CN112159770A - Method for preparing biocontrol bacterial fertilizer by fermenting decayable waste through Bacillus belgii - Google Patents
Method for preparing biocontrol bacterial fertilizer by fermenting decayable waste through Bacillus belgii Download PDFInfo
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Abstract
The invention discloses an application of Bacillus belgii in preparing a biocontrol bacterial fertilizer by fermenting decayable waste and a preparation method thereof, belonging to the technical field of biocontrol bacterial fertilizer preparation. The Bacillus belgii is classified and named as Bacillus belgii LT-2(Bacillus velezensis LT-2), is preserved in China center for type culture collection, and has a registration number of CCTCC No: M2019904 and a preservation date of 2019, 11 months and 07 days. The invention mainly comprises the following technical processes: preparing solid yeast, preparing compost fermentation seed liquid and preparing biocontrol bacterial manure by solid fermentation. The invention takes the organic corrodible waste as the raw material to prepare the high-efficiency agricultural biocontrol bacterial fertilizer, accelerates the process of biological products for replacing chemical pesticides, solves the problem of crop food safety caused by long-term dependence on the chemical pesticides, and has important significance for promoting the sustainable development of modern agriculture in China.
Description
Technical Field
The invention particularly relates to an application of Bacillus beleisi in preparation of a biocontrol bacterial fertilizer by fermenting decayable waste, belonging to the technical field of biocontrol bacterial fertilizers.
Background
Due to the fact that chemical fertilizers and pesticides are abused for a long time to seriously pollute cultivated lands, such as thinned plough layers, hardened and acidified soil, insufficient organic matter content, unbalanced nutrients, pesticide pollution, poor soil ecological function and the like, soil ecological systems are seriously damaged, and human health is directly threatened. The application of the organic fertilizer is reported to provide stable, sufficient and continuous nutrient supply for crops, so that the growth of the crops is promoted, the yield is increased, and the market prospect is good. Therefore, the natural and harmless and environment-friendly microbial organic biocontrol bacterial fertilizer is selected to replace pesticides and chemical fertilizers to prevent and treat insect pests and diseases in agriculture and forestry, promote healthy growth of crops and improve the yield of the crops, and becomes a current research hotspot.
The total amount of the compostable wastes in China is sufficient (mainly comprising residues after processing crop straws, vegetables, melons and fruits, animal skins, viscera, excrement and the like), organic matters in the compostable wastes are rich in cellulose, hemicellulose, sugars, protein, grease, nitrogen, phosphorus, potassium and other elements, and the compostable wastes are the first-selected cheap, high-quality, environment-friendly, green and healthy raw materials for preparing the organic bacterial manure.
Bacillus belgii Bacillus valescens is a microorganism which is commonly used in soil, and recently, the Bacillus belgii is reported to play an important role in biological control (plant growth, disease and insect resistance and induction system disease resistance) and crop yield increase as a novel biocontrol bacterium at home and abroad. Thus, B.velezensis has a great research prospect as a novel microbial resource in the biological control of plant diseases. Bacillus belgii as a biocontrol bacterium is widely applied to agricultural production, can promote plant growth, and simultaneously inhibits the infection of animal pathogenic bacteria and plant pathogenic bacteria by secreting secondary metabolites, so that the Bacillus belgii is considered to be a safe and environment-friendly strategy for replacing chemical fertilizers and pesticides.
Through retrieval, no report that the biocontrol bacterial fertilizer is prepared by fermenting organic waste serving as a raw material by using B.velezensis is found at present.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to solve the problems that: provides a Bacillus belgii B.velezensis LT-2 and a method for preparing biocontrol bacterial fertilizer by fermenting organic waste serving as a raw material.
The technical scheme is as follows: in order to solve the problems, the invention adopts the following technical scheme:
the invention obtains a strain of Bacillus velezensis B.velezensis through screening from distiller yeast, which is classified and named as Bacillus velezensis LT-2 and is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2019904, deposit address: wuhan city, Hubei province, eight flood mountain areas, Wuhan university China center for type culture Collection, zip code: 430072, preservation number is CCTCC No: m2019904, and the preservation date is 11/7/2019. The following all refer to this strain as a production strain.
Bacillus velezensis LT-2(Bacillus velezensis LT-2) has the following properties:
1. the morphological characteristics and physiological and biochemical characteristics of the colonies are shown in Table 1.
TABLE 1 morphological characteristics and physiological and biochemical characteristics of colonies
2. 16S rDNA sequence analysis:
the length of the nucleotide sequence of the 16S rDNA gene of the strain is 1396bp, and the gene sequence is shown as SEQID number 1: as shown. The sequences tested were compared for homology from the Gene Bank database using the BLAST program to construct a phylogenetic tree based on the 16S rDNA full sequence. The results show that: the strain achieves 100% homology with B.velezensis zjt 9. According to the results of morphological observation and analysis of physiological and biochemical experiments of strains, the bacillus subtilis B.velezensis LT-2 is determined to be used in the invention.
The method for preparing the biocontrol bacterial fertilizer by fermenting the Bacillus belief B.velezensis LT-2 by taking organic waste as a raw material also belongs to the protection scope of the invention.
The method for preparing the biocontrol bacterial fertilizer by fermenting the Bacillus belgii B.velezensis LT-2 by taking organic waste as a raw material sequentially comprises the following steps of:
(1) preparing solid yeast seeds:
(1a) selecting strains:
the deposited strain B.velezensis LT-2 is selected.
(1b) Activating strains:
inoculating a B.velezensis LT-2 strain to a slant culture medium, performing static culture at 24-36 ℃ for 16-20 h, picking a single colony again, streaking the single colony on the slant culture medium, and performing culture at 24-36 ℃ for 16-20 h to obtain an activated strain for later use;
wherein the bevel container is an eggplant bottle; the slant culture medium is LB culture medium: 5g/L of yeast powder, 10g/L of peptone and 10g/L of sodium chloride;
(1c) preparing a solid yeast seed solution:
inoculating the activated strain in the step (1b) into a fermentation tank filled with the solid yeast seed liquid under an aseptic condition, and culturing at the rpm of 150-220 and the temperature of 24-36 ℃ for 18-36 h to obtain the solid yeast seed liquid;
wherein the solid koji seed liquid culture medium is as follows: 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride and 1g/L of magnesium sulfate; the fermentation tank is a mechanical stirring type 1000L online sterilization fermentation system, and the liquid loading amount is 60-75%.
(1d) Preparing solid yeast seeds by fermentation:
inoculating the solid koji seed liquid in the step (1c) into a rotary drum type solid bioreactor filled with a koji culture medium, culturing at 24-36 ℃ for 24-48 h, pressing the koji culture medium containing Bacillus belgii into koji blanks by using an extruder, and naturally drying to obtain solid koji;
wherein the yeast culture medium is as follows: 10-20 parts of molasses, 35-65 parts of soybean meal, 5-10 parts of rice husk, 45-55 parts of water, 0.5-1.2 g/kg of manganese sulfate and 7.0-8.0 of pH value; wherein the drum type solid bioreactor 1 batch can ferment 3000kg of organic waste.
The most preferred koji culture medium comprises the following components: 10 parts of molasses, 40 parts of soybean meal, 5 parts of rice husk and 45 parts of water, wherein the pH value is 7.0-8.0. In addition, 0.5-1.2 g of manganese sulfate is added into each kilogram of yeast culture medium.
(2) Preparing a seed solution:
(2a) crushing solid yeast seeds:
crushing the solid yeast seeds in the step (1d) by using a crusher, and controlling the particle size of solid yeast seed particles to be below 0.3 cm;
(2b) preparing a fermented seed solution:
inoculating the solid yeast particles (2-5%, w/w) obtained in the step (2a) into a seed culture medium, controlling the temperature to be 70-80 ℃ for one hour, cooling to 24-36 ℃, and carrying out open mechanical stirring culture for 24-48 hours to obtain a fermented seed liquid;
wherein the temperature is controlled at 70-80 ℃ for one hour to ensure that the mixed bacteria die at high temperature, which is called as the 'impurity-removing bacteria' for short.
Wherein the seed culture medium is: 50-70 parts of kitchen waste, 20-40 parts of soybean meal, 2-10 parts of sodium cellulose, 0.5-1.2 g/kg of manganese sulfate, 15-30 parts of water and 7.0-8.0 of pH value. The optimal formula is as follows: 50 parts of kitchen waste, 20 parts of soybean meal powder, 5 parts of sodium cellulose, 25 parts of water, 0.5-1.2 g/kg of manganese sulfate and 7.0-8.0 of pH value.
(3) Preparing a biocontrol bacterial fertilizer by solid state fermentation:
(3a) drying/drying the organic corrodible waste until the moisture content is 15-25%, crushing the organic corrodible waste by using a crusher, and controlling the particle size of the organic corrodible waste to be below 0.3 cm;
wherein the optimum moisture is 17%.
(3b) Mixing the organic compostable waste particles in the step (3a) and the fermented seed liquid in the step (2b) and other auxiliary materials according to a certain proportion, controlling the water content to be 55-65%, and performing 'strip pile type' compost fermentation to prepare the biocontrol bacterial manure. And when the fermentation temperature is higher than 65 ℃, turning and cooling by using a turning machine.
Wherein the optimal proportion is 7: 2.4: 0.6;
wherein the optimal turning conditions are as follows: and starting turning after the internal temperature of the stack is stable, wherein turning is performed by using a walking stack turning machine, and the completion of one-time stack turning operation is based on that the measured internal temperature after turning is reduced by more than 20 ℃ than before turning.
(3c) Preparation of biocontrol bacterial fertilizer blank
And (4) pressing the fermentation product obtained in the step (3b) into a blank by using an extruder, and naturally drying in the air to obtain the biocontrol bacterial fertilizer blank.
The best time for making the blank is that the fermentation temperature is lower than 45 ℃, namely the compost fermentation is carried out after the post-fermentation period.
Has the advantages that: the invention has the following advantages:
(1) the invention obtains a strain B.velezensis LT-2 with biocontrol effect by screening for the first time.
(2) The invention replaces the commercial microbial inoculum by a mode of self-preparing solid Bacillus belgii B.velezensis LT-2 yeast, applies the biocontrol bacterial manure to crop cultivation and planting, greatly reduces the production cost of agricultural crops, realizes 'changing waste into valuable', and has very important social significance.
(3) The invention takes the kitchen waste as the main component of the seed culture medium and the decayable waste as the biological control bacterial fertilizer fermentation substrate, can promote the recycling of the decayable waste and has obvious economic and social benefits.
(4) The invention replaces the mode of 'fermentation after composting and mechanical drying' with the mode of 'bacterial manure blank + natural air drying', shortens the production period and reduces the production cost.
(5) The strain can prepare the biocontrol bacterial fertilizer by fermenting organic wastes as raw materials, has high fertility and good biological control effect, and has important significance for making up the production defects and the defects of the existing organic fertilizer, establishing an agricultural crop disease green control technology and promoting the sustainable development of modern agriculture in China.
(6) During the preparation process of the biocontrol bacterial fertilizer, the Bacillus bleekensis LT-2 synthesizes microbial polysaccharide which can be used as a novel biological stimulator to promote plant growth.
The novel method for preparing the biocontrol bacterial fertilizer by fermenting the organic waste serving as the raw material by using the Bacillus bleekensis LT-2 disclosed by the invention not only solves the problems of harmless treatment and resource utilization of the compostable waste, but also successfully prepares the organic biocontrol bacterial fertilizer antagonistic to pathogenic bacteria, and has important significance for making up the production defects and the defects of the existing organic fertilizer, establishing an agricultural crop disease green prevention and control technology, repairing the soil ecosystem in China, realizing self-protection of cultivated land in China and promoting sustainable development of modern agriculture in China.
Drawings
FIG. 1 shows a 16S rDNA PCR purification agarose gel electrophoresis of B.beijerinckii LT-2 (A) and a phylogenetic tree of B.beijerinckii LT-2 (B).
FIG. 2 Effect of manganese ions on spore production by Bacillus belgii LT-2
FIG. 3 shows the influence of rice husk addition on the preparation of biocontrol bacterial manure
FIG. 4 influence of substrate humidity on preparation of biocontrol bacterial manure
The specific implementation mode is as follows:
the present invention can be better understood from the following examples, however, those skilled in the art will readily appreciate that the descriptions of the examples are only for the purpose of illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.
Example 1: isolation and characterization of B.velezensis LT-2.
A strain which grows fast and has high cellulase activity is found in the early stage, in order to identify the strain, a bacterial genome DNA extraction kit is used for extracting the genome DNA of B.velezensis LT-2, the 16S rDNA sequence is amplified by PCR with an upstream primer 27F and a downstream primer 1492R as shown in figure 1A, the product after PCR amplification is subjected to gel recovery and purification, and the gel recovery and purification product is sent to Soujin Weizhi biotechnology limited to carry out sequencing. The nucleotide sequence length of the 16S rDNA gene of the strain obtained by sequencing is 1396bp, and the gene sequence is shown as SEQID No. 1. BLAST comparison of the sequencing results with known 16S rDNA sequences in GeneBank databases and homology comparison using the BLAST program resulted in the construction of phylogenetic trees based on the 16S rDNA full sequences. The results show that: this strain reached 100% homology with bacillus belgii B. velezensis zjt9 (fig. 1B). According to the results of strain morphology observation and physiological and biochemical experiment analysis, the Bacillus belgii is identified to be used in the invention, and is specifically named as Bacillus belgii B.
Example 2: optimization of manganese ion concentration in solid koji culture medium and seed liquid culture medium
Mn is reported2+The method is beneficial to the generation of the spore of the Bacillus belgii, so that the concentration of manganese ions in the solid koji culture medium and the seed liquid culture medium is optimized. B. velezensis LT-2 was inoculated to Mn in different amounts2+(0.25,0.50,0.75,1.00,1.25,1.50g/kg) in the solid koji culture medium and the seed liquid culture medium, the initial pH was 7.5, the culture temperature was 32 ℃ and the culture time was 48 hours, and then the number of spores was measured. It was found that 1.00g/kg of Mn was added to the solid koji culture medium2+The number of spores reaches the maximum, 8.2 multiplied by 109Per mL; 0.75g/kg Mn is added into a seed liquid culture medium2+The number of spores reaches the maximum, 8.5 multiplied by 109one/mL. Thus, optimal Mn in solid koji and seed liquid media2+The amounts of addition were 1.00g/kg and 0.75g/kg, respectively.
Example 3: optimization of rice hull content of bio-control bacterial fertilizer auxiliary material prepared by fermentation of Bacillus beleisi LT-2
The bacillus fermentation belongs to aerobic fermentation, a large amount of oxygen is needed in the fermentation process, and the addition of rice hulls can be increased in literature reportThe gap between the solid substrates ensures the fermentation by allowing air to enter. In this example, which illustrates the influence of different rice husk addition amounts on the preparation of biocontrol bacterial manure by bacterial strain fermentation, a seed culture solution is inoculated into a biocontrol bacterial manure preparation fermentation system with rice husk addition amounts of 2%, 4%, 6%, 8%, 10% and 12% (w/w) respectively in an inoculation amount of 25% (v/w) for solid state fermentation, and when the rice husk addition amount is 6%, the solid state fermentation temperature rise speed is fastest, and the required fermentation time is shortened. Therefore, 6% of rice hull is selected as the optimum amount. (method for measuring mechanism weight loss ratio comprises leaching fermented matrix with distilled water, filtering, repeating for 3 times, oven drying water insoluble matrix, and weighing the matrix before fermentation1When the weight loss ratio of the substrate is (1-m)2/m1)×100％)。
Example 3: optimization of substrate humidity of biocontrol bacterial fertilizer prepared by fermentation of Bacillus belgii LT-2
The literature reports that substrate humidity is one of the key factors for the success or failure of solid state fermentation. In this example, which illustrates the effect of different substrate humidity on the biocontrol bacterial manure prepared by bacterial strain fermentation, the seed culture solution is inoculated into substrates with humidity of 50%, 55%, 60%, 65%, 70% and 75% (w/w) respectively at an inoculation amount of 25% (v/w) for solid state fermentation, and when the substrate humidity is 60%, the solid state fermentation temperature rise speed is fastest, and the required fermentation time is shortened. Therefore, 60% is selected as the optimum solid state fermentation substrate humidity. (method for measuring mechanism weight loss ratio comprises leaching fermented matrix with distilled water, filtering, repeating for 3 times, oven drying water insoluble matrix, and weighing the matrix before fermentation1When the weight loss ratio of the substrate is (1-m)2/m1) ×100％)。
Example 4: biocontrol bacterial fertilizer prepared by fermenting Bacillus belgii LT-2
Inoculating Bacillus belgii B.velezensis LT-2 strain on LB slant culture medium, standing at 32 deg.C for 14 hr, selecting single colony again, streaking onto LB slant culture medium, and standing at 32 deg.C for 14 hr to obtain activated strain. Then inoculating the activated strain into a fermentation tank (1000L) filled with solid koji seed liquid under aseptic condition, and culturing at 200rpm and 36 deg.C for 24h to obtain solid koji seed liquid. Inoculating the solid yeast seed liquid into a rotary drum type solid bioreactor filled with a yeast culture medium, culturing at 32 ℃ for 48h, and pressing into a yeast blank by using an extruder. And then crushing the yeast blank by using a crusher until the particle size of the particles is below 0.3cm, inoculating the yeast blank into an open mechanical stirring fermentation tank containing a seed culture medium (50 parts of kitchen waste, 20 parts of soybean meal, 5 parts of sodium cellulose, 25 parts of water, 1.0g/kg of manganese sulfate and 7.0-8.0 of pH value), heating to 70-80 ℃ to kill mixed bacteria, cooling to 32-36 ℃ after 1 hour, and performing 24 hours to obtain a fermented seed solution. Fermenting the seed solution (1250 kg) again, the crushed organic rottable waste (3500kg) and the rice hulls (250kg) according to the following ratio of 2.5: 7: 0.5, controlling the moisture content of the fermentation substrate to be 55 percent by water, carrying out 'stack type' compost fermentation to prepare the biocontrol bacterial fertilizer, and turning and cooling by a turning machine when the fermentation temperature is higher than 65 ℃ to reduce the temperature by 25 ℃. And (3) when the natural fermentation temperature is lower than 45 ℃, pressing the solid fermentation substrate into a blank by using an extruder, and naturally drying to obtain the biocontrol bacterial fertilizer blank (3.6 tons). In addition, the content of the extracellular polysaccharide of the Bacillus beiLeisi LT-2 in the biocontrol bacterial fertilizer blank is measured to be 3% (dry basis) by an ethanol precipitation method (the content of the extracellular polysaccharide is measured by the method that the biocontrol bacterial fertilizer blank is leached by distilled water and then filtered, the filtrate is precipitated by ethanol after being concentrated, and the precipitate is dried to obtain the extracellular polysaccharide).
Example 5: growth promoting (fertility) effect of biocontrol bacterial manure on corn
This example illustrates the effect of biocontrol bacterial manure in promoting the growth of corn seedlings.
The corn culture experiments were performed in a plant tissue culture room. The planting medium used in the control group was: a mixture of 90% soil and 10% non-composted fermentation substrate. The planting matrix used for T1 is: a mixture of soil and a Bacillus belgii LT-2 cell dissolved out from a biocontrol bacterial fertilizer; the planting matrix used for T2 is: 99.5% soil and 0.5% extracellular polysaccharide of Bacillus belgii LT-2. The planting matrix used for T3 is: a mixture of 90% soil and 10% compost fermented substrate (i.e. biocontrol bacterial manure). The above groups were divided into 4 groups, and germination rate (96h), stem height (2 weeks), leaf length, leaf width (3 weeks), fresh weight of stem and leaf (2 weeks) were tested, and 3 replicates per group were performed. The results are shown in Table 2. Compared with a control group, the Bacillus belgii LT-2 can respectively improve the germination rate, stem height, leaf length, leaf width and fresh weight of corn by 9.61%, 6.38%, 12.45%, 27.00% and 36.84%, and shows that the Bacillus belgii LT-2 has the capacity of promoting the growth of corn. In addition, Bacillus belgii LT-2 exopolysaccharides were found to increase germination rate, stem height, leaf length, leaf width and fresh weight of corn by 7.68%, 12.07%, 19.90%, 13.00% and 29.82%, respectively, but their corn growth-promoting effects were inferior to those of Bacillus belgii LT-2. In addition, the synergistic effect of the bacteria and the exopolysaccharide ensures that the biocontrol bacterial manure (containing Bacillus belgii LT-2 and exopolysaccharide thereof) has the best effect of promoting the growth of corn (the germination percentage is improved by 11.57 percent, the stem height is improved by 33.97 percent, the leaf length is improved by 22.25 percent, the leaf width is improved by 37.00 percent, and the fresh weight is improved by 33.33 percent). Therefore, the biocontrol bacterial manure (containing Bacillus belgii LT-2 and exopolysaccharide thereof) is an ideal biological fertilizer or plant growth promoter.
TABLE 2 growth promoting effect of biocontrol bacterial manure on corn
Example 6: growth promoting (fertility) effect of biocontrol bacterial manure on wheat
This example illustrates the effect of biocontrol bacterial manure in promoting the growth of corn seedlings.
Wheat culture experiments were performed in a plant tissue culture room. The planting medium used in the control group was: a mixture of 90% soil and 10% non-composted fermentation substrate. The planting matrix used for T1 is: a mixture of soil and a Bacillus belgii LT-2 cell dissolved out from a biocontrol bacterial fertilizer; the planting matrix used for T2 is: 99.5% soil and 0.5% extracellular polysaccharide of Bacillus belgii LT-2. The planting matrix used for T3 is: a mixture of 90% soil and 10% compost fermented substrate (i.e. biocontrol bacterial manure). Each group was subdivided into 4 groups, and germination percentage (48h), number of grains and thousand grain weight were tested, 3 replicates per group. The results are shown in Table 2. Compared with the control group, the Bacillus belgii LT-2 can respectively improve the germination rate, the wheat grain number and the thousand grain weight of wheat by 14.54 percent, 24.10 percent and 19.11 percent, and shows that the Bacillus belgii LT-2 has the capability of promoting the growth of wheat. In addition, Bacillus belgii LT-2 exopolysaccharide was found to increase germination rate, number of grains and thousand-grain weight of wheat by 8.54%, 21.66% and 4.74%, respectively, but its effect of promoting wheat growth was inferior to that of Bacillus belgii LT-2. In addition, the synergistic effect of the bacteria and the exopolysaccharide ensures that the biocontrol bacterial manure (containing Bacillus belgii LT-2 and exopolysaccharide thereof) has relatively best promotion effect on the growth of wheat (the germination rate is improved by 16.37 percent, the number of wheat grains is improved by 44.54 percent, and the thousand grain weight is improved by 25.25 percent). Therefore, the biocontrol bacterial manure (containing the Bacillus belgii LT-2 and the exopolysaccharide thereof) is an ideal biological fertilizer, plant growth promoter or plant yield increasing agent.
TABLE 2 growth promoting effect of biocontrol bacterial manure on wheat
Example 7: growth promoting (fertility) effect of biocontrol bacterial manure on rice
This example illustrates the effect of biocontrol bacterial manure in promoting the growth of rice seedlings.
The rice culture test was performed in a plant tissue culture room. The planting medium used in the control group was: a mixture of 90% soil and 10% non-composted fermentation substrate. The planting matrix used for T1 is: a mixture of soil and a Bacillus belgii LT-2 cell dissolved out from a biocontrol bacterial fertilizer; the planting matrix used for T2 is: 99.5% soil and 0.5% extracellular polysaccharide of Bacillus belgii LT-2. The planting matrix used for T3 is: a mixture of 90% soil and 10% compost fermented substrate (i.e. biocontrol bacterial manure). Each group was subdivided into 4 groups, and germination percentage (10 days), stem height (35 days), leaf length, leaf width (35 days), fresh weight of stem and leaf (35 days) experiments were performed, 3 in each group. The results are shown in Table 2. Compared with a control group, the Bacillus belgii LT-2 can respectively improve the germination rate, stem height, leaf length, leaf width and fresh weight of rice by 6.12%, 5.67%, 3.19%, 4.88% and 6.56%, and shows that the Bacillus belgii LT-2 has the capability of promoting the growth of rice. In addition, it was found that Bacillus belgii LT-2 exopolysaccharides could increase the germination rate, stem height, leaf length, leaf width and fresh weight of rice by 12.24%, 5.35%, 3.93%, 9.76% and 4.92%, respectively, but their effect of promoting rice growth was inferior to that of Bacillus belgii LT-2. In addition, the bacteria and the exopolysaccharide act synergistically, so that the biocontrol bacterial manure (containing the Bacillus belgii LT-2 and the exopolysaccharide) has the best promotion effect on the growth of rice (the germination rate is improved by 16.32%, the stem height is improved by 9.72%, the leaf length is improved by 9.83%, the leaf width is improved by 12.20%, and the fresh weight is improved by 8.20%).
TABLE 2 growth promoting effect of biocontrol bacterial manure on rice
Example 8: biocontrol effect of biocontrol bacterial fertilizer on fusarium graminearum
This example illustrates the biocontrol effect of biocontrol bacterial manure on fusarium graminearum.
All the planting substrates for the experiments are soil, the experiments are totally divided into 6 groups, and 15 groups of the experiments are parallel. After all plants grow to the height of more than 25cm, inoculating liquid Fusarium graminearum (Fusarium graminearum) into the matrix of all experimental groups, and respectively adding biocontrol bacterial fertilizers with the total mass of 3%, 6% and 9% of the matrix into T1, T2 and T3 groups; and (3) adding sterilized biocontrol bacterial manure with the mass of 3%, 6% and 9% of the total mass of the matrix into the control group 1, the control group 2 and the control group 3 respectively, and continuously culturing for 3 weeks to count the disease rate of the rice. The results are shown in Table 3. The control effect of all the control groups is low and is maintained below 13.33 percent; compared with a control group, the test group shows good control effect on rice head blight after the biocontrol bacterial manure is used, and the biocontrol effect is more obvious along with the increase of the inoculation amount. Therefore, the biocontrol bacterial fertilizer prepared by the invention has good market application prospect.
TABLE 3 biocontrol effect of biocontrol bacterial manure
Example 9: biocontrol bacterial manure biocontrol effect on Alternaria alternata pathogenic bacteria
This example illustrates the biocontrol effect of biocontrol bacterial manure on fusarium graminearum.
All the planting substrates for the experiments are soil, the experiments are totally divided into 6 groups, and 15 groups of the experiments are parallel. After all strawberry plants grow to the height of more than 25cm, liquid Alternaria alternata (3%, 6% and 9% of the total mass of the matrix) is inoculated into the matrix of all experimental groups, respectively, biocontrol bacterial manure is added into the groups of T1, T2 and T3, 3%, 6% and 9% of the total mass of the matrix is added into the groups of a control group 1, a control group 2 and a control group 3, respectively, sterilized biocontrol bacterial manure is added into the groups, and the disease rate of the strawberries is counted after the strawberries are continuously cultured for 3 weeks.
TABLE 4 biocontrol effect of biocontrol bacterial manure
Finally, it should also be noted that the above-mentioned list is only a specific embodiment of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the teachings of the present invention are to be considered within the scope of the present invention.
Sequence listing
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Claims (10)
1. A method for preparing biocontrol bacterial fertilizer by fermenting decayable waste by using Bacillus belgii is characterized by comprising the following steps: the Bacillus belgii is classified and named as Bacillus velezensis LT-2, is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2019904 and the preservation date of 2019, 11 months and 7 days.
2. The method for preparing a bio-control bacterial fertilizer by fermenting a compostable waste with Bacillus belgii according to claim 1, wherein: the method comprises the following steps:
(1) drying the organic corrodible waste until the moisture content is 15-25% by mass, and crushing the organic corrodible waste to obtain organic corrodible waste particles;
(2) inoculating Bacillus belgii LT-2 fermentation seed liquid into organic compostable waste particles, adding auxiliary materials, uniformly stirring, controlling the water content to be 55-65%, performing 'strip-pile' composting fermentation to prepare a biocontrol bacterial fertilizer, and turning and cooling when the fermentation temperature is higher than 65 ℃;
(3) and (3) when the compost fermentation temperature in the step (2) is lower than 45 ℃, allowing the compost fermentation to enter a post-fermentation stage, pressing the fermentation product into a blank, and naturally drying to obtain the biocontrol bacteria fertilizer blank.
3. The method for preparing a bio-control bacterial fertilizer by fermenting a compostable waste with bacillus belief in claim 2, wherein the method comprises the steps of: in the step (2), the preparation method of the Bacillus belgii LT-2 fermentation seed liquid comprises the following steps:
(1a) activating strains:
inoculating a B.velezensis LT-2 strain to a slant culture medium, performing static culture at 24-36 ℃ for 16-20 h, picking a single colony again, streaking the single colony on the slant culture medium, and performing culture at 24-36 ℃ for 16-20 h to obtain an activated strain for later use;
(1b) preparing a solid yeast seed solution:
inoculating the activated strain obtained in the step (1a) into a fermentation tank filled with the solid yeast seed liquid under an aseptic condition, and culturing at the rpm of 150-220 and the temperature of 24-36 ℃ for 18-36 h to obtain the solid yeast seed liquid;
(1c) preparing solid yeast seeds by fermentation:
inoculating the solid koji seed liquid obtained in the step (1b) into a rotary drum type solid bioreactor filled with a koji culture medium according to the inoculation amount with the volume fraction of 2-5%, culturing at 24-36 ℃ for 24-48 h, pressing the koji culture medium containing Bacillus belgii into a koji blank by using an extruder, and air-drying to obtain a solid koji;
(1d) crushing solid yeast seeds:
crushing the solid yeast seeds in the step (1c) by using a crusher, and controlling the particle size of solid yeast seed particles to be below 0.3 cm;
(1e) preparing a fermented seed solution:
and (3) inoculating the solid yeast particles obtained in the step (1d) into a seed culture medium, controlling the temperature to be 70-80 ℃ for one hour, cooling to be 24-36 ℃, and carrying out open mechanical stirring culture for 24-48 hours to obtain a fermented seed liquid.
4. The method for preparing a bio-control bacterial fertilizer by fermenting a compostable waste with Bacillus belgii according to claim 3, wherein: in the step (1a), the slant culture medium is LB culture medium: 5g/L of yeast powder, 10g/L of peptone and 10g/L of sodium chloride;
in the step (1b), the solid koji seed liquid culture medium is: 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride and 1g/L of magnesium sulfate;
in the step (1c), the yeast culture medium is: 10-20 parts of molasses, 35-65 parts of soybean meal, 5-10 parts of rice hulls, 45-55 parts of water, 0.5-1.2 g/kg of manganese sulfate and 7.0-8.0 of pH value.
5. The method for preparing a bio-control bacterial fertilizer by fermenting a compostable waste with Bacillus belgii according to claim 3, wherein: in the step (1e), the formula of the seed culture medium comprises the following components in parts by mass: 50-70 parts of kitchen waste, 20-40 parts of soybean meal, 2-10 parts of sodium cellulose, 0.5-1.2 g/kg of manganese sulfate, 15-30 parts of water and 7.0-8.0 of pH value.
6. The method for preparing a bio-control bacterial fertilizer by fermenting a compostable waste with bacillus belief in claim 2, wherein the method comprises the steps of: in the step (1), the organic compostable waste comprises: straw, rice straw, weed, aquatic weed, tree leaf, vegetable leaf, branch, fruit peel, fruit shell, animal feces and one or more of animal skin, hair, scale and internal organs.
7. The method for preparing a bio-control bacterial fertilizer by fermenting a compostable waste with bacillus belief in claim 2, wherein the method comprises the steps of: in the step (2), the mass ratio of the organic corrodible waste particles to the fermentation seed liquid to the auxiliary materials is 5-8: 2-4: 0.2 to 0.8.
8. The method for preparing a bio-control bacterial fertilizer by fermenting a compostable waste with bacillus belief in claim 2, wherein the method comprises the steps of: in the step (2), the auxiliary material is one or a combination of more of soil, peanut shells and rice husks.
9. The biocontrol bacterial fertilizer prepared by the method for preparing the biocontrol bacterial fertilizer by fermenting the compostable waste through Bacillus belief in any one of claims 1 to 8.
10. Use of the biocontrol bacterial fertilizer of claim 9 for promoting plant growth, plant seed germination, or biocontrol.
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| CN115074295A (en) * | 2022-07-21 | 2022-09-20 | 华中农业大学 | Bacillus belgii growing by taking camphor as carbon source, high-temperature compost decomposing inoculant and application |
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