Preparation method and application of compound microbial functional bacterial fertilizer
By preparing composite microbial functional bacterial fertilizers and using specific microbial fermentation straw base materials, the prevention and treatment of banana wilt and jackfruit flow gel disease is solved, and the soil structure is improved, achieving efficient disease control and resource utilization.
Patent Information
- Application Number
- CN202510632833.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-16
- Publication Date
- 2025-07-18
AI Technical Summary
In the prior art, banana wilt and jackfruit swelling disease have poor control effects in subtropical and tropical regions, and the utilization rate of crop straw is low, resulting in environmental pollution and waste of resources.
Functional bacteria fertilizers are prepared by fermenting straw base materials by using the preparation method of compound microbial functional bacteria, leukoprotein, Bacillus amylose, Bacillus cellulose and Streptomyces aureus HN6, and functional bacteria fertilizers are prepared by fermenting straw base materials. They are used to prevent and treat banana blight and jackfruit flow disease and improve soil structure.
Effective prevention and treatment of banana wilt and jackfruit flowing disease were achieved, with the prevention effects being 75.97% and 77.07% respectively. At the same time, the soil structure was improved, crop growth was promoted, and straw utilization was improved.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of functional bacterial fertilizers, and specifically to a preparation method and application of a composite microbial functional bacterial fertilizer. Background Art
[0002] Bacterial fertilizers, also known as biological fertilizers, bio-fertilizers, etc. Bacterial fertilizers not only contain the nutrients necessary for crop growth, but also contain a large number of beneficial microorganisms. The beneficial microorganisms reproduce in the soil, playing functions such as improving the soil and preventing diseases. Especially after the concept of "reducing the use of chemical fertilizers and pesticides" was put forward, bacterial fertilizers have gradually become a research hotspot.
[0003] Crops are important carriers of energy, matter and nutrients. As a large agricultural country, China produces a large amount of crop straw every year. However, the comprehensive utilization level of straw has been low, seriously polluting the environment, and straw burning occurs from time to time. In southern China and subtropical regions of Southeast Asia and other areas, there are rich crop resources, and various straws are produced in large quantities every year but cannot be fully utilized. It is necessary to study appropriate development technologies and industrial systems for straw.
[0004] Some studies have pointed out that composting straw can obtain fertilizers that can not only improve the physical and chemical structure of the soil, but also supplement various organic and mineral nutrients to the soil. The high temperature generated during the composting process can effectively kill the pathogens and weed seeds in the straw, and at the same time promote the efficient degradation of the fermentation substrate by thermophilic microorganisms, ultimately realizing the composting, reduction, harmlessness, stabilization and resource utilization of the fermentation substrate. The mature compost contains various active microbial metabolites, humic acid, etc., and also contains a variety of beneficial microorganisms, which have a wide range of uses in agricultural production.
[0005] Bananas and jackfruits are important cash crops in southern China and Southeast Asian regions. Banana wilt disease has brought immeasurable losses to agriculture. And jackfruit stem bleeding (Jackfruit Stem Bleeding or Gummosis) is one of the common diseases in jackfruit cultivation, mainly caused by fungal or bacterial infections, manifested as the exudation of colloidal substances on the surface of the trunk, branches or fruits, and in severe cases, it will lead to weak tree vigor, reduced fruit yield and even death. If it is possible to develop straw compost to produce functional bacterial fertilizers in subtropical and tropical regions according to local conditions to control the diseases of the above crops, it will not only achieve a virtuous cycle of resource development and utilization, but also provide technical services for clean agricultural production, green development and ecological environment protection. Summary of the Invention
[0006] The object of the present invention is to provide a preparation method and application of a composite microbial functional bacterial fertilizer, aiming to develop a functional bacterial fertilizer that can not only prevent diseases and promote the growth of crops but also improve the soil structure of the planting area, and provide a technical solution for promoting the prevention and control of banana wilt and jackfruit gummosis. The object of the present invention is achieved by the following technical solutions:
[0007] A preparation method of a composite microbial functional bacterial fertilizer includes the following steps:
[0008] 1) Strain preparation: Prepare the mother strains of Thermoascus aurantiacus and Phanerochaete chrysosporium; the culture solutions of Bacillus amyloliquefaciens and Bacillus cellulosilyticus; and the mother strain of Streptomyces verticillatus HN6;
[0009] 2) Preparation of fermentation broth: Prepare the fermentation broth of Thermoascus aurantiacus, the fermentation broth of Phanerochaete chrysosporium, the fermentation broth of Bacillus amyloliquefaciens and the fermentation broth of Bacillus cellulosilyticus. The bacterial content of the above fermentation broths is 1×10 9 cfu / mL; Prepare the culture solution of Streptomyces verticillatus HN6, and the bacterial content is 1×10 7 cfu / mL;
[0010] 3) Preparation of fermentation substrate: Dry, crush and mix the substrate banana straw, corn straw, cowpea straw and rice straw according to the weight ratio of 1:0.8 - 1.2:0.8 - 1.2:0.8 - 1.2 to obtain a straw mixture, and then mix it with cow dung to obtain a fermentation substrate. The ratio of the straw mixture to cow dung is: 25 kg of cow dung / 1 ton of straw mixture;
[0011] 4) Preparation of primary functional bacterial fertilizer: Mix the fermentation broth of Thermoascus aurantiacus, the fermentation broth of Phanerochaete chrysosporium, the fermentation broth of Bacillus amyloliquefaciens and the fermentation broth of Bacillus cellulosilyticus in equal volume to obtain a mixed fermentation broth. Inoculate the mixed fermentation broth into the fermentation substrate at an inoculation amount of 10% by mass fraction, mix and pile it into a heap, then cover the heap with a black net film, and then carry out primary fermentation, manual turning and secondary fermentation to make a primary functional bacterial fertilizer;
[0012] 5) Composting and aging: When the temperature of the primary functional bacterial fertilizer is 35 - 40 °C, carry out composting and aging to form humus; the time for composting and aging is 20 days;
[0013] 6) Inoculating Streptomyces verticillatus HN6: When the temperature of the aged primary functional bacterial fertilizer drops to 26 - 35 °C, inoculate the culture solution of Streptomyces verticillatus HN6 at 10 7 cfu / mL again, and culture for 10 days to obtain the composite microbial functional bacterial fertilizer; the culture solution of Streptomyces verticillatus HN6 is inoculated according to 2% of the weight of the aged primary functional bacterial fertilizer.
[0014] For further optimization, the specific operation of step 1) is:
[0015] Thermophilic Sporotrichum thermophile and white-rot fungi were separately inoculated onto PDA plates and cultured for 3 days as mother cultures for standby; the composition of the PDA plates was as follows: 200 g of potatoes, 20 g of glucose, and 15 - 20 g of agar were added to each liter of distilled water; Bacillus amyloliquefaciens and Bacillus cellulolyticus were separately inoculated into nutrient broth and cultured with shaking at 28°C for 24 h to prepare seed culture solutions for standby; the functional bacterium Streptomyces aureus HN6 was inoculated onto Gause's No. 1 plate and cultured at 28°C for 3 days; for standby.
[0016] For further optimization, the specific operation in step 2) was as follows:
[0017] The cultured Thermophilic Sporotrichum thermophile and white-rot fungi were separately inoculated into PDB liquids and cultured with shaking at 400 rpm for 3 days to obtain Thermophilic Sporotrichum thermophile fermentation broth and white-rot enzyme fermentation broth; the seed culture solutions of Bacillus amyloliquefaciens and Bacillus cellulolyticus were inoculated into nutrient broth and cultured with shaking at 28°C for 24 h to obtain Bacillus amyloliquefaciens fermentation broth and Bacillus cellulolyticus fermentation broth; Streptomyces aureus HN6 was inoculated into Gause's No. 1 liquid fermentation medium and cultured with shaking at 28°C for 72 h to obtain Streptomyces aureus HN6 culture solution.
[0018] Furthermore, in step 2):
[0019] The composition of the PDB liquid was as follows: 200 g / L of potatoes and 20 g / L of glucose were added to each liter of water, and the pH was natural;
[0020] The formula composition of the nutrient broth was: 3 g of beef extract, 10 g of peptone, 5 g of sodium chloride, made up to 1000 mL with water, and the pH was 7.0 - 7.2;
[0021] The formula composition of the Gause's No. 1 liquid fermentation medium was: 20 g of soluble starch, 1 g of KNO3, 0.5 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.5 g of NaCl, 0.01 g of FeSO4·7H2O, made up to 1000 mL with water, and the pH was 7.0 - 7.2.
[0022] Furthermore, in step 3), the base materials of banana straw, corn straw, cowpea straw, and rice straw were dried in a weight ratio of 1:1:1:1.
[0023] Further, in step 4) of the preparation of the primary functional bacterial fertilizer: The total primary fermentation time is 13 days, and the temperature is monitored every day. The cone first undergoes temperature-raising fermentation for 5 days until the temperature rises to 60 - 65 °C, then undergoes high-temperature fermentation at 60 - 65 °C for 3 days, and then undergoes temperature-lowering fermentation for 5 days until the temperature drops to 40 °C; The total secondary fermentation time is 12 days. Specifically, after manual turning of the pile, the pile is formed into a cone again for fermentation. The temperature of the cone rises to 55 °C for fermentation, ferments at 55 °C for 7 days, and then undergoes temperature-lowering fermentation for 5 days until the temperature drops to 40 °C; The pile body is a cone with a height of 105 cm and a diameter of 100 cm.
[0024] The compound microbial functional bacterial fertilizer is used for the prevention and control of banana wilt in potted plants, the prevention and control of banana wilt in the field, and the growth promotion of field bananas. For the prevention and control of banana wilt in potted plants, the bacterial fertilizer is mixed into the soil at an addition rate of 0.5 kg per cubic meter; For the prevention and control of banana wilt in the field and the growth promotion of field bananas, the bacterial fertilizer is mixed into the soil at an addition rate of 20 kg per mu, applied once during the banana seedling planting period, and top-dressed once during the fruit growth and development period.
[0025] The compound microbial functional bacterial fertilizer is used for the prevention and control of jackfruit gummosis and the growth promotion of jackfruit. The compound microbial functional bacterial fertilizer is used for the prevention and control of jackfruit gummosis and the growth promotion of jackfruit at an addition rate of 20 kg per mu, applied 2 times per month three months before fruiting, and top-dressed once after fruit picking.
[0026] The advantages and beneficial effects of the present invention are:
[0027] The functional bacterial fertilizer of the present invention can prevent diseases and promote the growth of crops and improve the soil structure of the planting area. Among them, the nitrogen content is 2.65%; the phosphorus content is 2.13%; the potassium content is 1.66%; the viable spore count of Streptomyces HN6 is 1.45×10 10 cfu·g-1. It has a good growth-promoting effect on the roots and stems of the tested radish and rice seeds.
[0028] The control effect of the functional bacterial fertilizer on banana wilt in potted plants is 75.97%. The control effect on banana wilt in the field is 74.37%. The control effect on jackfruit gummosis in the field is 77.07%. It has a certain growth-promoting effect on field bananas and jackfruit plants. Description of the Drawings
[0029] The present invention will be further described below with reference to the drawings and embodiments.
[0030] Figure 1 They are physical pictures of the compost pile before and after composting; The left side is before composting, and the right side is after composting;
[0031] Figure 2 They are the effects of the compost leachate on the germination of radish seeds;
[0032] Figure 3 Effect of compost leachate on rice seed germination. Detailed implementation mode
[0033] Example 1:
[0034] A preparation method of a composite microbial functional bacterial fertilizer, comprising the following steps:
[0035] 1) Strain preparation: Thermophilic Sporotrichum thermophile (ACCC 30346, purchased from China Center for Type Culture Collection of Agricultural Microorganisms) was inoculated on a PDA plate at a culture temperature of 37 °C, and white rot fungus (ACCC 30942, purchased from China Center for Type Culture Collection of Agricultural Microorganisms) was inoculated on a PDA plate at a culture temperature of 25 °C and cultured for 3 days as stock culture for standby. The composition of the PDA plate is: 200 g of potato, 20 g of glucose, and 15 - 20 g of agar are added to each liter of distilled water; Bacillus amyloliquefaciens (CICC 10063, purchased from China Center for Industrial Culture Collection of Microorganisms) and Bacillus cellulolyticus (ATCC 21833, purchased from American Type Culture Collection) were respectively inoculated into nutrient broth and cultured with shaking at 28 °C for 24 h to prepare seed culture solutions for standby; The functional bacterium Streptomyces aureoverticillatus HN6 was inoculated on a Gause's No. 1 plate and cultured at 28 °C for 3 days for standby.
[0036] 2) Preparation of fermentation broth: After the PDB liquid medium was prepared, it was filled into a 5000 mL Erlenmeyer flask with a loading amount of 2500 mL and sterilized at 121 °C for 30 min. The cultured Thermophilic Sporotrichum thermophile and white rot fungus were respectively inoculated into 2500 mL / bottle of PDB liquid in a laminar flow cabinet and cultured with shaking at 400 rpm for 3 days to obtain Thermophilic Sporotrichum thermophile fermentation broth and white rot fungus fermentation broth. The fermentation broths were respectively poured into sterilized plastic buckets and sealed for standby in the laminar flow cabinet; The seed culture solutions of Bacillus amyloliquefaciens and Bacillus cellulolyticus were inoculated into 2500 mL of nutrient broth and cultured with shaking at 28 °C for 24 h to obtain Bacillus amyloliquefaciens fermentation broth and Bacillus cellulolyticus fermentation broth. The bacterial content of the fermentation broths was 1×10 9 cfu / mL; Streptomyces aureoverticillatus HN6 was inoculated into Gause's No. 1 liquid fermentation medium and cultured with shaking at 28 °C for 72 h to obtain Streptomyces aureoverticillatus HN6 culture solution. The bacterial content of the Streptomyces aureoverticillatus HN6 culture solution was 1×10 7 cfu / mL.
[0037] The composition of the PDB liquid is: 200 g / L of potato and 20 g / L of glucose are added to each liter of water, and the pH is natural;
[0038] The formula components of the beef extract peptone culture medium are: 3 g of beef extract, 10 g of peptone, 5 g of sodium chloride, and make up to 1000 mL with water, pH 7.0 - 7.2;
[0039] The formula components of the Gao's No. 1 liquid fermentation medium are: 20 g of soluble starch, 1 g of KNO3, 0.5 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.5 g of NaCl, 0.01 g of FeSO4·7H2O, and make up to 1000 mL with water, pH 7.0 - 7.2.
[0040] 3) Preparation of the fermentation base material: The base materials, banana straw, corn straw, cowpea straw, and rice straw, are dried, crushed, and mixed according to the weight ratio of 1:1:1:1 to obtain a straw mixture, and then mixed with cow dung to obtain the fermentation base material. The ratio of the straw mixture to cow dung is: 25 kg of cow dung / 1 ton of straw mixture; The composting site should be selected on a flat, sunny and windward place near the water source and can be made outdoors.
[0041] 4) Preparation of the primary functional bacterial fertilizer: Mix the fermentation broths of Thermomyces lanuginosus, white rot enzyme, Bacillus amyloliquefaciens, and Cellulomonas cellulans in equal volumes to obtain a mixed fermentation broth. Inoculate the mixed fermentation broth into the fermentation base material at an inoculation amount of 10% by mass fraction. After mixing, pile it into a pile body. The pile body is a cone with a height of 105 cm and a diameter of 100 cm. Then, after covering the cone with a black net membrane, carry out primary fermentation, manual turning, and secondary fermentation in sequence to make the primary functional bacterial fertilizer.
[0042] The inoculation amount of the functional bacterial liquid is inoculated at 2% of the weight of the mixed base material; The total primary fermentation time is 13 days, and the temperature is monitored every day. The temperature of the cone rises to 60 - 65 °C after 5 days of fermentation, then undergoes high-temperature fermentation at 60 - 65 °C for 3 days, and then the temperature drops to 40 °C after another 5 days of fermentation; The total secondary fermentation time is 12 days. Specifically, after manual turning, it is piled into a cone with a height of 105 cm and a diameter of 100 cm again for fermentation. The temperature of the cone rises to 55 °C for fermentation, ferments at 55 °C for 7 days, and then the temperature drops to 40 °C after another 5 days of fermentation.
[0043] 5) Composting and aging: When the temperature of the primary functional bacterial fertilizer is between 36 and 39 °C, carry out composting and aging to form humus; The time for composting and aging is 20 days;
[0044] 6) Inoculating Streptomyces verticillatus HN6: When the temperature of the aged primary functional bacterial fertilizer drops to 28 - 30 °C, inoculate again with 10 7The culture solution of Streptomyces aureofaciens HN6 at cfu / mL was cultured for 10 days to obtain the composite microbial functional bacterial fertilizer; the culture solution of Streptomyces aureofaciens HN6 was inoculated at 2% of the weight of the aged primary functional bacterial fertilizer.
[0045] Detection of bacterial fertilizer:
[0046] 1. Detection of composting index
[0047] (1) Apparent change
[0048] The straw turns brown or dark brown, is soft and elastic when held by hand when wet, is very brittle and easy to break when dry, and the temperature inside the pile no longer rises.
[0049] (2) Detection of physical and chemical indexes
[0050] ① pH detection
[0051] Mix 2 g of the base material sample with 20 mL of distilled water, culture at 25 °C and 120 rpm for 2 h, after the culture ends, centrifuge at 8000 rpm in a centrifuge, and obtain the supernatant after 10 min for pH determination.
[0052] ② Determine the total nitrogen content in the bacterial fertilizer by a continuous chemical flow analyzer
[0053] Place the collected sample on a newspaper and air-dry it naturally for 5 d, then place the dried soil sample in a mortar and grind it to a diameter of <0.15 mm. Weigh 1 g of the soil sample and place it at the bottom of a dry digestion tube, moisten it with a small amount of water, add 5 mL of concentrated sulfuric acid and a trace amount of catalyst for digestion until the solution in the digestion tube turns grayish white with a slightly green color, and then continue to digest for a period of time. Remove the digestion tube. Use a continuous chemical flow analyzer to determine the nitrogen content in each culture solution.
[0054] ③ Determine the available phosphorus content in the bacterial fertilizer by the molybdenum-antimony anti-colorimetric method.
[0055] Preparation of the test solution: Accurately weigh 2.0 g of the <0.15 mm soil sample, place it in a 50 mL digestion tube, moisten it with a small amount of water, add 8 mL of concentrated sulfuric acid, shake well, then add 10 drops of 70% - 72% HClO4, shake well, add a small funnel to the tube mouth, place it on an electric furnace for digestion for 40 - 60 min, and do a blank control at the same time. Transfer the cooled digestion solution to a 100 mL volumetric flask with a small amount of distilled water, and make up the volume after cooling.
[0056] Determination: Accurately pipette 5 mL of the filtrate into a 50 mL volumetric flask, dilute it with water to 30 mL, add 2 drops of 2,4-dinitrophenol indicator, then add 4 mol / L NaOH solution dropwise until the solution turns yellow, and finally add concentrated sulfuric acid solution dropwise until the yellow color of the solution just fades. Then add 5 mL of molybdenum antimony anti-color reagent, and finally make up the volume to 50 mL and shake well. After 30 min, measure the absorbance at a wavelength of 700 nm on a spectrophotometer. At the same time, prepare phosphorus solutions with final concentrations of 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 μg﹒mL -1 in a 50 mL volumetric flask using a phosphorus standard solution. The other steps are the same as above. Finally, obtain a phosphorus standard curve with phosphorus concentration as the abscissa and absorbance as the ordinate. And calculate the content of available phosphorus in the sample using the following formula:
[0057]
[0058] where: C - mass concentration of phosphorus in the test solution (μg﹒mL -1 ); V - volume of the sample preparation solution (mL); V1 - volume of the filtrate pipetted (mL); V2 - volume of the test solution (mL); m - mass of the sample (g).
[0059] ④ Determine the content of available potassium using a flame spectrophotometer
[0060] Pipette a certain amount of potassium solution from the potassium standard solution, make up the volume, and prepare potassium solutions of 0.0, 1.0, 2.0, 4.0, 8.0, 12.0, 16.0 μg / mL. After measurement with a flame spectrophotometer, obtain a potassium standard curve with potassium content as the abscissa and absorbance as the ordinate. Place the collected soil sample on a newspaper and air-dry it naturally for 5 d. Then place the dried soil sample in a mortar and grind it. Weigh 2.0 g of the soil sample into a dry plastic bottle. Add 20 mL of 1 mol / L neutral ammonium acetate solution to the plastic bottle, cover the bottle cap, shake it on a shaker for 30 min, filter it, and measure the filtrate on a flame spectrophotometer. Calculate the content of available potassium in the sample using the following formula:
[0061]
[0062] where:
[0063] C - mass concentration of K in the test solution obtained from the standard curve (μg﹒mL -1 ); V - volume of the leaching agent added (mL); m - mass of the sample (g).
[0064] (3) Determination of the germination index (GI) of the compost leachate:
[0065] Mix 5 g of the sample with 50 mL of distilled water in a 250 mL conical flask, place it on a shaker at 120 rpm for 2 h, and the filtrate is the compost extract. Lay a layer of filter paper in a petri dish with a diameter of 9 cm, evenly place 10 radish seeds or rice seeds, and add 10 mL of the compost extract. Add 10 mL of sterilized tap water as a control, with three replicates for each treatment. Incubate the petri dishes in the dark at 28 °C for 4 d and check the germination rate. The calculation formula is: GI(%) = (germination rate of treated seeds × average root length of treated seeds) ÷ (germination rate of control seeds × average root length of control seeds) × 100
[0066] 2. Determination of the content of Streptomyces HN6
[0067] Mix 2 g of the compost sample with 20 mL of distilled water, shake at 25 °C and 120 rpm for 2 h, and centrifuge at 8000 rpm / min for 10 min. Take 1 mL of the supernatant and dilute it successively to 10 -7 、10 -8 、10 -9 . Pipette 25 μL onto the Gao's No. 1 solid medium, spread it evenly with a spreader, culture for 5 d, and count the number of bacteria in the unit sample.
[0068] Example 2
[0069] The difference between this example and Example 1 is as follows:
[0070] 5) Composting and aging: When the temperature of the primary functional bacterial fertilizer is between 35 and 38 °C, carry out composting and aging to form humus; the time for composting and aging is 20 days;
[0071] 6) Inoculating Streptomyces aureoverticillatus HN6: When the temperature of the aged primary functional bacterial fertilizer drops to 29 °C, inoculate 10 7 cfu / mL of Streptomyces aureoverticillatus HN6 culture solution again.
[0072] Example 3
[0073] The difference between this example and Example 1 is as follows:
[0074] 5) Composting and aging: When the temperature of the primary functional bacterial fertilizer is between 37 and 40 °C, carry out composting and aging to form humus; the time for composting and aging is 20 days;
[0075] 6) Inoculating Streptomyces aureoverticillatus HN6: When the temperature of the aged primary functional bacterial fertilizer drops to 34 °C, inoculate 10 7 cfu / mL of Streptomyces aureoverticillatus HN6 culture solution again.
[0076] Example 4
[0077] The difference between this example and Example 1 is as follows:
[0078] 3) Dry, crush, and mix the base materials of banana straw, corn straw, cowpea straw, and rice straw according to a weight ratio of 1:0.8:1.2:1 to obtain a straw mixture.
[0079] Comparative Example 1
[0080] The difference between this example and Example 1 lies in
[0081] In step 2), the bacterial content of the fermentation broth of Thermophilic Sporotrichum thermophile, white rot enzyme fermentation broth, Bacillus amyloliquefaciens fermentation broth, and Cellulolytic Bacillus fermentation broth is 1×10 8 cfu / mL. After inoculation at this concentration, the situation where the growth of bacteria is fast and affects the growth of fungi may occasionally occur.
[0082] Comparative Example 2
[0083] The difference between this example and Example 1 lies in
[0084] In step 2), the bacterial content of the fermentation broth of Thermophilic Sporotrichum thermophile, white rot enzyme fermentation broth, Bacillus amyloliquefaciens fermentation broth, and Cellulolytic Bacillus fermentation broth is 1×10 10 cfu / mL. With the inoculation at this concentration, increasing the bacterial content will shorten the initial decay process, resulting in the inappropriate growth of individual bacteria and prolonging the decay process.
[0085] Comparative Example 3
[0086] The difference between this example and Example 1 lies in
[0087] In step 6), inoculate the culture solution of Streptomyces verticillatus HN6 at 10 6 cfu / mL. A decrease in the strain content will cause the effective bacteria in the final fermentation product, that is, the fertilizer end product, to fail to meet the content requirements of the fertilizer.
[0088] Comparative Example 4
[0089] The difference between this example and Example 1 lies in
[0090] In step 6), inoculate the culture solution of Streptomyces verticillatus HN6 at 10 8 cfu / mL. An increase in the strain concentration will have a positive effect compared to Example 1, but there is no significant improvement.
[0091] Application Example 1
[0092] The control effect of the bacterial fertilizer on potted bananas
[0093] Collect soil samples from non-pathogenic host planting areas, sieve them through a sieve with a pore size of 85 μm, sterilize them at 160 °C for 2 h, and mix the functional bacterial fertilizer treatment in Example 1 into the sterilized soil at an addition rate of 0.5 kg per cubic meter. Divide the mixed soil into sterilized seedling-raising cups, and use no application of compost as a blank control. Rinse the roots of banana seedlings with clean water, plant them in the seedling-raising cups, and inoculate them with 10 7 cfu / mL banana wilt spore suspension by the root injury method. Then, investigate the disease index of each treatment on the 14th, 22nd, and 30th days after inoculating the pathogenic bacteria, and statistically calculate the final control effect with reference to the disease grading standard. Disease index = [Σ (number of diseased plants × disease grade) / (total number of plants × highest disease grade)] × 100; Control effect (%) = (disease index of sterile water treatment - disease index of other treatments) / disease index of sterile water treatment × 100.
[0094] Application Example 2
[0095] Control effect of bacterial fertilizer on bananas in the field
[0096] The test field was selected in Shanghongxing Village, Baisuo Town, Hainan Province, where banana wilt often occurs. Promote and apply the functional bacterial fertilizer obtained in Example 1 at 20 kg per mu in banana planting. Apply it once during the seedling planting period of bananas and supplement it once during the fruit growth and development period. Use no treatment as a blank control. Each treatment has 10 plants and is repeated 3 times. Measure the control effect 30 days after the last application, and measure the growth-promoting effect of the bacterial fertilizer on bananas.
[0097] Application Example 3
[0098] Prevention and control of gummosis of jackfruit by bacterial fertilizer
[0099] The test field was selected in the jackfruit gummosis-prone base (4-year-old trees) of Xiqin Farm, Danzhou, Hainan Province. Promote and apply the obtained functional bacterial fertilizer at 20 kg per mu on jackfruit plants. Apply it 2 times per month for three months before fruiting and supplement it once after fruit picking. Use no treatment as a blank control. Statistically calculate the control effect of gummosis 30 days after the last application, and measure the growth-promoting effect of applying the bacterial fertilizer on jackfruit. The disease grading standard is as follows:
[0100] Table 1 Disease grading standard of jackfruit gummosis
[0101]
[0102] Note: Primary branches, secondary branches, and tertiary branches refer to the main trunk, main branches, and lateral branches respectively.
[0103] Measurement results and analysis of the above Example 1
[0104] 1. Detection of composting indicators
[0105] (1) Apparent changes
[0106] After 14 days of composting in the first stage, the straw turned brown. When holding the substrate by hand, it felt soft and elastic, brittle and easy to break when dry, and the temperature inside the compost pile was constantly around 35 - 38 °C.
[0107] (2) Detection of physical and chemical indexes
[0108] Table 2 Determination of physical and chemical parameters of the bacterial fertilizer
[0109]
[0110] (3) Determination of the germination index (GI) of the compost leachate on seeds
[0111] It can be seen from the determination of the effect on the germination of radish seeds that the compost leachate and its dilution solutions do not affect the germination of radish seeds, but can significantly promote the growth of radish root length and stem length. The 50-fold dilution solution has the most prominent promoting effect on radish root length and stem length.
[0112] Table 3 Effect of the compost leachate on seed germination
[0113]
[0114] It can be seen from the determination of the effect on the germination of rice seeds that the compost leachate and its dilution solutions do not affect the germination of radish seeds, but can significantly promote the growth of radish root length and stem length. The undiluted leachate has the most obvious promoting effect on rice root length, and the 50-fold dilution solution has the most prominent promoting effect on rice stem length.
[0115] Table 4 Effect of the compost leachate on the germination of rice seeds
[0116]
[0117]
[0118] 2. Determination of the content of Streptomyces HN6
[0119] After detection, the bacteria-carrying amount of this bacterial fertilizer was 1.45×10 10 cfu·g -1 , and the effective bacteria content exceeded 1×10 9 cfu·g -1 , meeting the standards of biological fertilizers in China.
[0120] 3. Application of the bacterial fertilizer in the prevention and control of potted banana wilt
[0121] It can be seen from Table 5 that the functional bacterial fertilizer prepared in Example 1 can well control the occurrence and development of banana wilt, and the final control effect measured was 75.97%.
[0122] Table 5 Pot experiment control effect of the functional bacterial fertilizer on banana wilt
[0123]
[0124] 4. Disease prevention effect on field bananas
[0125] As can be seen from Table 6, the prepared functional bacterial fertilizer can well control the occurrence and development of banana wilt disease in the field, and the finally measured control effect is 74.37%.
[0126] Table 6 Field control effect of functional bacterial fertilizer on banana wilt disease
[0127]
[0128] 5. Growth promotion effect on field banana plants
[0129] Table 7 Growth promotion effect of functional bacterial fertilizer on banana plants
[0130]
[0131] Note: Different capital and lowercase letters indicate significant differences between strains in different treatments (a: P < 0.05; A: P < 0.01)
[0132] As can be seen from Table 7, the functional bacterial fertilizer in Example 1 can have an obvious growth promotion effect on banana plants, especially on the enhancement of root activity of the plants, showing a highly significant difference from other indicators. Secondly, it has a relatively large impact on fresh weight, showing a highly significant difference from the impacts on plant height and stem diameter.
[0133] 6. Control effect of bacterial fertilizer on jackfruit gummosis
[0134] As can be seen from Table 8, the prepared functional bacterial fertilizer can well control the occurrence and development of jackfruit gummosis, and the finally measured control effect is 77.07%.
[0135] Table 8 Field control effect of functional fertilizer on jackfruit gummosis
[0136]
[0137] 7. Growth promotion effect of bacterial fertilizer on jackfruit
[0138] Table 9 Growth promotion effect of functional fertilizer on jackfruit plants
[0139]
[0140] Note: Different capital and lowercase letters indicate significant differences between strains in different treatments (a: P < 0.05; A: P < 0.01)
[0141] As can be seen from Table 9, the functional fertilizer in Example 1 can significantly affect the physiological indexes of jackfruit leaves, increase the contents of chlorophyll, soluble sugar and soluble protein in the plant leaves, especially having the greatest impact on chlorophyll and a relatively large impact on soluble sugar.
[0142] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention and not to limit it. Although the present invention has been described in detail with reference to the preferred arrangement, those of ordinary skill in the art should understand that the technical solution of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solution of the present invention.
Claims
1. A preparation method of a composite microbial functional bacterial fertilizer, characterized in that: It includes the following steps: 1) Strain preparation: Prepare the mother strains of Thermoascus aurantiacus and Phanerochaete chrysosporium; the culture solutions of Bacillus amyloliquefaciens and Bacillus cellulosilyticus; and the mother strain of Streptomyces verticillus HN6; 2) Preparation of fermentation broth: Prepare Thermomyces lanuginosus fermentation broth, white rot enzyme fermentation broth, Bacillus amyloliquefaciens fermentation broth and Bacillus cellulolyticus fermentation broth. The bacterial content of the above fermentation broths is 1×10 9 cfu / mL; Prepare the culture solution of Streptomyces verticillatus HN6, and the bacterial content is 1×10 7 cfu / mL; 3) Preparation of fermentation substrate: Dry, crush and mix the substrate banana straw, corn straw, cowpea straw and rice straw according to the weight ratio of 1:0.8 - 1.2:0.8 - 1.2:0.8 - 1.2 to obtain a straw mixture, and then mix it with cow dung to obtain the fermentation substrate. The ratio of the straw mixture to cow dung is: 25 kg of cow dung / 1 ton of straw mixture; 4) Preparation of primary functional bacterial fertilizer: Mix the fermentation broths of Thermoascus aurantiacus, Phanerochaete chrysosporium, Bacillus amyloliquefaciens and Bacillus cellulosilyticus in equal volumes to obtain a mixed fermentation broth. Inoculate the mixed fermentation broth into the fermentation substrate at an inoculation amount of 10% by mass fraction. After mixing, pile it into a heap, and then cover the cone with a black mesh net. After primary fermentation, manual turning and secondary fermentation in sequence, the primary functional bacterial fertilizer is made; 5) Composting and aging: When the temperature of the primary functional bacterial fertilizer is 35 - 40 °C, carry out composting and aging to form humus; the time for composting and aging is 20 days; 6) Inoculate Streptomyces aureoverticillatus HN6: When the temperature of the aged primary functional bacterial fertilizer drops to 26 - 35 °C, inoculate the culture solution of Streptomyces aureoverticillatus HN6 at 10 7 cfu / mL again and culture for 10 days to obtain the compound microbial functional bacterial fertilizer; the culture solution of Streptomyces aureoverticillatus HN6 is inoculated at 2% of the weight of the aged primary functional bacterial fertilizer.
2. The preparation method of the compound microbial functional bacterial fertilizer according to claim 1, characterized in that: The specific operation of step 1) is: Respectively inoculate Thermoascus aurantiacus and Phanerochaete chrysosporium onto PDA plates and culture for 3 d as mother strains for standby; the composition of the PDA plate is: add 200 g of potato, 20 g of glucose, and 15 - 20 g of agar to each liter of distilled water; Inoculate Bacillus amyloliquefaciens and Bacillus cellulosilyticus into beef extract peptone culture solution respectively and shake culture at 28 °C for 24 h to prepare seed culture solutions for standby; Inoculate the functional strain Streptomyces verticillus HN6 onto Gao's No. 1 plate and culture at 28 °C for 3 d; for standby.
3. The preparation method of the compound microbial functional bacterial fertilizer according to claim 2, characterized in that: The specific operation of step 2) is: Respectively inoculate the cultured Thermoascus aurantiacus and Phanerochaete chrysosporium into PDB liquids and shake culture at 400 rpm for 3 d to obtain the fermentation broths of Thermoascus aurantiacus and Phanerochaete chrysosporium; Inoculate the seed culture solutions of Bacillus amyloliquefaciens and Bacillus cellulosilyticus into beef extract peptone culture solution and shake culture at 28 °C for 24 h to obtain the fermentation broths of Bacillus amyloliquefaciens and Bacillus cellulosilyticus; Inoculate Streptomyces verticillus HN6 into Gao's No. 1 liquid fermentation medium and shake culture at 28 °C for 72 h to obtain the culture solution of Streptomyces verticillus HN6.
4. The preparation method of the compound microbial functional bacterial fertilizer according to claim 3, characterized in that: In step 2), the composition of the PDB liquid is: add 200 g / L of potato and 20 g / L of glucose to each liter of water, and the pH is natural; the formula composition of the beef extract peptone culture solution is: 3 g of beef extract, 10 g of peptone, 5 g of sodium chloride, make up to 1000 mL with water, and the pH is 7.0 - 7.2; the formula composition of the Gao's No. 1 liquid fermentation medium is: 20 g of soluble starch, 1 g of KNO3, 0.5 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.5 g of NaCl, 0.01 g of FeSO4·7H2O, make up to 1000 mL with water, and the pH is 7.0 - 7.
2.
5. The preparation method of the compound microbial functional bacterial fertilizer according to claim 1, characterized in that: In step 3), the base materials, namely banana straw, corn straw, cowpea straw and rice straw, are dried in a weight ratio of 1:1:1:
1.
6. The preparation method of the compound microbial functional bacterial fertilizer according to claim 1, characterized in that: In the preparation of the primary functional bacterial fertilizer in step 4): The total primary fermentation time is 13 days, and the temperature is monitored every day. The cone first undergoes a 5-day temperature-rising fermentation until the temperature rises to 60-65°C, then undergoes a 3-day high-temperature fermentation at 60-65°C, and then undergoes a 5-day temperature-lowering fermentation until the temperature drops to 40°C; The total secondary fermentation time is 12 days. Specifically, after manual turning of the pile, it is piled up into a cone again for fermentation. The temperature of the cone rises to 55°C for fermentation, ferments at 55°C for 7 days, and then undergoes a 5-day temperature-lowering fermentation until the temperature drops to 40°C; The pile body is a cone with a height of 105 cm and a diameter of 100 cm.
7. Use of the compound microbial functional bacterial fertilizer according to any one of claims 1 to 6, characterized in that: The compound microbial functional bacterial fertilizer is used for the prevention and control of banana wilt in potted plants, the prevention and control of banana wilt in fields, and the growth promotion of field bananas.
8. Use of the compound microbial functional bacterial fertilizer according to claim 7, characterized in that: For the prevention and control of banana wilt in potted plants, the bacterial fertilizer is mixed into the soil at an application rate of 0.5 kg per cubic meter; For the prevention and control of banana wilt in fields and the growth promotion of field bananas, the bacterial fertilizer is mixed into the soil at an application rate of 20 kg per mu, applied once during the banana seedling planting period and top-dressed once during the fruit growth and development period.
9. Use of the compound microbial functional bacterial fertilizer according to any one of claims 1 to 6, characterized in that: The compound microbial functional bacterial fertilizer is used for the prevention and control of jackfruit gummosis and the growth promotion of jackfruit.
10. Use of the compound microbial functional bacterial fertilizer according to claim 9, characterized in that: The compound microbial functional bacterial fertilizer is used for the prevention and control of jackfruit gummosis and the growth promotion of jackfruit. The bacterial fertilizer is mixed into the soil at an application rate of 20 kg per mu, applied 2 times per month three months before fruiting, and top-dressed once after fruit picking.