AU2020101394A4

AU2020101394A4 - Microbial fertilizer with double effects of fertilization and disease-resistance, and preparation method and use thereof

Info

Publication number: AU2020101394A4
Application number: AU2020101394A
Authority: AU
Inventors: Lihua Jiang; Hongmei Li; Ni LI; Jing Shi; Mei Wang; Yu Xu; Yan Yang
Original assignee: Institute of Agricultural Resources and Environment of Shandong Academy of Agricultural Sciences
Current assignee: Institute of Agricultural Resources and Environment of Shandong Academy of Agricultural Sciences
Priority date: 2020-07-17
Filing date: 2020-07-17
Publication date: 2020-08-20
Anticipated expiration: 2028-07-17

Abstract

MICROBIAL FERTILIZER WITH DOUBLE EFFECTS OF FERTILIZATION AND DISEASE-RESISTANCE, AND PREPARATION METHOD AND USE THEREOF ABSTRACT The present invention provides a microbial fertilizer, and a preparation method and use thereof, and belongs to the technical field of fertilizer production. The microbial fertilizer is prepared from the following raw materials: crop stalks, livestock and poultry manure, a first bacterial agent and a second bacterial agent, where the first bacterial agent includes Bacillus megaterium and Bacillus subtilis, and the second bacterial agent includes Pseudomonas fluorescens and Streptomyces jingyangensis. The microbial fertilizer of the present invention includes Bacillus megaterium, Bacillus subtilis, Pseudomonas fluorescens and Streptomyces jingyangensis, and this strain combination can achieve the nutrient, disease-resistance and stress-resistance efficacies. In addition, the present invention adopts an absorbent prepared from raw materials of crop stalks and livestock and poultry manure as the adsorption matrix for bacterial agents, making the product have stable performance, extended shelf life and better effect.

Description

MICROBIAL FERTILIZER WITH DOUBLE EFFECTS OF FERTILIZATION AND DISEASE-RESISTANCE, AND PREPARATION METHOD AND USE THEREOF

TECHNICAL FIELD The present invention relates to the technical field of fertilizer production, and in particular to a microbial fertilizer, and a preparation method and use thereof.

BACKGROUND Microbial fertilizers, also known as biological fertilizers, inoculants, bacterial manures or the like, refer to a class of fertilizer products that focus on the life activities of microorganisms and enable crops to have a specific fertilizer response. Microbial fertilizer has a gradually-expanded market in China, and becomes an important member of the fertilizer family. There are three main development stages for the development of microbial fertilizers in China. During the first development stage, rhizobium inoculants and azotobacter agents are developed, including rhizobium preparations. Subsequently, significant progress is made in researches on non-legume free-living nitrogen fixation, associative symbiotic nitrogen fixation, and potassium bacteria and phosphobacteria, and then a variety of microbial fertilizer products with single-trophic bacteria (such as rhizobia, azotobacter, phosphate-solubilizing bacteria, potassium-solubilizing bacteria, and actinomyces) as a principal part emerge successively, and exhibit huge market application value. The research on microbial fertilizers at this stage focuses on increasing the content of major nutrients in soil, especially the nitrogen content, using the characteristics of microbes. The typical feature is that the product has a single strain as an effective strain, which is called the first generation microbial fertilizer (single-nutrient type). The emphasis of the second development stage is to improve the utilization efficiency of nutrients and fertilizers in soil using characteristics of microbes of multiple trophic types. According to the principle of a chemical compound fertilizer, single-trophic bacteria, such as azotobacter, phosphate-solubilizing bacteria and potassium-solubilizing bacteria, are compounded into microbial fertilizers, and a series of "nutrient-complementary" microbial fertilizers (the second generation microbial fertilizer) are developed. Such fertilizers provide a variety of nutrients for crops by decomposing insoluble phosphorus or potassium in soil and fixing nitrogen in the air through the life activities of microorganisms. The focus of the third development stage is to impart microbial fertilizers new functions, such as overcoming the stress of chemical pollution on the agricultural environment and the agricultural product quality, when the soil quality of farmland is decreasing and chemical fertilizers, organic fertilizers and pesticides cause increasingly-heavy pollution. The research at this stage mainly focuses on biocontrol and growth-promoting bacteria and microbial fertilizer strains that have a remediation effect on the soil. At present, according to functions, microbial fertilizers can be divided into two types. One type can increase the nutrient supply for plants through the life activities of microorganisms, including increasing the total nutrient supply for plants in the soil and the production environment, and thus improve the nutrient status of plants, thereby increasing the output. The other type, such as 5406 bacterial agent, Paecilomyces lilacinus and other products that inhibit nematodes, can antagonize the pathogenic effects of some pathogenic microorganisms and reduce pests and diseases for crops. However, the existing microbial fertilizers have disadvantages, such as a single strain, incomplete functions and unstable use effects. There is a lack of a microbial fertilizer with double effects of fertilization and disease-resistance that can promote the nutrient absorption, increase the yield, and effectively reduce the occurrence of diseases for crops. Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

OBJECT OF THE INVENTION It is the object of the present invention to substantially overcome or at least ameliorate one or more of the disadvantages of the prior art or to provide a useful alternative.

SUMMARY In one embodiment, the present invention relates to a microbial fertilizer, and a preparation method and used thereof. The microbial fertilizer can promote the nutrient uptake of a crop, increase the yield, and effectively reduce the occurrence of diseases. In one aspect, the present invention provides a microbial fertilizer prepared from the following raw materials: crop stalks, livestock and poultry manure, a first bacterial agent and a second bacterial agent, wherein the first bacterial agent includes Bacillus megaterium and Bacillus subtilis; the second bacterial agent includes Pseudomonas fluorescens and Streptomyces jingyangensis; the total mass of the crop stalks and the livestock and poultry manure, the volume of the first bacterial agent and the volume of the second bacterial agent have a ratio of (200-350) kg (6-10) L: (12-20) L; and the first bacterial agent and the second bacterial agent have an effective total viable count of 1 x 108cfu/ml to 120 x 108cfu/ml. Preferably, in the first bacterial agent, the Bacillus megaterium has an effective viable count of 60 x 108 cfu/ml to 100 x 108 cfu/ml, and the Bacillus subtilis has an effective viable count of 80 x 108 cfu/ml to 120 x 108 cfu/ml; and in the second bacterial agent, the Pseudomonasfluorescenshas an effective viable count of 40 x 108 cfu/ml to 60 x 108 cfu/ml, and the Streptomycesjingyangensis has an effective viable count of 1 x 108 cfu/ml to 5 x 108 cfu/ml. In one embodiment, the microbial fertilizer has an effective viable count of 1x 108 cfu/g to x 108 cfu/g. In one embodiment, the microbial fertilizer has a pH value of 5.5 to 7.5. In one embodiment, the microbial fertilizer has a moisture content of 20% to 30%. In one embodiment, the microbial fertilizer has an organic matter content > 40% In one embodiment, the microbial fertilizer has a dosage form of powder or granule, and the granular microbial fertilizer has a fineness < 5.6 mm. In one embodiment, the microbial fertilizer has a mold (microbial contaminant) count < 3.0 x 106 cfu/g. In a further aspect, the present invention provides a method for preparing the above microbial fertilizer, including the following steps: 1) inoculating Bacillus megaterium and Bacillus subtilis in a first fermentation medium, and conducting a first fermentation at 28°C to 32°C and 180 rpm to 200 rpm for 30 h to 40 h to obtain a first bacterial agent, wherein the first fermentation medium includes the following components, in parts by mass: 1,000 to 1,100 parts of starch, 3,200 to 3,800 parts of soybean cake powder, 120 to 180 parts of dipotassium phosphate, 320 to 380 parts of ammonium sulfate, 150 to 200 parts of magnesium sulfate, 3 to 4 parts of ferric chloride, 8 to 12 parts of manganese sulfate, 30 to 40 parts of calcium carbonate, and 110 to 130 parts of yeast powder; 2) inoculating Pseudomonas fluorescens and Streptomyces jingyangensis in a second fermentation medium, and conducting a second fermentation at 28°C to 32°C and 180 rpm to 200 rpm for 30 h to 40 h to obtain a second bacterial agent, wherein the second fermentation medium includes the following components, in parts by mass: 20,500 to 21,500 parts of starch, ,500 to 6,500 parts of soybean cake powder, 200 to 250 parts of dipotassium phosphate, 120 to 150 parts of yeast powder, 120 to 180 parts of peptone, 5 to 10 parts of ferric chloride, 55 to 65 parts of calcium sulfate, and 250 to 300 parts of magnesium sulfate; 3) mixing the crop stalks with livestock and poultry manure, and conducting aerobic composting fermentation to obtain an adsorbent; and 4) mixing the first bacterial agent, the second bacterial agent and the adsorbent to obtain a microbial fertilizer; where steps 1), 2) and 3) can be performed in any order. In a further aspect, the present invention provides use of the above microbial fertilizer in increasing crop yield and reducing crop diseases. The present invention has the following beneficial effects: The present invention provides a microbial fertilizer prepared from the following raw materials: crop stalks, livestock and poultry manure, a first bacterial agent and a second bacterial agent. The first bacterial agent includes Bacillus megaterium and Bacillus subtilis, and the second bacterial agent includes Pseudomonasfluorescensand Streptomycesjingyangensis. The total mass of the crop stalks and the livestock and poultry manure, the volume of the first bacterial agent and the volume of the second bacterial agent have a ratio of (200-350) kg : (6-10) L : (12-20) L. The first bacterial agent and the second bacterial agent have an effective total viable count of 1 x 108 cfu/ml to 120 x 108 cfu/ml. The microbial fertilizer of the present invention includes Bacillus megaterium, Bacillus subtilis, Pseudomonas fluorescens and Streptomyces jingyangensis, and this strain combination can achieve the nutrient, disease-resistance and stress-resistance efficacies. In addition, the present invention adopts an absorbent prepared from raw materials of crop stalks and livestock and poultry manure as the adsorption matrix for bacterial agents, making the product have stable performance, extended shelf life and better effect. Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

DETAILED DESCRIPTION A preferred embodiment of the present invention will now be described by way of specific embodiments with reference to the examples. The following examples are only illustrative and not restrictive, and cannot be used to limit the protection scope of the present invention. The present disclosure provides a microbial fertilizer prepared from the following raw materials: crop stalks, livestock and poultry manure, a first bacterial agent and a second bacterial agent. The first bacterial agent includes Bacillus megaterium and Bacillus subtilis. The second bacterial agent includes Pseudomonas fluorescens and Streptomyces jingyangensis. The total mass of the crop stalks and the livestock and poultry manure, the volume of the first bacterial agent, and the volume of the second bacterial agent have a ratio of (200-350) kg :

(6-10) L: (12-20) L, and preferably of (250-300) kg: 10 L: 15 L. The first bacterial agent and the second bacterial agent have an effective total viable count of 1 x 108 cfu/g to 120 x 108cfu/ml, and preferably of 30 x 108 cfu/g to 70 x 108cfu/ml. In the present disclosure, the Bacillus megaterium in the first bacterial agent has an effective viable count preferably of 60 x 108 cfu/ml to 100 x 108 cfu/ml, and more preferably of x 108 cfu/ml to 70 x 108cfu/ml, and is a conventional product commercially available. In a specific implementation of the present disclosure, the Bacillus megaterium is purchased from Agricultural Culture Collection of China (ACCC), with a deposit number of ACCC10011. The Bacillus megaterium plays a role in biocontrol and improving fertilizer utilization, which antagonizes pathogenic bacteria by secreting antibacterial substances and competing for nutrients, and improves the phosphorus effect by decomposing organic phosphorus in soil that is difficult to be utilized. In the present disclosure, the Bacillus subtilis in the first bacterial agent has an effective viable count preferably of 80 x 108 cfu/ml to 120 x 108 cfu/ml, and more preferably of 40 x108 cfu/ml to 80 x 108 cfu/ml, and is a conventional product commercially available. In a specific implementation of the present disclosure, the Bacillus subtilis is purchased from Agricultural Culture Collection of China (ACCC), with a deposit number of Ku-3. The Bacillus subtilis plays a role in nutrient supply and disease resistance. The active substances produced during the growth of Bacillus subtilis can exhibit a specific fertilizer effect and have a significant inhibitory effect on pathogenic bacteria. In the present disclosure, the Pseudomonasfluorescensin the second bacterial agent has an effective viable count preferably of 40 x 108 cfu/ml to 60 x 108 cfu/ml, and is a conventional product commercially available. In a specific implementation of the present disclosure, the Pseudomonasfluorescenscomes from deposited strains screened by the present research group, with a deposit number of NKYWM0003. The Pseudomonas fluorescens can produce auxin substances to promote the growth of crop roots and facilitate the disease-resistance and growth of a plant. In the present disclosure, the Streptomycesjingyangensis in the second bacterial agent has an effective viable count preferably of 1 x 108 cfu/ml to 5 x 108 cfu/ml, and is a conventional product commercially available. In a specific implementation of the present disclosure, the Streptomyces jingyangensis is purchased from Agricultural Culture Collection of China (ACCC), with a deposit number of ACCC40021. The Streptomycesjingyangensisplays a role in biocontrol by producing antimicrobial and insecticidal substances during the metabolic process. In the present disclosure, the first bacterial agent and the second bacterial agent include trophic, disease-resistant and stress-resistant strains, thereby exerting multiple functions of nutrient, disease-resistance and stress-resistance. In the present disclosure, the microbial fertilizer has a pH value preferably of 5.5 to 7.5, and more preferably of 6 to 6.5; a moisture content preferably of 20% to 30%, and more preferably of 25%; an organic matter content preferably > 40%; a dosage form preferably of powder or granule; a fineness < 5.6 mm; a mold (microbial contaminant) count preferably < 3.0 x 106 cfu/g; and a microbial contamination rate preferably of 20% to 30%. The present disclosure also provides a method for preparing the microbial fertilizer according to the above solution, including the following steps: 1) inoculating Bacillus megaterium and Bacillus subtilis in a first fermentation medium, and conducting a first fermentation at 28°C to 32°C and 180 rpm to 200 rpm for 30 h to 40 h to obtain a first bacterial agent, where the first fermentation medium includes the following components, in parts by mass: 1,000 to 1,100 parts of starch, 3,200 to 3,800 parts of soybean cake powder, 120 to 180 parts of dipotassium phosphate, 320 to 380 parts of ammonium sulfate, 150 to 200 parts of magnesium sulfate, 3 to 4 parts of ferric chloride, 8 to 12 parts of manganese sulfate, 30 to 40 parts of calcium carbonate, and 110 to 130 parts of yeast powder; 2) inoculating Pseudomonas fluorescens and Streptomyces jingyangensis in a second fermentation medium, and conducting a second fermentation at 28°C to 32°C and 180 rpm to 200 rpm for 30 h to 40 h to obtain a second bacterial agent, where the second fermentation medium includes the following components, in parts by mass: 20,500 to 21,500 parts of starch, ,500 to 6,500 parts of soybean cake powder, 200 to 250 parts of dipotassium phosphate, 120 to 150 parts of yeast powder, 120 to 180 parts of peptone, 5 to 10 parts of ferric chloride, 55 to 65 parts of calcium sulfate, and 250 to 300 parts of magnesium sulfate; 3) mixing the crop stalks with livestock and poultry manure, and conducting aerobic composting fermentation to obtain an adsorbent; and 4) mixing the first bacterial agent, the second bacterial agent and the adsorbent to obtain a microbial fertilizer; where steps 1), 2) and 3) can be performed in any order. Before Bacillus megaterium and Bacillus subtilis are inoculated in a first fermentation medium, the present disclosure preferably further includes subjecting Bacillus megaterium and Bacillus subtilis to activated cultivation and expanded cultivation in sequence. The present disclosure has no special limitation on methods for activated cultivation and expanded cultivation, and conventional methods in the art may be used. In the present disclosure, after expanded cultivation, Bacillus megaterium and Bacillus subtilis are co-inoculated in a first fermentation medium, and a first fermentation is conducted at 28°C to 32°C and 180 rpm to 200 rpm for 30 h to 40 h to obtain a first bacterial agent. The first fermentation is conducted preferably at 30°C and preferably for 36 h. The first fermentation medium includes the following components, in parts by mass: 1,000 to 1,100 parts of starch, 3,200 to 3,800 parts of soybean cake powder, 120 to 180 parts of dipotassium phosphate, 320 to 380 parts of ammonium sulfate, 150 to 200 parts of magnesium sulfate, 3 to 4 parts of ferric chloride, 8 to 12 parts of manganese sulfate, 30 to 40 parts of calcium carbonate, and 110 to 130 parts of yeast powder; and preferably includes the following components, in parts by mass: 1,050 parts of starch, 3,500 parts of soybean cake powder, 150 parts of dipotassium phosphate, 350 parts of ammonium sulfate, 180 parts of magnesium sulfate, 3.5 parts of ferric chloride, 10 parts of manganese sulfate, 35 parts of calcium carbonate, and 120 parts of yeast powder. In the present disclosure, synergistic fermentation of Bacillus megaterium and Bacillus subtilis is adopted, which can increase the spore production rate of the two functional strains, reduce fermentation time, improve production efficiency, and decrease production cost. Before Pseudomonas fluorescens and Streptomyces jingyangensis are inoculated in a second fermentation medium, the present disclosure preferably further includes subjecting Pseudomonasfluorescensand Streptomycesjingyangensis to activated cultivation and expanded cultivation in sequence. The present disclosure has no special limitation on the method for activated cultivation, and a conventional method in the art may be used. In the present disclosure, after expanded cultivation, Pseudomonas fluorescens and Streptomyces jingyangensis are inoculated in a second fermentation medium, and a second fermentation is conducted at 28°C to 32°C and 180 rpm to 200 rpm for 30 h to 40 h to obtain a second bacterial agent. The second fermentation is conducted preferably at 30°C and preferably for 36 h. The second fermentation medium includes the following components, in parts by mass: ,500 to 21,500 parts of starch, 5,500 to 6,500 parts of soybean cake powder, 200 to 250 parts of dipotassium phosphate, 120 to 150 parts of yeast powder, 120 to 180 parts of peptone, 5 to parts of ferric chloride, 55 to 65 parts of calcium sulfate, and 250 to 300 parts of magnesium sulfate; and preferably includes the following components, in parts by mass: 20,100 parts of starch, 6,000 parts of soybean cake powder, 220 parts of dipotassium phosphate, 140 parts of yeast powder, 150 parts of peptone, 8 parts of ferric chloride, 60 parts of calcium sulfate, and 280 parts of magnesium sulfate. In the present disclosure, synergistic fermentation of Pseudomonas fluorescens and Streptomyces jingyangensis is adopted, which can increase the hypha content of Streptomyces jingyangensis. In the present disclosure, the crop stalks are mixed with livestock and poultry manure, and aerobic composting fermentation is conducted to obtain an adsorbent. The present disclosure has no special limitation on sources of the crop stalks and livestock and poultry manure, and conventional sources in the art may be adopted. In a specific implementation of the present disclosure, after the crop stalks are mixed with livestock and poultry manure, the method further includes adding a starter culture. The crop stalks, livestock and poultry manure and starter culture are used at a mass ratio preferably of (30-50):(50-70):(0.2-0.5). The present disclosure has no special limitation on the starter culture, and conventional organic matter-decomposing inoculant commercially available may be used (refer to GB20287-2006 for technical indicators). The present disclosure has no special limitation on the temperature and time for the aerobic composting fermentation, and conventional temperature and time in the art may be adopted. In the present disclosure, after a first bacterial agent, a second bacterial agent and an adsorbent are obtained, the first bacterial agent, the second bacterial agent and the adsorbent are mixed to obtain a microbial fertilizer. In a specific implementation of the present disclosure, the first bacterial agent and the second bacterial agent are mixed with the adsorbent separately to obtain a first material and a second material, and the first material and the second material are dried and mixed to obtain a microbial fertilizer. The volume of the first bacterial agent and the mass of the adsorbent have a ratio preferably of (6-10) L : 100 kg, and more preferably of 8 L: 100 kg. The volume of the second bacterial agent and the mass of the adsorbent have a ratio preferably of (12-20) L : 100 kg, and more preferably of 15 L : 100 kg. The first material and the second material are used at a ratio preferably of (40-120):(60-160), and more preferably of :100. The drying is conducted preferably by air drying. The present disclosure has no special limitation on the time and temperature of the drying, provided that the moisture content in the dried microbial fertilizer reaches 20% to 30%. After a microbial fertilizer is obtained, the present disclosure preferably includes crushing and sieving the microbial fertilizer, and the sieving is conducted at a sieve mesh number preferably of 18. The present disclosure also provides use of the microbial fertilizer according to the above solution in improving crop yield. The present disclosure also provides use of the microbial fertilizer according to the above solution in reducing crop diseases. The technical solutions provided by the present disclosure will be described in detail below with reference to examples, but the examples should not be construed as limiting the claimed scope of the present disclosure.

Example 1 A method for preparing a microbial fertilizer

1. Strains Bacillus megaterium, with a deposit number of ACCC10011; Bacillus subtilis, with a deposit number of Ku-3 in Agricultural Culture Collection of China (ACCC); Pseudomonas fluorescens, with a deposit number of NKYWM0003 from the present research group; Streptomyces jingyangensis, with a deposit number of ACCC40021 in Agricultural Culture Collection of China (ACCC). 2. Fermentation process Bacillus subtilis (one strain)--agar slants (4)-- 500 ml shake flasks (4)--class A seed tank (70 L)--fermentation tank (700 L) Line 1# Bacillus megaterium (one strain)--agar slants (4)-- 500 ml shake flasks (4)--class A seed tank (70 L)--fermentation tank (700 L)

Pseudomonasfluorescens (one strain)--agar slants (4)-- 500 ml shake flasks (4)- class A seed tank (70 L)--fermentation tank (700 L) Line 2# Streptomycesjingyangensis (one strain)--agar slants (4)-- 500 ml shake flasks (4)- class A seed tank (70 L)--fermentation tank (700 L)

3. Fermentation method 1) Shake-flask fermentation Bacillus megaterium, 0.2% of glucose, 0.5% of beef extract, 0.8% of peptone, 0.03% of MgSO4-7H20, 0.3% of NaCl, and 0.15% of K 2HPO4; pH 7.0-7.5, 33.5°C, 220 rpm, 26 h. Bacillus subtilis, 5 g of beef extract, 5 g of peptone, 5 g of sodium chloride, 1 g of yeast powder, 0.2 g of calcium carbonate, 0.5 g of MgSO4, and 1,000 mL of tap water; PH 7.0-7.5, °C, 220 rpm, 24 h. Pseudomonasfluorescens, 1.5 g/L monopotassium phosphate, 20 g/L peptone, and 1.5 g/L magnesium sulfate; pH 7.2, 30°C, 200 rpm, 30 h. Streptomyces jingyangensis, 15 g/L soluble starch, 1.2 g/L potassium nitrate, 0.5 g/L magnesium sulfate, 0.5 g/L sodium chloride, 0.02 g/L ferrous sulfate, 0.5 g/L dipotassium phosphate, and 0.3 g/L yeast powder; pH 7.0, 28°C, 200 rpm, 36 h. 2) Liquid fermentation medium and fermentation process parameters Si: Production process line 1# (synergistic fermentation of Bacillus megaterium and Bacillus subtilis) Seed tank: On the basis of the medium shown in Table 1, 0.05% to 0.1% (in percentage by volume) of defoamer was additionally added for the cultivation of the two strains. The resulting media were sterilized, cooled to 30°C, and then inoculated with the shake-flask cultures. Cultivation conditions: cultivation (fermentation) temperature: 30°C; cultivation (fermentation) time: 24 h; rotational speed: 180 rpm; and ventilation: 1:1 (v : v/min). Fermentation tank: low cost of culture medium, and high spore production rate during fermentation. On the basis of the medium shown in Table 1, 0.05% to 0.1% (in percentage by volume) of defoamer was additionally added. The resulting media were sterilized, cooled to °C, and then added with the fermentation broth that had been cultivated in seed tank for 24 h. Cultivation conditions: 30°C, 36 h, 180 rpm, and ventilation of 1:1 (v : v/min).

Table 1 Medium formula for the fermentation of Bacillus megaterium and Bacillus subtilis Reagent Bacillus subtilis Reagent Bacillus Reagent Fermentation

seed tank (70 L)/g megaterium tank

seed tank (70 L)/g (700 L)/g

Ammonium 140 Ammonium 120 Starch 1050

sulfate sulfate

Glucose 600 Peptone 250 Soybean cake 3500

powder

Dipotassium 20 Dipotassium 11 Dipotassium 150

phosphate phosphate phosphate

Sodium chloride 15 Sodium 70 Ammonium 350

chloride sulfate

Potassium 15 Beef extract 100 Magnesium 175

chloride sulfate

Magnesium 50 Magnesium 28 Ferric chloride 3.5

sulfate sulfate

Manganese 1.5 Manganese 1.5 Manganese 10

sulfate sulfate sulfate

Ferrous sulfate 2.0 Ferrous 1.5 Calcium 35

sulfate carbonate

Yeast powder 12 Yeast powder 15 Yeast powder 120

Notes: 1. Before added to the tank, yeast powder is dissolved in water to form a paste. 2. Starch should be added to the tank filled with some water under the slow stirring of a stirrer to avoid agglomeration.

S2: Production process line 2# (synergistic fermentation of Streptomycesjingyangensis and Pseudomonasfluorescens) Seed tank: On the basis of the medium shown in Table 2, 0.05% to 0.1% (in percentage by volume) of defoamer was additionally added for the cultivation of the two strains. The resulting media were sterilized, cooled to 30°C, and then inoculated with the shake-flask cultures.

Cultivation conditions: 30°C, 30 h, 180 rpm, and ventilation of 1:1 (v : v/min). Fermentation tank: On the basis of the medium shown in Table 2, 0.05% to 0.1% (in percentage by volume) of defoamer was additionally added. The resulting media were sterilized, cooled to 30°C, and then added with the fermentation broth that had been cultivated in seed tank for 30 h. Cultivation conditions: 30°C, 36 h, 180 rpm, and ventilation of 1:1 (v : v/min).

Table 2 Medium formula for the fermentation of Streptomyces jingyangensis and Pseudomonasfluorescens Reagent Pseudomonas Reagent Streptomyces Reagent Fermentation

fluorescens class A jingyangensis tank

tank (70 L)/g class A tank (700 L)/g

(70 L)/g

Monopotassium 14 Potato 700 Starch 21,000

phosphate starch

Dipotassium 56 Brown 1400 Soybean 6000

phosphate sugar cake powder

Soybean cake 350 Yeast 360 Dipotassium 220

powder powder phosphate

Yeast powder 46 Potassium 76 Yeast powder 140

nitrate

Sodium 0.4 Dipotassium 36 Peptone 150

molybdate phosphate

Ferricchloride 0.36 Magnesium 36 Ferric 7

Sulfate chloride

Calcium sulfate 7.2 Sodium 36 Calcium 60

chloride sulfate

Magnesium 14 Ferrous 0.75 Magnesium 280

sulfate sulfate sulfate

Peptone 25

Notes: 1. Before added to the tank, yeast powder is dissolved in water to form a paste. 2. Ferric chloride, bulk solids, should be prepared into a 1% solution, and then added to the tank in a proportion. 3. Starch should be added to the tank filled with some water under the slow stirring of a stirrer to avoid agglomeration.

3) Adsorption of fermentation broths Crop stalks and livestock and poultry manure: Crop stalks were derived from organic waste of agricultural production, and livestock and poultry manure was derived from faeces of poultry raising enterprises. According to the weight percentage, 30% of crop stalks and 69.5% of livestock and poultry manure were mixed, and then 0.5% of starter culture (organic matter-decomposing inoculant commercially available) was added. Aerobic composting fermentation was conducted at 40°C to 70°C under excellent ventilation, and a temperature of over 50°C was held for 5 to 10 days to obtain a material having a carbon-to-nitrogen ratio of (25-35):1 and a moisture content of 60% to 7 5 %, and the pH and moisture content of the material were adjusted to obtain an adsorbent. The organic material obtained by aerobic fermentation of crop stalks and livestock and poultry manure was adopted as an adsorbent instead of traditional peat. In line 1, the bacterial solution was absorbed onto the matrix at a ratio of 5 L/100 kg to obtain a first material. In line 2, the bacterial solution was absorbed on matrix at a ratio of 10 L/100 kg to obtain a second material. The first material and the second material were air dried to have a moisture content of % to 30% and then mixed at a mass ratio of 70: 100. The resulting mixture was crushed, sieved through an 18-mesh sieve, and packaged or granulated and packaged.

Example 2 Products 1, 2 and 3 obtained according to the method of Example 1 are as follows: Product 1: total viable count: 150 million cfu/g, with 50 million cfu/g of Bacillus megaterium, 100 million cfu/g of Bacillus subtilis, 8 million cfu/g of Pseudomonasfluorescens, and 1 million cfu/g of Streptomycesjingyangensis; mold (microbial contaminant) count < 3.0 x 106 cfu/g; microbial contamination rate < 20%; moisture content < 30%; fineness < 5.6 mm; pH 7.0; shelf-life: 12 months; product dosage form: powder; and organic matter content > 40%. Product 2: total viable count: 500 million cfu/g, with 100 million cfu/g of Bacillus megaterium, 400 million cfu/g of Bacillus subtilis, 20 million cfu/g of Pseudomonas fluorescens, and 20 million cfu/g of Streptomycesjingyangensis; mold (microbial contaminant) count < 3.0 x 106cfu/g; microbial contamination rate < 20%; moisture content < 30%; fineness

< 5.6 mm; pH 7.0; shelf-life: 12 months; product dosage form: powder; and organic matter content > 40%. Product 3: total viable count: 150 million cfu/g, with 50 million cfu/g of Bacillus megaterium, 100 million cfu/g of Bacillus subtilis, 8 million cfu/g of Pseudomonasfluorescens, and 1 million cfu/g of Streptomycesjingyangensis; mold (microbial contaminant) count < 3.0 x 106 cfu/g; microbial contamination rate < 30%; moisture content < 20%; fineness 5 5.6 mm; pH 7.0; shelf-life: 12 months; product dosage form: granule; and organic matter content > 20%.

Example 3 The use effect of the microbial fertilizer prepared in Example 1 In 2016 to 2019, a multi-site field test demonstration was set up in Jinxiang, Shandong, China, for the application of the microbial fertilizer prepared in Example 1 to garlic. 3 treatments were set as follows: optimized fertilization (garlic-specific (18-10-17) compound fertilizer was applied as a basic fertilizer at 80 kg/mu; and the special fertilizer was applied again before the Qingming Festival in the next year at 20 kg/mu, with 1 kg of copper calcium sulphate and 0.6 kg of thiophanate-methyl being well mixed with the chemical fertilizer); the microbial fertilizer prepared in Example 1; and a similar microbial agent commercially available (effective viable count (cfu) > 500 million/g, with microbial strains of Bacillus megaterium, Bacillus subtilis and Paenibacilluspolymyxa; and total nutrients (N + P2 05 + K20) > 5%). For treatments of the microbial fertilizer prepared in Example 1 and a similar microbial agent commercially available, on the basis of optimized fertilization, 5 kg of fertilizer was applied when garlic was sown, 5 kg of fertilizer was applied before winter, and 5 kg of fertilizer was applied at the regreening stage; and during the whole growth period, agricultural chemicals were applied at an amount halved compared to that of the optimized fertilization treatment. It can be seen from Table 4 that, compared with the optimized fertilization, treatments with the microbial fertilizer prepared in Example 1 and the commercially-available bacterial agent both lead to an increased garlic bulb yield, and the microbial fertilizer prepared in Example 1 exhibits a significant difference from the commercially-available product and the optimized fertilization treatment. Treatments with the microbial fertilizer prepared in Example 1 and the commercially-available bacterial agent lead to garlic sprout yield increases of 10.0% and 18.8% respectively compared with the optimized fertilization, and there are significant differences among the three treatments.

Table 4 Comparison of the use effect of the microbial fertilizer prepared in Example 1 with that of other treatments Treatment Garlic bulb yield Garlic bulb yield Garlic sprout Garlic sprout yield

(kg/mu) increase(%) yield (kg/mu) increase(%)

Optimized fertilization 1896.32b - 316.16c

Microbial fertilizer 1997.43a 5.33 347.73b 10.0

prepared in Example 1

Bacterial agent 1923.98b 1.46 375.74a 18.8

commercially available

It can be seen from Table 5 that the microbial fertilizer prepared in Example 1 and the commercially-available bacterial agent both can mitigate the root rot of garlic bulbs, and the microbial fertilizer prepared in Example 1 exhibits a more significant disease-resistance than the commercially-available bacterial agent.

Table 5 Survey of garlic root rot disease index Treatment The number of garlic The number of diseased garlic Disease index

bulbs bulbs

(garlic bulbs/2 m 2 ) (diseased garlic bulbs/2 m2 ) (%)

Optimized fertilization 82 7 8.53

Microbial fertilizer prepared 95 3 3.15

in Example 1

Bacterial agent 98 5 5.1

commercially available

The above descriptions are merely preferred implementations of the present disclosure. It should be noted that a person of ordinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the present disclosure and the appended claims.

Claims

What is claimed is:

1. A microbial fertilizer prepared from the following raw materials: crop stalks, livestock and poultry manure, a first bacterial agent and a second bacterial agent, wherein the first bacterial agent comprises Bacillus megaterium and Bacillus subtilis; the second bacterial agent comprises Pseudomonas fluorescens and Streptomyces jingyangensis; the total mass of the crop stalks and the livestock and poultry manure, the volume of the first bacterial agent and the volume of the second bacterial agent have a ratio of (200-350) kg (6-10) L: (12-20) L; and the first bacterial agent and the second bacterial agent have an effective total viable count of I x 108 cfu/g to 120 x 108 cfu/ ml.

2. The microbial fertilizer according to claim 1, wherein, in the first bacterial agent, the Bacillus megaterium has an effective viable count of 60 x 108 cfu/ ml to 100 x 108 cfu/ ml, and the Bacillus subtilis has an effective viable count of 80 x 108 cfu/ ml to 120 x 108 cfu/ ml; and in the second bacterial agent, the Pseudomonasfluorescenshas an effective viable count of 40 x 108 cfu/ ml to 60 x 108 cfu/ ml, and the Streptomyces jingyangensis has an effective viable count of I x 108 cfu/g to 5 x 108 cfu/ ml.

3. The microbial fertilizer according to claim 1 or 2, wherein the microbial fertilizer has a dosage form of powder or granule, and the granular microbial fertilizer has a fineness < 5.6 mm, a pH value of 5.5 to 7.5, a moisture content of 20% to 30%, an organic matter content > 40%, an effective viable count of 1x 108 cfu/g to 5 x 108cfu/g, and a mold (microbial contaminant) count < 3.0 x 106 cfu/g.

4. A method for preparing the microbial fertilizer according to any one of claims I to 3, comprising the following steps: 1) inoculating Bacillus megaterium and Bacillus subtilis in a first fermentation medium, and conducting a first fermentation at 28°C to 32°C and 180 rpm to 200 rpm for 30 h to 40 h to obtain a first bacterial agent, wherein the first fermentation medium comprises the following components, in parts by mass: 1,000 to 1,100 parts of starch, 3,200 to 3,800 parts of soybean cake powder, 120 to 180 parts of dipotassium phosphate, 320 to 380 parts of ammonium sulfate, 150 to 200 parts of magnesium sulfate, 3 to 4 parts of ferric chloride, 8 to 12 parts of manganese sulfate, 30 to 40 parts of calcium carbonate, and 110 to 130 parts of yeast powder; 2) inoculating Pseudomonasfluorescens and Streptomyces jingyangensis in a second fermentation medium, and conducting a second fermentation at 28°C to 32°C and 180 rpm to 200 rpm for 30 h to 40 h to obtain a second bacterial agent, wherein the second fermentation medium comprises the following components, in parts by mass: 20,500 to 21,500 parts of starch, 5,500 to 6,500 parts of soybean cake powder, 200 to 250 parts of dipotassium phosphate, 120 to 150 parts of yeast powder, 120 to 180 parts of peptone, 5 to parts of ferric chloride, 55 to 65 parts of calcium sulfate, and 250 to 300 parts of magnesium sulfate; 3) mixing the crop stalks with livestock and poultry manure, and conducting aerobic composting fermentation to obtain an adsorbent; and 4) mixing the first bacterial agent, the second bacterial agent and the adsorbent to obtain a microbial fertilizer; wherein steps 1), 2) and 3) can be performed in any order.