Microbial fertilizer for preventing and treating citrus huanglongbing as well as preparation method and application of microbial fertilizer

Abstract

The invention discloses a microbial fertilizer for preventing and treating citrus liberobacter asiaticum as well as a preparation method and application thereof, belongs to the technical field of prevention and treatment of agricultural diseases and pests, and particularly relates to the microbial fertilizer for preventing and treating the citrus liberobacter asiaticum. Comprising 5-10 parts of bacillus subtilis, 5-10 parts of bacillus amyloliquefaciens, 3-8 parts of bacillus megatherium, 3-8 parts of bacillus licheniformis, 3-8 parts of photosynthetic bacteria, 3-8 parts of nitrogen-fixing bacteria, 3-8 parts of phosphate solubilizing bacteria, 3-8 parts of potassium bacteria, 30-50 parts of decomposed organic fertilizer, 10-20 parts of humic acid, 5-10 parts of sodium alginate, 5-10 parts of compound amino acid and 1-5 parts of compound trace elements. The microbial fertilizer disclosed by the invention can effectively inhibit the growth of pathogenic bacteria of the citrus huanglongbing, improve the disease resistance of citrus trees, improve the soil environment and promote the growth and development of the citrus trees, and the preparation method is mature and perfect in process and suitable for large-scale production and application.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of agricultural pest control, and particularly relates to a microorganism fertilizer for preventing and treating citrus Huanglongbing and a preparation method and application thereof. BACKGROUND

[0002] Citrus Huanglongbing, also known as Huangshao disease or Huangku disease, is a kind of devastating disease caused by Asian Liberibacter infection, which occurs on citrus. It seriously affects yield and quality, and even causes citrus tree to die. It is more serious in spring and summer with more rain and in autumn with drought. It is more serious in orchards with insufficient fertilization, low-lying terrain, poor drainage, and closed tree canopy. Young trees are more resistant than old trees, and 4-8 year-old trees are more susceptible. Citrus psylla occurs more, and citrus Huanglongbing also occurs more. The pathogen is Asian Liberibacter (Liberbacter asiaticum jagoueix.), which belongs to the genus Liberibacter. The pathogen is round or oval, with a size of 50-600 microns x 170-1600 microns. The optimal temperature for growth of the bacterial mycelium is about 20℃, and the growth is slow below 10℃ or above 35℃. The temperature for ovate conidial spore germination is 5-35℃, and the optimum temperature is 15-25℃. This kind of pathogen can infect citrus trees. The transmission modes of citrus Huanglongbing mainly include two kinds of citrus psylla transmission and graft transmission. Citrus psylla is the transmission medium of citrus Huanglongbing. As the main pest of new shoots of citrus, adult psylla mostly lay eggs on infected tender shoots, and after the nymphs hatch, they start to suck the juice of the tender shoots, thereby producing a large number of infected adult psylla, which then fly to new plants to spread citrus Huanglongbing.

[0003] In recent years, microbial fertilizer as a new type of biological control method has shown significant potential in preventing and controlling citrus Huanglongbing. Studies have shown that specific microbial combinations can inhibit or destroy the development process of nematode eggs, reduce the adult nematode rate, and thus cut off the transmission medium of Huanglongbing. For example, the combination of Paecilomyces lilacinus, Bacillus licheniformis and Trichoderma has been proven to effectively reduce the basic occurrence of nematodes in orchards, achieving the effect of prevention and control. The beneficial microorganisms in microbial fertilizer can inhibit the growth of pathogenic bacteria through various mechanisms, such as competition for nutrients and living space, production of antibacterial substances, and induction of plant resistance, etc. At the same time, they can also improve soil structure, increase soil fertility, promote plant growth, and enhance the disease resistance of plants. These findings provide theoretical support for the technology based on soil remediation and improvement combined with biological control. The present application is committed to providing a new and effective microbial fertilizer for preventing and treating citrus Huanglongbing, providing a new direction for the prevention and treatment of citrus Huanglongbing. SUMMARY

[0004] To solve the above technical problems, the present application provides a microbial fertilizer for preventing and treating citrus Huanglongbing and a preparation method and application thereof, the microbial fertilizer can effectively inhibit the growth of citrus Huanglongbing pathogen, improve the disease resistance of citrus trees, improve the soil environment, and promote the growth and development of citrus trees, and the preparation method is mature and perfect, suitable for large-scale production and application.

[0005] To achieve the above object, the present application provides a microbial fertilizer for preventing and treating citrus Huanglongbing, comprising the following components by weight:

[0006] Bacillus subtilis 5-10 parts, Bacillus amyloliquefaciens 5-10 parts, Bacillus megaterium 3-8 parts, Bacillus licheniformis 3-8 parts, photosynthetic bacteria 3-8 parts, nitrogen-fixing bacteria 3-8 parts, phosphorus-solubilizing bacteria 3-8 parts, potassium-solubilizing bacteria 3-8 parts, composted organic fertilizer 30-50 parts, humic acid 10-20 parts, sodium alginate 5-10 parts, complex amino acid 5-10 parts, and complex trace elements 1-5 parts.

[0007] The present application also provides a preparation method of the microbial fertilizer, comprising the following steps:

[0008] 1) Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus megaterium, Bacillus licheniformis, photosynthetic bacteria, nitrogen-fixing bacteria, phosphorus-solubilizing bacteria, and potassium-solubilizing bacteria are respectively activated and cultured to obtain activated Bacillus subtilis, activated Bacillus amyloliquefaciens, activated Bacillus megaterium, activated Bacillus licheniformis, activated photosynthetic bacteria, activated nitrogen-fixing bacteria, activated phosphorus-solubilizing bacteria, and activated potassium-solubilizing bacteria;

[0009] 2) The activated Bacillus subtilis, activated Bacillus amyloliquefaciens, activated Bacillus megaterium, activated Bacillus licheniformis, activated photosynthetic bacteria, activated nitrogen-fixing bacteria, activated phosphorus-solubilizing bacteria, and activated potassium-solubilizing bacteria are respectively inoculated into liquid culture medium according to an inoculation amount of 4-6% v / v, and respectively fermented and cultured to obtain Bacillus subtilis fermentation broth, Bacillus amyloliquefaciens fermentation broth, Bacillus megaterium fermentation broth, Bacillus licheniformis fermentation broth, photosynthetic bacteria fermentation broth, nitrogen-fixing bacteria fermentation broth, phosphorus-solubilizing bacteria fermentation broth, and potassium-solubilizing bacteria fermentation broth;

[0010] 3) The Bacillus subtilis fermentation broth, Bacillus amyloliquefaciens fermentation broth, Bacillus megaterium fermentation broth, Bacillus licheniformis fermentation broth, photosynthetic bacteria fermentation broth, nitrogen-fixing bacteria fermentation broth, phosphorus-solubilizing bacteria fermentation broth, and potassium-solubilizing bacteria fermentation broth are mixed with composted organic fertilizer, humic acid, sodium alginate, complex amino acid, and complex trace elements, granulated, dried, and microbial fertilizer is obtained.

[0011] Preferably, the liquid medium formula in step 2) is as follows: maltose 15-25 g, lactose 10-20 g, tryptone 5-15 g, yeast extract 4-6 g, sodium chloride 1.5-3.5 g, magnesium sulfate 1-3 g, L-cysteine hydrochloride 0.4-0.6 g, tomato powder 0.4-0.6 g, anhydrous calcium chloride 0.1-0.2 g, and water to 1 L.

[0012] Preferably, the temperature of the activated Bacillus subtilis fermentation culture in step 2) is 30-37℃, and the time is 12-20 h; the temperature of the activated Bacillus amyloliquefaciens fermentation culture is 28-32℃, and the time is 12-20 h; the temperature of the activated Bacillus megaterium fermentation culture is 25-30℃, and the time is 12-20 h; and the temperature of the activated Bacillus licheniformis fermentation culture is 28-30℃, and the time is 12-20 h.

[0013] Preferably, the temperature of the activated photosynthetic bacteria fermentation culture in step 2) is 35-37℃, and the time is 24-48 h; and the temperature of the activated nitrogen-fixing bacteria fermentation culture is 28-30℃, and the time is 24-48 h.

[0014] Preferably, the temperature of the activated phosphorus-dissolving bacteria fermentation culture in step 2) is 25-28℃, and the time is 44-48 h; and the temperature of the activated potassium-dissolving bacteria fermentation culture is 25-28℃, and the time is 44-48 h.

[0015] The application further provides application of the microbial fertilizer in preventing and treating citrus Huanglongbing.

[0016] Preferably, the microbial fertilizer inhibits growth and reproduction of the pathogen of citrus Huanglongbing.

[0017] Preferably, the application specifically refers to using the microbial fertilizer as base fertilizer and / or topdressing, and applying the microbial fertilizer around the roots of the citrus trees by ditching, hole digging or spreading, and then watering.

[0018] Further preferably, the dosage of the microbial fertilizer is calculated as 10-15 kg per citrus tree.

[0019] Compared with the prior art, the application has the following advantages and technical effects:

[0020] The present application provides a kind of microbial fertilizer for preventing and treating citrus Huanglongbing and its preparation method and application, various microorganisms in the microbial fertilizer of the present application are compounded, dominant flora is formed around the root of citrus tree, through the secretion of the growth of citrus beneficial gamma-polyglutamic acid, lycopene and the enzyme that can effectively decompose nutrient material in soil, nitrogen element in soil is fixed, ineffective phosphorus and potassium in soil are transformed, while inhibiting the growth and reproduction of citrus Huanglongbing pathogen, provide nitrogen, phosphorus, potassium and other nutrient elements, improve soil fertility, promote the growth of root system of citrus tree, enhance the photosynthesis of citrus tree, improve the resistance and yield quality of citrus tree;Meanwhile it can also improve soil structure, increase soil aeration and water retention, provide a good soil environment for the growth of citrus tree, effectively reduce the incidence and disease index of Huanglongbing.The microbial fertilizer of the present application uses biological control method, does not contain harmful chemical substances, has no pollution to environment, is harmless to human health, meets the requirements of green agricultural development.The preparation method of the microbial fertilizer of the present application is easy to operate, the required equipment and raw materials are suitable for large-scale production and application. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments. Obviously, the drawings described in the following only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0022] Figure 1 Statistical analysis chart of Ct value of plants in different groups;

[0023] Figure 2 Statistical analysis chart of yield per plant of plants in different groups;

[0024] Figure 3 Statistical analysis chart of single fruit weight of plants in different groups. DETAILED DESCRIPTION

[0025] Now, various exemplary embodiments of the present application will be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of some aspects, characteristics and embodiments of the present application.

[0026] It should be understood that the terms described in the present application are only for describing the specific embodiments, and are not used to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value in the stated range and any other stated value or intermediate value in the stated range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0027] Unless otherwise indicated, all technical and scientific terms have the same meaning as those one of ordinary skill in the art of the invention would understand. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the present specification and any document incorporated by reference, the present specification controls.

[0028] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples herein. The description and examples are illustrative only.

[0029] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0030] The "parts" described in the present application are measured by mass unless otherwise specified.

[0031] The Bacillus subtilis used in the application is Bacillus subtilis CICC 24713, which is purchased from the China Industrial Microbial Culture Collection Center; the Bacillus amyloliquefaciens is Bacillus amyloliquefaciens CICC 20164, which is purchased from the China Industrial Microbial Culture Collection Center; the Bacillus megaterium is Bacillus megaterium CICC 23692, which is purchased from the China Industrial Microbial Culture Collection Center; the Bacillus licheniformis is Bacillus licheniformis CICC 10099, which is purchased from the China Industrial Microbial Culture Collection Center; the photosynthetic bacteria is Rhodopseudomonas palustris CICC 23812, which is purchased from the China Industrial Microbial Culture Collection Center; the nitrogen-fixing bacteria is Azotobacter chroococcum CICC 21686, which is purchased from the China Industrial Microbial Culture Collection Center; the phosphorus-solubilizing bacteria is Bacillus mucilaginosus CICC 22524, which is purchased from the China Industrial Microbial Culture Collection Center; the potassium-solubilizing bacteria is Bacillus mucilaginosus CICC 23575, which is purchased from the China Industrial Microbial Culture Collection Center; the Paenibacillus polymyxa is Paenibacillus polymyxa CICC 10494, which is purchased from the China Industrial Microbial Culture Collection Center; the rotten organic fertilizer is purchased from Hebei Wolmei Biological Technology Co., Ltd.; the compound amino acid is purchased from Henan Huaxing Biological Technology Co., Ltd.; and the compound trace element is purchased from Sichuan Xingda Heng Technology Co., Ltd.

[0032] The culture medium used in the activated culture described in the application is LB liquid culture medium, which is purchased from Beijing Solaybao Technology Co., Ltd.

[0033] Example 1

[0034] 1) The Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus megaterium, Bacillus licheniformis, photosynthetic bacteria, nitrogen-fixing bacteria, phosphorus-solubilizing bacteria and potassium-solubilizing bacteria are respectively activated and cultured by using LB liquid culture medium, and the temperature requirement of the activated culture is 37℃, and the time of the activated culture is 48h, so as to obtain activated Bacillus subtilis, activated Bacillus amyloliquefaciens, activated Bacillus megaterium, activated Bacillus licheniformis, activated photosynthetic bacteria, activated nitrogen-fixing bacteria, activated phosphorus-solubilizing bacteria and activated potassium-solubilizing bacteria.

[0035] 2) Activated Bacillus subtilis was inoculated at an inoculum rate of 5% v / v into a liquid culture medium (20 g maltose, 15 g lactose, 10 g tryptone, 5 g yeast extract, 2.5 g sodium chloride, 2 g magnesium sulfate, 0.5 g L-cysteine ​​hydrochloride, 0.5 g tomato powder, 0.15 g anhydrous calcium chloride, and water was added to 1 L), and fermented at 34° C. for 16 h to obtain a Bacillus subtilis fermentation liquid. Activated Bacillus amyloliquefaciens was inoculated at an inoculum rate of 5% v / v into a liquid culture medium (20 g maltose, 15 g lactose, 10 g tryptone, 5 g yeast extract, 2.5 g sodium chloride, 2 g magnesium sulfate, 0.5 g L-cysteine ​​hydrochloride, 0.5 g tomato powder, 0.15 g anhydrous calcium chloride, and water was added to 1 L), and fermented at 30° C. for 18 h to obtain a Bacillus amyloliquefaciens fermentation liquid. The activated Bacillus megaterium was inoculated into a liquid culture medium (20 g maltose, 15 g lactose, 10 g tryptone, 5 g yeast extract, 2.5 g sodium chloride, 2 g magnesium sulfate, 0.5 g L-cysteine ​​hydrochloride, 0.5 g tomato powder, 0.15 g anhydrous calcium chloride, and water was added to 1 L) at an inoculum amount of 5% v / v, and the culture was fermented at 28° C. for 18 h to obtain a Bacillus megaterium fermentation liquid. The activated Bacillus licheniformis was inoculated into a liquid culture medium (20 g maltose, 15 g lactose, 10 g tryptone, 5 g yeast extract, 2.5 g sodium chloride, 2 g magnesium sulfate, 0.5 g L-cysteine ​​hydrochloride, 0.5 g tomato powder, 0.15 g anhydrous calcium chloride, and water was added to 1 L) at an inoculum amount of 5% v / v, and the culture was fermented at 29° C. for 18 h to obtain a Bacillus licheniformis fermentation liquid. The activated photosynthetic bacteria were inoculated into a liquid culture medium (20 g maltose, 15 g lactose, 10 g tryptone, 5 g yeast extract, 2.5 g sodium chloride, 2 g magnesium sulfate, 0.5 g L-cysteine ​​hydrochloride, 0.5 g tomato powder, 0.15 g anhydrous calcium chloride, and water was added to 1 L) at an inoculum rate of 5% v / v, and fermented at 36°C for 36 h to obtain a photosynthetic bacteria fermentation liquid. The activated nitrogen-fixing bacteria were inoculated into a liquid culture medium (20 g maltose, 15 g lactose, 10 g tryptone, 5 g yeast extract, 2.5 g sodium chloride, 2 g magnesium sulfate, 0.5 g L-cysteine ​​hydrochloride, 0.5 g tomato powder, 0.15 g anhydrous calcium chloride, and water was added to 1 L) at an inoculum rate of 5% v / v, and fermented at 29°C for 36 h to obtain a nitrogen-fixing bacteria fermentation liquid. The activated phosphate-solubilizing bacteria were inoculated into a liquid culture medium (20 g maltose, 15 g lactose, 10 g tryptone, 5 g yeast extract, 2.5 g sodium chloride, 2 g magnesium sulfate, 0.5 g L-cysteine ​​hydrochloride, 0.5 g tomato powder, 0.15 g anhydrous calcium chloride, and water was added to 1 L) at an inoculum rate of 5% v / v, and fermented at 26° C. for 46 h to obtain a phosphate-solubilizing bacteria fermentation broth.The activated potassium-lysing bacteria is inoculated in liquid culture medium (maltose 20 g, lactose 15 g, tryptone 10 g, yeast extract 5 g, sodium chloride 2.5 g, magnesium sulfate 2 g, L-cysteine hydrochloride 0.5 g, tomato powder 0.5 g, anhydrous calcium chloride 0.15 g, and water to 1 L) at 5% v / v, and is fermented at 26°C for 46 h to obtain a potassium-lysing bacteria fermentation liquor.

[0036] 3) 7.5 parts of the Bacillus subtilis fermentation liquor, 7.5 parts of the Bacillus amyloliquefaciens fermentation liquor, 5.5 parts of the Bacillus megaterium fermentation liquor, 5.5 parts of the Bacillus licheniformis fermentation liquor, 5.5 parts of the photosynthetic bacteria fermentation liquor, 5.5 parts of the nitrogen-fixing bacteria fermentation liquor, 5.5 parts of the phosphorus-lysing bacteria fermentation liquor, and 5.5 parts of the potassium-lysing bacteria fermentation liquor are mixed with 40 parts of decomposed organic fertilizer, 15 parts of humic acid, 7.5 parts of sodium alginate, 7.5 parts of compound amino acid, and 3 parts of compound trace element, granulated to a particle size of 3 mm, dried, and a microbial fertilizer is obtained.

[0037] Example 2

[0038] 1) The Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus megaterium, Bacillus licheniformis, photosynthetic bacteria, nitrogen-fixing bacteria, phosphorus-lysing bacteria, and potassium-lysing bacteria are respectively activated and cultured in LB liquid culture medium, and the activation culture temperature is 37°C, and the activation culture time is 48 h, to obtain activated Bacillus subtilis, activated Bacillus amyloliquefaciens, activated Bacillus megaterium, activated Bacillus licheniformis, activated photosynthetic bacteria, activated nitrogen-fixing bacteria, activated phosphorus-lysing bacteria, and activated potassium-lysing bacteria.

[0039] 2) Activate Bacillus subtilis according to the inoculation amount 4% v / v inoculated in liquid medium (maltose 15 g, lactose 20 g, tryptone 5 g, yeast extract 6 g, sodium chloride 1.5 g, magnesium sulfate 3 g, L-cysteine hydrochloride 0.4 g, tomato powder 0.6 g, anhydrous calcium chloride 0.1 g, add water to 1 L), 30℃ fermentation culture 20h, get Bacillus subtilis fermentation broth. Activate Bacillus amyloliquefaciens according to the inoculation amount 4% v / v inoculated in liquid medium (maltose 15 g, lactose 20 g, tryptone 5 g, yeast extract 6 g, sodium chloride 1.5 g, magnesium sulfate 3 g, L-cysteine hydrochloride 0.4 g, tomato powder 0.6 g, anhydrous calcium chloride 0.1 g, add water to 1 L), 28℃ fermentation culture 20h, get Bacillus amyloliquefaciens fermentation broth. Activate Bacillus megaterium according to the inoculation amount 4% v / v inoculated in liquid medium (maltose 15 g, lactose 20 g, tryptone 5 g, yeast extract 6 g, sodium chloride 1.5 g, magnesium sulfate 3 g, L-cysteine hydrochloride 0.4 g, tomato powder 0.6 g, anhydrous calcium chloride 0.1 g, add water to 1 L), 25℃ fermentation culture 20h, get Bacillus megaterium fermentation broth. Activate Bacillus licheniformis according to the inoculation amount 4% v / v inoculated in liquid medium (maltose 15 g, lactose 20 g, tryptone 5 g, yeast extract 6 g, sodium chloride 1.5 g, magnesium sulfate 3 g, L-cysteine hydrochloride 0.4 g, tomato powder 0.6 g, anhydrous calcium chloride 0.1 g, add water to 1 L), 28℃ fermentation culture 20h, get Bacillus licheniformis fermentation broth. Activate photosynthetic bacteria according to the inoculation amount 4% v / v inoculated in liquid medium (maltose 15 g, lactose 20 g, tryptone 5 g, yeast extract 6 g, sodium chloride 1.5 g, magnesium sulfate 3 g, L-cysteine hydrochloride 0.4 g, tomato powder 0.6 g, anhydrous calcium chloride 0.1 g, add water to 1 L), 35℃ fermentation culture 48h, get photosynthetic bacteria fermentation broth. Activate nitrogen-fixing bacteria according to the inoculation amount 4% v / v inoculated in liquid medium (maltose 15 g, lactose 20 g, tryptone 5 g, yeast extract 6 g, sodium chloride 1.5 g, magnesium sulfate 3 g, L-cysteine hydrochloride 0.4 g, tomato powder 0.6 g, anhydrous calcium chloride 0.1 g, add water to 1 L), 28℃ fermentation culture 48h, get nitrogen-fixing bacteria fermentation broth. Activate phosphorus solubilizing bacteria according to the inoculation amount 4% v / v inoculated in liquid medium (maltose 15 g, lactose 20 g, tryptone 5 g, yeast extract 6 g, sodium chloride 1.5 g, magnesium sulfate 3 g, L-cysteine hydrochloride 0.4 g, tomato powder 0.6 g, anhydrous calcium chloride 0.1 g, add water to 1 L), 25℃ fermentation culture 48h, get phosphorus solubilizing bacteria fermentation broth.The activated potassium-lysing bacteria is inoculated in liquid culture medium (maltose 15 g, lactose 20 g, tryptone 5 g, yeast extract 6 g, sodium chloride 1.5 g, magnesium sulfate 3 g, L-cysteine hydrochloride 0.4 g, tomato powder 0.6 g, anhydrous calcium chloride 0.1 g, and water to 1 L) at 4% v / v, and is fermented at 25°C for 48 h to obtain a potassium-lysing bacteria fermentation liquor.

[0040] 3) 5 parts of the Bacillus subtilis fermentation liquor, 5 parts of the Bacillus amyloliquefaciens fermentation liquor, 3 parts of the Bacillus megaterium fermentation liquor, 3 parts of the Bacillus licheniformis fermentation liquor, 3 parts of the photosynthetic bacteria fermentation liquor, 3 parts of the nitrogen-fixing bacteria fermentation liquor, 3 parts of the phosphorus-lysing bacteria fermentation liquor, and 3 parts of the potassium-lysing bacteria fermentation liquor are mixed with 30 parts of the decomposed organic fertilizer, 10 parts of the humic acid, 5 parts of the sodium alginate, 5 parts of the compound amino acid, and 1 part of the compound trace element, granulated with a particle size of 3 mm, dried at 40°C to constant weight, to obtain the microbial fertilizer.

[0041] Example 3

[0042] 1) The Bacillus subtilis, the Bacillus amyloliquefaciens, the Bacillus megaterium, the Bacillus licheniformis, the photosynthetic bacteria, the nitrogen-fixing bacteria, the phosphorus-lysing bacteria, and the potassium-lysing bacteria are respectively activated and cultured in LB liquid culture medium, and the temperature requirement for the activation culture is 37°C, and the time for the activation culture is 48 h, to obtain the activated Bacillus subtilis, the activated Bacillus amyloliquefaciens, the activated Bacillus megaterium, the activated Bacillus licheniformis, the activated photosynthetic bacteria, the activated nitrogen-fixing bacteria, the activated phosphorus-lysing bacteria, and the activated potassium-lysing bacteria.

[0043] 2) Activate Bacillus subtilis according to the inoculation amount 6% v / v inoculated in liquid medium (maltose 25 g, lactose 10 g, tryptone 15 g, yeast extract 4 g, sodium chloride 3.5 g, magnesium sulfate 1 g, L-cysteine hydrochloride 0.6 g, tomato powder 0.4 g, anhydrous calcium chloride 0.2 g, add water to 1 L), 37℃ fermentation culture 12 h, get Bacillus subtilis fermentation broth. Activate Bacillus amyloliquefaciens according to the inoculation amount 6% v / v inoculated in liquid medium (maltose 25 g, lactose 10 g, tryptone 15 g, yeast extract 4 g, sodium chloride 3.5 g, magnesium sulfate 1 g, L-cysteine hydrochloride 0.6 g, tomato powder 0.4 g, anhydrous calcium chloride 0.2 g, add water to 1 L), 32℃ fermentation culture 12 h, get Bacillus amyloliquefaciens fermentation broth. Activate Bacillus megaterium according to the inoculation amount 6% v / v inoculated in liquid medium (maltose 25 g, lactose 10 g, tryptone 15 g, yeast extract 4 g, sodium chloride 3.5 g, magnesium sulfate 1 g, L-cysteine hydrochloride 0.6 g, tomato powder 0.4 g, anhydrous calcium chloride 0.2 g, add water to 1 L), 30℃ fermentation culture 12 h, get Bacillus megaterium fermentation broth. Activate Bacillus licheniformis according to the inoculation amount 6% v / v inoculated in liquid medium (maltose 25 g, lactose 10 g, tryptone 15 g, yeast extract 4 g, sodium chloride 3.5 g, magnesium sulfate 1 g, L-cysteine hydrochloride 0.6 g, tomato powder 0.4 g, anhydrous calcium chloride 0.2 g, add water to 1 L), 30℃ fermentation culture 12 h, get Bacillus licheniformis fermentation broth. Activate photosynthetic bacteria according to the inoculation amount 6% v / v inoculated in liquid medium (maltose 25 g, lactose 10 g, tryptone 15 g, yeast extract 4 g, sodium chloride 3.5 g, magnesium sulfate 1 g, L-cysteine hydrochloride 0.6 g, tomato powder 0.4 g, anhydrous calcium chloride 0.2 g, add water to 1 L), 37℃ fermentation culture 24 h, get photosynthetic bacteria fermentation broth. Activate nitrogen-fixing bacteria according to the inoculation amount 6% v / v inoculated in liquid medium (maltose 25 g, lactose 10 g, tryptone 15 g, yeast extract 4 g, sodium chloride 3.5 g, magnesium sulfate 1 g, L-cysteine hydrochloride 0.6 g, tomato powder 0.4 g, anhydrous calcium chloride 0.2 g, add water to 1 L), 30℃ fermentation culture 24 h, get nitrogen-fixing bacteria fermentation broth. Activate phosphorus solubilizing bacteria according to the inoculation amount 6% v / v inoculated in liquid medium (maltose 25 g, lactose 10 g, tryptone 15 g, yeast extract 4 g, sodium chloride 3.5 g, magnesium sulfate 1 g, L-cysteine hydrochloride 0.6 g, tomato powder 0.4 g, anhydrous calcium chloride 0.2 g, add water to 1 L), 28℃ fermentation culture 44 h, get phosphorus solubilizing bacteria fermentation broth.The activated potassium-lysing bacteria is inoculated into liquid culture medium (maltose 25 g, lactose 10 g, tryptone 15 g, yeast extract 4 g, sodium chloride 3.5 g, magnesium sulfate 1 g, L-cysteine hydrochloride 0.6 g, tomato powder 0.4 g, anhydrous calcium chloride 0.2 g, and water to 1 L) at an inoculation amount of 6% v / v, and is fermented at 28°C for 44 h to obtain a potassium-lysing bacteria fermentation liquor.

[0044] 3) 10 parts of the Bacillus subtilis fermentation liquor, 10 parts of the Bacillus amyloliquefaciens fermentation liquor, 8 parts of the Bacillus megaterium fermentation liquor, 8 parts of the Bacillus licheniformis fermentation liquor, 8 parts of the photosynthetic bacteria fermentation liquor, 8 parts of the nitrogen-fixing bacteria fermentation liquor, 8 parts of the phosphorus-lysing bacteria fermentation liquor, and 8 parts of the potassium-lysing bacteria fermentation liquor are mixed with 50 parts of decomposed organic fertilizer, 20 parts of humic acid, 10 parts of sodium alginate, 10 parts of compound amino acid, and 5 parts of compound trace element, granulated to a particle size of 3 mm, and dried to obtain a microbial fertilizer.

[0045] Comparative Example 1

[0046] 1) The Bacillus subtilis, Bacillus polymyxa, Bacillus megaterium, Bacillus licheniformis, photosynthetic bacteria, nitrogen-fixing bacteria, phosphorus-lysing bacteria, and potassium-lysing bacteria are respectively activated and cultured using LB liquid medium, and the activation and culture temperature is 37°C, and the activation and culture time is 48 h to obtain activated Bacillus subtilis, activated Bacillus polymyxa, activated Bacillus megaterium, activated Bacillus licheniformis, activated photosynthetic bacteria, activated nitrogen-fixing bacteria, activated phosphorus-lysing bacteria, and activated potassium-lysing bacteria.

[0047] 2) Activate Bacillus subtilis according to the inoculation amount 5% v / v inoculated in liquid medium (maltose 20 g, lactose 15 g, tryptone 10 g, yeast extract 5 g, sodium chloride 2.5 g, magnesium sulfate 2 g, L-cysteine hydrochloride 0.5 g, tomato powder 0.5 g, anhydrous calcium chloride 0.15 g, add water to 1 L), 34℃ fermentation culture 16h, get Bacillus subtilis fermentation broth. Activate Bacillus polymyxa according to the inoculation amount 5% v / v inoculated in liquid medium (maltose 20 g, lactose 15 g, tryptone 10 g, yeast extract 5 g, sodium chloride 2.5 g, magnesium sulfate 2 g, L-cysteine hydrochloride 0.5 g, tomato powder 0.5 g, anhydrous calcium chloride 0.15 g, add water to 1 L), 30℃ fermentation culture 18h, get Bacillus polymyxa fermentation broth. Activate Bacillus megaterium according to the inoculation amount 5% v / v inoculated in liquid medium (maltose 20 g, lactose 15 g, tryptone 10 g, yeast extract 5 g, sodium chloride 2.5 g, magnesium sulfate 2 g, L-cysteine hydrochloride 0.5 g, tomato powder 0.5 g, anhydrous calcium chloride 0.15 g, add water to 1 L), 28℃ fermentation culture 18h, get Bacillus megaterium fermentation broth. Activate Bacillus licheniformis according to the inoculation amount 5% v / v inoculated in liquid medium (maltose 20 g, lactose 15 g, tryptone 10 g, yeast extract 5 g, sodium chloride 2.5 g, magnesium sulfate 2 g, L-cysteine hydrochloride 0.5 g, tomato powder 0.5 g, anhydrous calcium chloride 0.15 g, add water to 1 L), 29℃ fermentation culture 18h, get Bacillus licheniformis fermentation broth. Activate photosynthetic bacteria according to the inoculation amount 5% v / v inoculated in liquid medium (maltose 20 g, lactose 15 g, tryptone 10 g, yeast extract 5 g, sodium chloride 2.5 g, magnesium sulfate 2 g, L-cysteine hydrochloride 0.5 g, tomato powder 0.5 g, anhydrous calcium chloride 0.15 g, add water to 1 L), 36℃ fermentation culture 36h, get photosynthetic bacteria fermentation broth. Activate nitrogen-fixing bacteria according to the inoculation amount 5% v / v inoculated in liquid medium (maltose 20 g, lactose 15 g, tryptone 10 g, yeast extract 5 g, sodium chloride 2.5 g, magnesium sulfate 2 g, L-cysteine hydrochloride 0.5 g, tomato powder 0.5 g, anhydrous calcium chloride 0.15 g, add water to 1 L), 29℃ fermentation culture 36h, get nitrogen-fixing bacteria fermentation broth. Activate phosphorus solubilizing bacteria according to the inoculation amount 5% v / v inoculated in liquid medium (maltose 20 g, lactose 15 g, tryptone 10 g, yeast extract 5 g, sodium chloride 2.5 g, magnesium sulfate 2 g, L-cysteine hydrochloride 0.5 g, tomato powder 0.5 g, anhydrous calcium chloride 0.15 g, add water to 1 L), 26℃ fermentation culture 46h, get phosphorus solubilizing bacteria fermentation broth.The activated potassium-lysing bacteria were inoculated into liquid medium (maltose 20 g, lactose 15 g, tryptone 10 g, yeast extract 5 g, sodium chloride 2.5 g, magnesium sulfate 2 g, L-cysteine hydrochloride 0.5 g, tomato powder 0.5 g, anhydrous calcium chloride 0.15 g, and water to 1 L) at an inoculation amount of 5% v / v, and then fermented at 26°C for 46 h to obtain a potassium-lysing bacteria fermentation liquor.

[0048] 3) 7.5 parts of the Bacillus subtilis fermentation liquor, 7.5 parts of the Paenibacillus polymyxa fermentation liquor, 5.5 parts of the Bacillus megaterium fermentation liquor, 5.5 parts of the Bacillus licheniformis fermentation liquor, 5.5 parts of the photosynthetic bacteria fermentation liquor, 5.5 parts of the nitrogen-fixing bacteria fermentation liquor, 5.5 parts of the phosphorus-lysing bacteria fermentation liquor, and 5.5 parts of the potassium-lysing bacteria fermentation liquor were mixed with 40 parts of decomposed organic fertilizer, 15 parts of humic acid, 7.5 parts of sodium alginate, 7.5 parts of compound amino acid, and 3 parts of compound trace element, granulated to a particle size of 3 mm, and dried to obtain the microbial fertilizer.

[0049] Comparative Example 2

[0050] 1) The Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus megaterium, Bacillus licheniformis, photosynthetic bacteria, nitrogen-fixing bacteria, phosphorus-lysing bacteria, and potassium-lysing bacteria were respectively activated and cultured using LB liquid medium, and the activation and culture temperature was 37°C, and the activation and culture time was 48 h to obtain activated Bacillus subtilis, activated Bacillus amyloliquefaciens, activated Bacillus megaterium, activated Bacillus licheniformis, activated photosynthetic bacteria, activated nitrogen-fixing bacteria, activated phosphorus-lysing bacteria, and activated potassium-lysing bacteria.

[0051] 2) Activate Bacillus subtilis according to the inoculation amount 5% v / v inoculated in liquid medium (maltose 20 g, lactose 15 g, tryptone 10 g, yeast extract 5 g, sodium chloride 2.5 g, magnesium sulfate 2 g, L-cysteine hydrochloride 0.5 g, tomato powder 0.5 g, anhydrous calcium chloride 0.15 g, add water to 1 L), 34℃ fermentation culture 16h, get Bacillus subtilis fermentation broth. Activate Bacillus amyloliquefaciens according to the inoculation amount 5% v / v inoculated in liquid medium (maltose 20 g, lactose 15 g, tryptone 10 g, yeast extract 5 g, sodium chloride 2.5 g, magnesium sulfate 2 g, L-cysteine hydrochloride 0.5 g, tomato powder 0.5 g, anhydrous calcium chloride 0.15 g, add water to 1 L), 30℃ fermentation culture 18h, get Bacillus amyloliquefaciens fermentation broth. Activate Bacillus megaterium according to the inoculation amount 5% v / v inoculated in liquid medium (maltose 20 g, lactose 15 g, tryptone 10 g, yeast extract 5 g, sodium chloride 2.5 g, magnesium sulfate 2 g, L-cysteine hydrochloride 0.5 g, tomato powder 0.5 g, anhydrous calcium chloride 0.15 g, add water to 1 L), 28℃ fermentation culture 18h, get Bacillus megaterium fermentation broth. Activate Bacillus licheniformis according to the inoculation amount 5% v / v inoculated in liquid medium (maltose 20 g, lactose 15 g, tryptone 10 g, yeast extract 5 g, sodium chloride 2.5 g, magnesium sulfate 2 g, L-cysteine hydrochloride 0.5 g, tomato powder 0.5 g, anhydrous calcium chloride 0.15 g, add water to 1 L), 29℃ fermentation culture 18h, get Bacillus licheniformis fermentation broth. Activate photosynthetic bacteria according to the inoculation amount 5% v / v inoculated in liquid medium (maltose 20 g, lactose 15 g, tryptone 10 g, yeast extract 5 g, sodium chloride 2.5 g, magnesium sulfate 2 g, L-cysteine hydrochloride 0.5 g, tomato powder 0.5 g, anhydrous calcium chloride 0.15 g, add water to 1 L), 36℃ fermentation culture 36h, get photosynthetic bacteria fermentation broth. Activate nitrogen-fixing bacteria according to the inoculation amount 5% v / v inoculated in liquid medium (maltose 20 g, lactose 15 g, tryptone 10 g, yeast extract 5 g, sodium chloride 2.5 g, magnesium sulfate 2 g, L-cysteine hydrochloride 0.5 g, tomato powder 0.5 g, anhydrous calcium chloride 0.15 g, add water to 1 L), 29℃ fermentation culture 36h, get nitrogen-fixing bacteria fermentation broth. Activate phosphorus solubilizing bacteria according to the inoculation amount 5% v / v inoculated in liquid medium (maltose 20 g, lactose 15 g, tryptone 10 g, yeast extract 5 g, sodium chloride 2.5 g, magnesium sulfate 2 g, L-cysteine hydrochloride 0.5 g, tomato powder 0.5 g, anhydrous calcium chloride 0.15 g, add water to 1 L), 26℃ fermentation culture 46h, get phosphorus solubilizing bacteria fermentation broth.The activated potassium-lysing bacteria were inoculated into liquid medium (maltose 20 g, lactose 15 g, tryptone 10 g, yeast extract 5 g, sodium chloride 2.5 g, magnesium sulfate 2 g, L-cysteine hydrochloride 0.5 g, tomato powder 0.5 g, anhydrous calcium chloride 0.15 g, and water to 1 L) at 5% v / v, and then fermented at 26°C for 46 h to obtain the potassium-lysing bacteria fermentation liquor.

[0052] 3) 3 parts of the Bacillus subtilis fermentation liquor, 3 parts of the Bacillus amyloliquefaciens fermentation liquor, 10 parts of the Bacillus megaterium fermentation liquor, 10 parts of the Bacillus licheniformis fermentation liquor, 10 parts of the photosynthetic bacteria fermentation liquor, 10 parts of the nitrogen-fixing bacteria fermentation liquor, 10 parts of the phosphorus-lysing bacteria fermentation liquor, and 10 parts of the potassium-lysing bacteria fermentation liquor were mixed with 40 parts of the decomposed organic fertilizer, 15 parts of the humic acid, 7.5 parts of sodium alginate, 7.5 parts of the compound amino acid, and 3 parts of the compound trace element, granulated to a particle size of 3 mm, and dried to obtain the microbial fertilizer.

[0053] Experimental Example 1

[0054] The plants infected with citrus Huanglongbing disease were confirmed by PCR technology, and the microbial fertilizer was applied as a top dressing, which was applied around the roots of the citrus trees, and a total of 3 times of top dressing was performed, with an interval of 45 days each time, and the amount of the microbial fertilizer was 12.5 kg per citrus tree each time. Water was poured after each application, and the disease severity of Huanglongbing was determined 7 days after each top dressing, and the yield was determined after the fruits were matured.

[0055] The plants infected with citrus Huanglongbing disease were divided into the Example 1 group, the Comparative Example 1 group, and the Comparative Example 2 group, and 10 plants were randomly allocated to each group.

[0056] The treatment effect of Huanglongbing disease was calculated by the disease course index. The disease severity of citrus Huanglongbing disease was classified, and the disease severity was classified into 0 to 4 levels. Level 0: Ct value > 36.0; Level 1: 32.0 < Ct value < 36.0; Level 2: 28.0 < Ct value < 32.0; Level 3: 24.0 < Ct value < 28.0; Level 4: Ct value < 24.0. Each plant was classified by the Ct value and the content of the pathogenic bacteria.

[0057] Table 1 Statistics of the amount of CLas bacteria (Ct value) of plants in different groups

[0058]

[0059]

[0060] As shown in Table 1 and Figure 1As shown in Table 2, the Ct value of the diseased plants was increased to a certain extent after applying the microbial fertilizer prepared in Example 1 and Comparative Examples 1-2, indicating that the amount of CLas in the plants was decreased. In particular, the application of the microbial fertilizer prepared in Example 1 to the plants in Example 1 had obvious effect on the regulation of the amount of CLas, the pathogenic bacteria of HLB.

[0061] The fruits of the same plant were placed in the same container for weighing and recording at harvest. The weight of a single fruit was determined by randomly selecting fruits from different parts of the plant, and the mass of 3 randomly selected fruits was weighed and averaged.

[0062] Table 2: Yield per plant and weight of a single fruit in different groups

[0063]

[0064] As shown in Table 2, Figure 2 and Figure 3 Compared with the plants to which the microbial fertilizers of Comparative Examples 1-2 were applied, the yield per plant and the weight of a single fruit of the plants to which the microbial fertilizer prepared in Example 1 was applied were significantly increased.

[0065] The above-described examples are only used to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A microbial fertilizer for preventing and treating citrus Huanglongbing disease, characterized in that: It comprises the following components in parts by weight: 5-10 parts of Bacillus subtilis, 5-10 parts of Bacillus amyloliquefaciens, 3-8 parts of Bacillus megaterium, 3-8 parts of Bacillus licheniformis, 3-8 parts of photosynthetic bacteria, 3-8 parts of nitrogen-fixing bacteria, 3-8 parts of phosphate-solubilizing bacteria, 3-8 parts of potassium-solubilizing bacteria, 30-50 parts of decomposed organic fertilizer, 10-20 parts of humic acid, 5-10 parts of sodium alginate, 5-10 parts of complex amino acids, and 1-5 parts of complex trace elements.

2. The method for preparing the microbial fertilizer according to claim 1, wherein The following steps are involved: 1) Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus megaterium, Bacillus licheniformis, photosynthetic bacteria, nitrogen-fixing bacteria, phosphate-solubilizing bacteria, and potassium-solubilizing bacteria are activated and cultured respectively to obtain activated Bacillus subtilis, activated Bacillus amyloliquefaciens, activated Bacillus megaterium, activated Bacillus licheniformis, activated photosynthetic bacteria, activated nitrogen-fixing bacteria, activated phosphate-solubilizing bacteria, and activated potassium-solubilizing bacteria; 2) inoculating the activated Bacillus subtilis, activated Bacillus amyloliquefaciens, activated Bacillus megaterium, activated Bacillus licheniformis, activated photosynthetic bacteria, activated nitrogen-fixing bacteria, activated phosphate-solubilizing bacteria, and activated potassium-solubilizing bacteria into a liquid culture medium at an inoculum rate of 4-6% v / v, and fermenting and culturing them respectively to obtain Bacillus subtilis fermentation broth, Bacillus amyloliquefaciens fermentation broth, Bacillus megaterium fermentation broth, Bacillus licheniformis fermentation broth, photosynthetic bacteria fermentation broth, nitrogen-fixing bacteria fermentation broth, phosphate-solubilizing bacteria fermentation broth, and potassium-solubilizing bacteria fermentation broth; 3) mixing the Bacillus subtilis fermentation liquid, Bacillus amyloliquefaciens fermentation liquid, Bacillus megaterium fermentation liquid, Bacillus licheniformis fermentation liquid, photosynthetic bacteria fermentation liquid, nitrogen-fixing bacteria fermentation liquid, phosphate-solubilizing bacteria fermentation liquid and potassium-solubilizing bacteria fermentation liquid with decomposed organic fertilizer, humic acid, sodium alginate, compound amino acids and compound trace elements, granulating and drying to obtain microbial fertilizer.

3. The preparation method according to claim 2, characterized in that: The liquid culture medium in step 2) is formulated as follows: 15-25 g maltose, 10-20 g lactose, 5-15 g tryptone, 4-6 g yeast extract, 1.5-3.5 g sodium chloride, 1-3 g magnesium sulfate, 0.4-0.6 g L-cysteine ​​hydrochloride, 0.4-0.6 g tomato powder, and 0.1-0.2 g anhydrous calcium chloride. Add water to 1 L.

4. The preparation method according to claim 2, characterized in that In step 2), the temperature of the activated Bacillus subtilis fermentation culture is 30-37° C. and the time is 12-20 hours; the temperature of the activated Bacillus amyloliquefaciens fermentation culture is 28-32° C. and the time is 12-20 hours; the temperature of the activated Bacillus megaterium fermentation culture is 25-30° C. and the time is 12-20 hours; the temperature of the activated Bacillus licheniformis fermentation culture is 28-30° C. and the time is 12-20 hours.

5. The preparation method according to claim 2, characterized in that: In step 2), the temperature for activating photosynthetic bacteria fermentation culture is 35-37° C. for 24-48 hours; the temperature for activating nitrogen-fixing bacteria fermentation culture is 28-30° C. for 24-48 hours.

6. The preparation method according to claim 2, characterized in that: In step 2), the temperature for activating the phosphate-solubilizing bacteria fermentation culture is 25-28° C., and the time is 44-48 hours; the temperature for activating the potassium-solubilizing bacteria fermentation culture is 25-28° C., and the time is 44-48 hours.

7. Use of the microbial fertilizer as claimed in claim 1 in preventing and treating citrus Huanglongbing disease.

8. The application according to claim 7, characterized in that: The microbial fertilizer inhibits the growth and reproduction of the citrus Huanglongbing pathogen.

9. The application according to claim 7, characterized in that: The application is specifically as follows: using the microbial fertilizer as base fertilizer and / or topdressing, applying it around the roots of the citrus trees by furrow application, hole application or broadcast application, and watering after application.

10. The use according to claim 9, characterized in that: The amount of the microbial fertilizer is calculated based on 10 to 15 kg per citrus tree.

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