round brown nitrogen-fixing bacteria is added on the 6th to 10th day, Bacillus megaterium is added on the 11th to 30th day, and Bacillus subtilis is added on the 31st to 60th day; (3) After planting and harvesting, the discarded strawberry substrate is recycled. (4) Repeat the above steps.

2. The method for recycling waste strawberry substrate according to claim 1, characterized in that: The fresh strawberry substrate also includes other commonly used substrate raw materials required for strawberry planting.

3. The method for recycling waste strawberry substrate as claimed in claim 2, characterized in that: The other commonly used substrate raw materials include but are not limited to at least one of peat soil, perlite, vermiculite, coconut bran, earthworm castings, and biochar.

4. The method for recycling waste strawberry substrate as claimed in claim 2, characterized in that: The other commonly used substrate raw materials are at least one of peat soil, earthworm castings and biochar.

5. The method for recycling waste strawberry substrate according to claim 1, characterized in that: The mass composition of the fresh strawberry matrix is: waste strawberry matrix: composted organic fertilizer = 6-7: 1-2.

6. The method for recycling waste strawberry substrate according to claim 1, characterized in that: The mass ratios of the thermophilic Bacillus stearothermophilus: brown nitrogen-fixing bacteria: Bacillus megaterium: Bacillus subtilis are 1-2:1-2:1-2:1-2 respectively.

7. The method for recycling waste strawberry substrate according to claim 1, characterized in that: Add 0.1-0.2% of Bacillus stearothermophilus by mass ratio of the organic fertilizer matrix raw material, add 0.1-0.2% of round brown nitrogen-fixing bacteria by mass ratio of the organic fertilizer matrix raw material, add 0.1-0.2% of Bacillus megaterium by mass ratio of the organic fertilizer matrix raw material, and add 0.1-0.2% of Bacillus subtilis by mass ratio of the organic fertilizer matrix raw material.

8. The method for recycling waste strawberry substrate according to claim 1, characterized in that: The organic fertilizer matrix includes but is not limited to at least one of sheep manure, rapeseed cake, and rice husk.

9. The method for recycling waste strawberry substrate according to claim 1, characterized in that: The Bacillus subtilis is a Bacillus subtilis rich in nano-organic selenium, which is prepared by the following method: (1) Activating Bacillus subtilis; (2) inoculating the activated Bacillus subtilis into LB liquid medium containing sodium selenite for cultivation; (3) Add the multilayer graphene oxide to the culture solution obtained in step (2) and culture on a shaking table.

10. The method for recycling waste strawberry substrate according to claim 9, characterized in that: The amount of multilayer graphene oxide added is 0.5-1% of the mass of the culture solution obtained in step (2).

11. The method for recycling waste strawberry substrate according to claim 1, characterized in that: The brown nitrogen-fixing bacteria are loaded on multi-layer graphene oxide.

12. The method for recycling waste strawberry substrate according to claim 1, characterized in that: The Bacillus megaterium is supported on multi-layer graphene oxide.