CN111197017B - Bacillus amyloliquefaciens CGMCC No.17844 and application thereof - Google Patents
Bacillus amyloliquefaciens CGMCC No.17844 and application thereof Download PDFInfo
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Abstract
The invention relates to the field of microorganisms, and relates to a bacillus amyloliquefaciens and application thereof. The Bacillus amyloliquefaciens is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 17844. The bacillus amyloliquefaciens can be prepared into a microbial agent, can also be prepared into a compound microbial agent together with other microbial agents, and can also be prepared into a fertilizer for preventing and treating root rot and improving crop yield.
Description
Technical Field
The invention relates to the field of microorganisms and fertilizers, in particular to bacillus amyloliquefaciens and application thereof, and specifically relates to bacillus amyloliquefaciens (preservation number CGMCC No.17844), a microbial agent, a compound microbial agent, a fertilizer and a method for preventing and treating root rot.
Background
The strawberries are easy to cultivate, bear fruits in the same year, have good taste, high yield and good economic benefit, and have important economic status in domestic and international markets. However, with the expansion of the planting area of strawberries, the number of cultivated varieties is large, the phenomena of continuous cropping and year-by-year aggravation of diseases occur for a long time, and the livestock and poultry manure which is not enough or completely decomposed for a long time is used as a base fertilizer, so that pathogenic bacteria are brought in, and the soil nutrient balance is influenced. Meanwhile, the strawberry seedlings of farmers are variable in source, only part of aseptic seedlings are purchased, most of the aseptic seedlings are subjected to detoxification treatment, the problem that the seedlings are infected with diseases cannot be scientifically prevented, continuous cropping diseases are serious, especially root rot is easy to break out and difficult to cure, leaves and root systems are damaged, and the yield and the quality are affected. Continuous cropping causes the change of the physical and chemical properties of soil, the content of soil salt is increased, the soil is hardened seriously, and the growth of root systems is influenced. The incidence of strawberry root rot is influenced by the continuous cropping age, planting region and strawberry seedling quality, and the strawberry root rot is controlled biologically mainly by physical means such as greenhouse closing, soil turning, temperature control and humidity control, chemical means such as benzimidazole protective bactericides and biological agents with functions of heavy parasitism resistance, disease resistance and induction generation of disease resistance. But the physical means has poor effect, the chemical means is easy to cause secondary pollution, and the biological means is an excellent means for preventing and treating the strawberry root rot disease due to environmental protection and strong disease resistance pertinence.
However, further improvements are needed for the biological treatment of root rot.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, the invention aims to provide the bacillus amyloliquefaciens, the microbial agent, the compound microbial agent, the fertilizer and the method for preventing and treating the root rot.
The bacillus amyloliquefaciens is a gram-positive bacterium widely distributed in nature and has strong application potential. The bacillus amyloliquefaciens is applied to soil or water bodies, can degrade toxic substances in the nature and purify polluted soil, rivers and lakes. The bacillus amyloliquefaciens is a typical plant growth promoting bacterium and can secrete active substances such as antibacterial protein, antibiotics, enzymes or polypeptides and the like, promote plant growth and prevent and treat plant fungal diseases. The bacillus amyloliquefaciens can secrete various antibacterial active substances, has wide resistance to germs on the surfaces of fruits and vegetables, and can solve the problems of fruit and vegetable fresh keeping and long-term storage. The excellent characteristics of the bacillus amyloliquefaciens have great application potential in the aspects of environmental protection and agricultural production. The bacillus is separated from strawberry root rot plants, the bacillus is identified as bacillus amyloliquefaciens (B.amyloliquefaciens) through morphological, physiological and biochemical characteristics and 16S rRNA genes, and the bacillus amyloliquefaciens shows good effects of promoting the growth of strawberries and preventing the root rot of the strawberries through laboratory plate antagonism experiments, strawberry potting experiments and field experiments and can be used for improving the yield and the quality of the strawberries.
Specifically, the invention provides the following technical scheme:
in a first aspect of the invention, the invention provides a bacillus amyloliquefaciens which is preserved in China general microbiological culture collection center with the preservation number of CGMCC No. 17844. The bacillus amyloliquefaciens provided by the invention is obtained by screening greenhouse soil for continuously planting strawberries for many years. Through the verification of a laboratory plate antagonism experiment, a strawberry potting experiment and a field experiment, the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is identified through morphological, physiological and biochemical characteristics and a 16S rRNA gene. The composition has good prevention and treatment effects on P.clavispora, and the inhibition rate on the pathogenic bacteria is over 80 percent. The microbial agent can inhibit the growth of pathogenic microorganism pestalotiopsis, reduce the generation of spores, can be used for preventing and treating root rot caused by pestalotiopsis, and meets the disease-resistant requirement in the strawberry planting process. The provided bacillus amyloliquefaciens can be used as a microbial agent and can also be compounded with other microbial agents to prepare a compound microbial agent, and the microbial agent or the compound microbial agent can be used as a fertilizer or added into the fertilizer to prevent and treat root rot, so that the yield of crops is remarkably increased, the quality is remarkably improved, and the taste is better. Taking strawberry as an example, the yield and quality of the crop can be greatly improved in the aspects of single fruit weight, acre yield, sugar degree and the like.
In a second aspect of the invention, the invention provides a microbial agent comprising a bacillus amyloliquefaciens according to the first aspect of the invention.
In some embodiments of the present invention, the microbial agent is in a dry powder form, and the effective viable count of the bacillus amyloliquefaciens in each gram of the microbial agent is at least 8 x 1010CFU, preferably 1X 1011And (4) CFU. The microbial inoculum is prepared into dry powder, and the effective viable count in each gram of microbial inoculum is 8 multiplied by 1010CFU or above, e.g. 1X 1011CFU or more, preferably 2X 1011More than CFU, so that the microbial agent can be applied to prevent and treat root rot of crops, and is rapid and efficientTreating root rot of crops and improving the quality of crops.
In a third aspect of the present invention, there is provided a method of preparing a microbial agent according to the second aspect of the present invention, the method comprising: fermenting the bacillus amyloliquefaciens; obtaining the microbial preparation based on the product of the fermentation process. The microbial agent is obtained by fermenting the bacillus amyloliquefaciens, can be used for preventing and treating root rot of crops, and simultaneously improves the quality and the yield of the crops.
According to an embodiment of the present invention, the method for preparing a microbial agent described above may further include the following technical features:
in some embodiments of the invention, the fermentation process comprises: carrying out liquid fermentation culture on the bacillus amyloliquefaciens so as to obtain a seed culture solution; and carrying out amplification fermentation culture on the seed culture solution so as to obtain a product of the fermentation treatment.
In some embodiments of the invention, the expanded fermentation culture is performed using a liquid fermentation medium or a solid fermentation medium; the liquid fermentation medium comprises: 8-15 parts of peptone, 1-5 parts of ammonium sulfate, 2-5 parts of beef extract powder, 3-8 parts of sodium chloride, 1-5 parts of dipotassium hydrogen phosphate, 3-8 parts of polyvinyl alcohol and 1000 parts of water, wherein the pH value of the liquid fermentation medium is 7-7.5; the solid fermentation medium comprises: 3-10 parts of wheat bran, 0.5-5 parts of soybean meal, 0.05-0.5 part of magnesium sulfate, 0.05-0.5 part of sodium chloride and water. The liquid fermentation culture medium or the solid fermentation culture medium can be used for rapid fermentation to obtain the microbial agent.
In some embodiments of the invention, the method further comprises: and drying and crushing the product of the fermentation treatment to obtain the powdery microbial agent. Through drying and crushing treatment, the powdery microbial agent can be obtained, and is convenient to store and use.
In a fourth aspect of the invention, the invention provides a complex microbial inoculant, which comprises a first microbial inoculant and a second microbial inoculant different from the first microbial inoculant, wherein the first microbial inoculant is the microbial inoculant in the second aspect of the invention. Different microbial agents are compounded, different microbial agents can have different or same or similar functions, and the compound microbial agent can play the aims of preventing and treating various crop diseases and improve the quality of crops.
In some embodiments of the invention, the second microbial agent comprises at least one selected from the group consisting of a bacillus mucilaginosus agent, a wood mold green agent, an aspergillus terreus agent, and a bacillus amyloliquefaciens agent. The microbial agents have great promotion effect on plant growth and better control effect on pathogenic bacteria, so the microbial agents are compounded with the first microbial agent to further improve the quality of crops.
In some embodiments of the present invention, the complex microbial inoculant comprises 1-3 parts by weight of the first microbial inoculant, and at least one selected from the following components: 1-3 parts by weight of a bacillus mucilaginosus microbial agent; 1-3 parts by weight of a trichoderma viride agent; 1-3 parts by weight of an aspergillus terreus microbial inoculum; 1-3 parts by weight of a bacillus amyloliquefaciens microbial agent.
In some embodiments of the present invention, the complex microbial inoculant comprises 1-1.5 parts by weight of the first microbial inoculant, and at least one selected from the following components: 1-1.5 parts by weight of a Bacillus mucilaginosus microbial agent; 1-1.5 parts by weight of a trichoderma viride agent; 1-1.5 parts by weight of an aspergillus terreus microbial inoculum; 1-1.5 parts by weight of a bacillus amyloliquefaciens microbial inoculum.
In a fifth aspect of the present invention, the present invention provides a fertilizer comprising the microbial agent of the second aspect of the present invention or the complex microbial agent of the fourth aspect of the present invention.
In some embodiments of the present invention, the fertilizer further comprises a base fertilizer, the base fertilizer comprises at least one selected from a biological organic fertilizer, an organic compound fertilizer, an inorganic compound fertilizer or an organic-inorganic compound fertilizer, and the content of the microbial agent or the compound microbial agent is 1 to 5 per mill of the content of the base fertilizer.
In some embodiments of the invention, the bio-organic fertilizer comprises 30-50 parts by weight of fermented cow dung, 20-40 parts by weight of a carbon-based fertilizer, 10-30 parts by weight of a small molecule organic material, and 5-15 parts by weight of calcium silicate.
In some embodiments of the present invention, the bio-organic fertilizer comprises 40 parts by weight of fermented cow dung, 30 parts by weight of a carbon-based fertilizer and 20 parts by weight of a small molecule organic material, and 10 parts by weight of calcium silicate.
In a sixth aspect of the invention, the invention provides an application of bacillus amyloliquefaciens in preparation of a microbial agent, a compound microbial agent or a fertilizer, wherein the bacillus amyloliquefaciens is the bacillus amyloliquefaciens in the first aspect of the invention.
In a seventh aspect of the present invention, the present invention provides a method for controlling root rot, comprising: applying an effective amount of bacillus amyloliquefaciens, a microbial agent, a compound microbial agent or a fertilizer to a plant, wherein the bacillus amyloliquefaciens is the bacillus amyloliquefaciens of the first aspect of the invention, the microbial agent is the microbial agent of the second aspect of the invention, the compound microbial agent is the compound microbial agent of the fourth aspect of the invention, and the fertilizer is the fertilizer of the fifth aspect of the invention.
In some embodiments of the invention the plant is a strawberry and the root rot is a root rot caused by pestalotiopsis.
Information on strain preservation
Bacillus amyloliquefaciens with the preservation number of CGMCC No.17844, the preservation unit is the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is the microorganism research institute of China academy of sciences No. 3 of Xilu No.1 of Beijing Korean, and the preservation date is 2019, 05 and 22 days.
Drawings
FIG. 1 is a photograph showing the morphology of a rod-shaped pestalotiopsis colony according to an embodiment of the present invention.
FIG. 2 is a phylogenetic tree of the sequence of the ITS of Pestalotiopsis clavuligerus (ZH-G01) according to an embodiment of the present invention.
FIG. 3 is a picture of the colony morphology of Bacillus amyloliquefaciens CGMCC No.17844 provided by the embodiment of the invention.
FIG. 4 is a picture of the effect of CGMCC No.17844 on antagonizing pathogenic bacteria according to the embodiment of the present invention.
FIG. 5 is a phylogenetic tree of the 16S rRNA sequence of Bacillus amyloliquefaciens (CGMCC No.17844) provided according to an embodiment of the present invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the accompanying drawings, it being noted that the described embodiments are exemplary and are intended to be illustrative of the present invention, and should not be construed as limiting the present invention.
Herein, when the content of a certain substance is expressed, the mass of the substance is referred to as a percentage of the total substance content, unless otherwise specified.
The bacillus amyloliquefaciens strain CGMCC NO.17844 capable of preventing and treating various plant diseases of strawberry root rot is obtained through a large amount of screening work. Experiments prove that the bacillus amyloliquefaciens shows a very high-efficient prevention and treatment effect in preventing and treating the strawberry root rot, and the bacillus amyloliquefaciens CGMCC NO.17844 strain also has a certain growth promoting effect, so that the yield of the strawberry is obviously increased. For example, the amylolytic strain of Bacillus amyloliquefaciens of the present invention can be used in the preparation of a microbial fertilizer for controlling multiple plant diseases. Therefore, the Bacillus amyloliquefaciens CGMCC NO.17844 has wide application prospect.
The provided bacillus amyloliquefaciens CGMCC No.17844 can be prepared into a microbial agent and used as a single microbial agent. To this end, in one aspect of the present invention, the present invention provides a microbial agent comprising Bacillus amyloliquefaciens CGMCC No. 17844. The provided microbial agent can be used as a fertilizer or a disease treatment drug to be applied to the prevention and treatment of root rot, can prevent and treat plant diseases and insect pests, and improves the crop yield.
The provided microbial agent can be obtained by fermentation. To this end, in another aspect of the present invention, the present invention provides a method for preparing the above microbial agent, comprising: fermenting bacillus amyloliquefaciens CGMCC No. 17844; obtaining the microbial agent based on a product of the fermentation process.
In at least some embodiments of the invention, the fermentation process comprises: carrying out liquid fermentation culture on the bacillus amyloliquefaciens so as to obtain a seed culture solution; inoculating the seed culture solution into a solid fermentation culture medium for carrying out amplification fermentation culture so as to obtain a product of the fermentation treatment. In at least some embodiments, the liquid fermentation culture can be performed by using an LB culture solution, and the time of the liquid fermentation culture is more than 10 hours, preferably 10 to 16 hours. In at least some embodiments of the present invention, the fermentation culture fluid is inoculated into the solid fermentation culture medium in an inoculation amount (the fermentation culture fluid accounts for 5-10% of the solid fermentation culture medium by mass) for fermentation culture. The solid fermentation medium comprises 3-10 parts by weight of wheat bran, 0.5-5 parts by weight of soybean meal, 0.05-0.5 part by weight of magnesium sulfate, 0.05-0.5 part by weight of sodium chloride and water, wherein the content of the water in the solid fermentation medium is 50-60%. In some preferred embodiments, the solid fermentation medium comprises 4 parts by weight of wheat bran, 1 part by weight of soybean meal, 0.1 part by weight of magnesium sulfate, 0.1 part by weight of sodium chloride and water, wherein the content of water in the solid fermentation medium is 55%.
In other embodiments of the present invention, the fermentation process comprises: carrying out liquid fermentation culture on the bacillus amyloliquefaciens so as to obtain a seed culture solution; inoculating the seed culture solution into a liquid fermentation culture medium for amplification fermentation culture so as to obtain a product of the fermentation treatment. In at least some embodiments, the liquid fermentation culture can be performed by using an LB culture solution, and the time of the liquid fermentation culture is more than 10 hours, preferably 10 to 16 hours. In at least some embodiments of the present invention, the seed culture fluid is inoculated in a liquid fermentation medium in an inoculum size of 5% to 10% for expanded fermentation culture. The liquid fermentation medium comprises 8-15 parts by weight of peptone, 1-5 parts by weight of ammonium sulfate, 2-5 parts by weight of beef extract powder, 3-8 parts by weight of sodium chloride, 1-5 parts by weight of dipotassium hydrogen phosphate, 3-8 parts by weight of polyvinyl alcohol and 1000 parts by weight of water, and the pH value of the liquid fermentation medium is 7-7.5. In some preferred embodiments, the liquid fermentation medium comprises 10 parts by weight of peptone, 2.5 parts by weight of ammonium sulfate, 3 parts by weight of beef extract powder, 5 parts by weight of sodium chloride, 2 parts by weight of dipotassium hydrogenphosphate, 5 parts by weight of polyvinyl alcohol and 1000 parts by weight of water, and the pH of the liquid fermentation medium is 7.2.
Drying and pulverizing the obtained fermentation product, wherein the viable count of Bacillus amyloliquefaciens in each gram of microbial agent is at least 8 × 1010CFU, preferably 1X 1011CFU or more, for example, 1.5X 1011CFU，2×1011CFU。
The provided bacillus amyloliquefaciens CGMCC No.17844 can be prepared into a microbial agent which can be used as a single microbial agent, and can also be compounded with other microbial agents to prepare a composite microbial agent. Therefore, in another aspect of the invention, the invention provides a complex microbial inoculant, which comprises a first microbial inoculant and a second microbial inoculant, wherein the first microbial inoculant is the microbial inoculant (namely the microbial inoculant containing bacillus amyloliquefaciens CGMCC No.17844), and the second microbial inoculant is different from the first microbial inoculant. The second microbial agent can be other bacillus amyloliquefaciens agents, or bacillus mucilaginosus agents, trichoderma viride agents, aspergillus terreus agents and the like as long as the purpose of preventing and treating plant diseases and insect pests or diseases can be achieved. Herein, the bacillus mucilaginosus agent refers to a microbial agent containing first bacillus, the trichoderma viride agent refers to a microbial agent containing trichoderma viride, and similarly, other microbial agents are also understood similarly.
In the process of preparing the complex microbial inoculum, various microorganisms can be prepared into corresponding microbial agents under appropriate conditions, and then the corresponding microbial agents are compounded. For example, Bacillus mucilaginosus can be fermented under conditions similar to Bacillus amyloliquefaciens CGMCC No.17844 to prepare Bacillus mucilaginosus preparation. In at least some embodiments, the bacillus mucilaginosus agent is obtained by fermentation of: carrying out liquid fermentation culture on the bacillus mucilaginosus to obtain a seed culture solution, and carrying out amplification fermentation culture on the seed culture solution so as to obtain a product after fermentation treatment. The expanded fermentation culture can be carried out by utilizing a solid fermentation culture medium or a liquid fermentation culture medium used by the bacillus amyloliquefaciens CGMCC number 17844.
The preparation conditions of the trichoderma viride and the aspergillus terreus are similar. In at least some embodiments of the present invention, trichoderma viride and aspergillus terreus, respectively, may be fermented in PDA liquid broth, and the fermentation broth then inoculated onto a second solid fermentation medium to yield fermentation products of aspergillus terreus and trichoderma viride, respectively. Among the useful second solid fermentation media are: 2-5 parts of wheat bran, 1-5 parts of corn starch, 0.5-3 parts of broken rice residues, 0.5-3 parts of millet, 0.3-1.5 parts of straw residues and water, wherein the content of the water in the solid fermentation medium is 55% -65%. In some preferred embodiments, the second solid fermentation medium comprises: 2 parts by weight of wheat bran, 1.5 parts by weight of corn starch, 1 part by weight of rice trash, 1 part by weight of millet, 0.5 part by weight of straw residue and water, wherein the content of water in the solid fermentation medium is 60%. Respectively drying and crushing the obtained fermentation products of the aspergillus terreus and the trichoderma viride to obtain powdery aspergillus terreus microbial inoculum and powdery trichoderma viride microbial inoculum, wherein each gram of the aspergillus terreus microbial inoculum contains 3 multiplied by 10 effective viable bacteria of the aspergillus terreus10Above, preferably 5 × 1010(ii) a The effective viable count of trichoderma viride contained in each gram of trichoderma viride is 2 multiplied by 1010Preferably 4X 1010。
In at least some embodiments of the present invention, the present invention provides a complex microbial inoculant comprising the first microbial inoculant and at least one of the bacillus mucilaginosus inoculant, trichoderma viride inoculant, and aspergillus terreus inoculant. In some embodiments, the first microbial agent can be blended with any one or more of a bacillus mucilaginosus microbial agent, a trichoderma viride microbial agent and an aspergillus terreus microbial agent according to a ratio of 1-3: 1-3. The obtained composite microbial inoculum contains more than 1000 hundred million viable bacteria per gram, preferably more than 2000 million viable bacteria per gram, such as 3000 million viable bacteria per gram.
In at least some embodiments of the invention, the provided composite microbial inoculum comprises 1-1.5 parts by weight of bacillus amyloliquefaciens CGMCC No.17844 microbial inoculum, 1-1.5 parts by weight of bacillus mucilaginosus microbial inoculum, 1-1.5 parts by weight of trichoderma viride microbial inoculum and 1-1.5 parts by weight of aspergillus terreus microbial inoculum. In other embodiments of the invention, the provided composite microbial inoculum comprises 1-1.5 parts by weight of bacillus amyloliquefaciens CGMCC No.17844 microbial inoculum and 1-1.5 parts by weight of bacillus mucilaginosus microbial inoculum. In still other embodiments of the present invention, the provided complex microbial inoculum comprises 1-1.5 parts by weight of bacillus amyloliquefaciens CGMCC No.17844 microbial inoculum, 1-1.5 parts by weight of trichoderma viride microbial inoculum and 1-1.5 parts by weight of aspergillus terreus microbial inoculum. In still other embodiments of the present invention, the provided complex microbial inoculum comprises 1-1.5 parts by weight of bacillus amyloliquefaciens CGMCC No.17844 microbial inoculum and 1-1.5 parts by weight of trichoderma viride microbial inoculum.
The microbial agent or the compound microbial agent can be used as a fertilizer or a disease treatment drug independently, and can also be added into the fertilizer for use. For example, the microbial agent or the compound microbial agent can be added into an organic compound fertilizer, an organic-inorganic compound fertilizer, an inorganic compound fertilizer and a biological organic fertilizer, the addition amount can be 1-5 per mill, and the obtained microbial fertilizer can be used for plant growth promotion or root rot control. To this end, in still another aspect of the present invention, the present invention provides a fertilizer comprising the above microbial agent or complex microbial agent. In at least some embodiments, the fertilizer is produced to contain more than 1 billion, such as 1.2 billion, of viable bacteria per gram of fertilizer.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are conventional products which are commercially available, and are not known to manufacturers.
Example 1 isolation and screening of Bacillus amyloliquefaciens (accession number CGMCC No.17844)
(1) A pathogenic bacterium of strawberry root rot, namely the rod-shaped pestalotiopsis, is obtained by separating a strawberry root rot disease strain, a colony morphology chart of the pathogenic bacterium is shown in figure 1, the colony morphology chart is determined through an ITS sequence, and MEGA7.0 is used for phylogenetic tree analysis and is shown in figure 2 (the rod-shaped pestalotiopsis is abbreviated as ZH-G01), and the result shows that the separated strain is the rod-shaped pestalotiopsis.
The ITS sequence of Peptosphaerella clavuligerus ZH-G01:
TACCTTTTGTTGCCTCGGCAGAAGTTATAGGTCTTCTTATAGCTGCTGCCGGTGGACCA TTAAACTCTTGTTATTTTATGTAATCTGAGCGTCTTATTTTAATAAGTCAAAACTTTCAA CAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATG TGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCATTAGTAT TCTAGTGGGCATGCCTGTTCGAGCGTCATTTCAACCCTTAAGCCTAGCTTAGTGTTGGG AATCTACTTCTTTTATTAGTTGTAGTTCCTGAAATACAACGGCGGATTTGTAGTATCCTC TGAGCGTAGTAATTTTTTTTCTCGCTTTTGTTAGGTGCTATAACTCCCAGCCGCTAAAC CCCCAATTTTTTGTGGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAA(SEQ ID NO:1)
(2) separating pathogenic bacteria and simultaneously separating and screening a plurality of strains with antagonistic effect on rod-shaped pestalotiopsis (ZH-G01) in diseased strains by a plate coating method and a plate marking method, wherein the strain comprises the following steps:
selecting a diseased part tissue from a greenhouse root rot disease plant of Acorus sibiricus Franch in Shandong, cleaning with clear water, sterilizing the surface, grinding with a mortar, taking 1ml of ground tissue sample, putting into 100ml of sterile water, oscillating at 150rpm and 30 ℃ for 30min, and then performing gradient dilution to 10%4Selecting 3 gradients for coating, wherein each gradient is 3 in parallel, culturing in an incubator at 30 ℃ for 2d, selecting strains with different colony morphologies, streaking on an NA culture medium, and regularly observing the colony growth condition. Then purifying by plate-scribingAnd (5) strains are respectively numbered and stored.
Primary screening: preparing a PDA culture medium by adopting a plate confronting method, punching a fungus cake with the diameter of 5mm on the edge of a plate of a rodlike pestalotiopsis strain by using a puncher, transplanting the fungus cake into the center of the plate, inoculating the strain around the plate by using a toothpick, culturing at a constant temperature of 25 ℃, and observing the inhibition effect of the strain on pathogenic bacteria day by day.
Re-screening: and comparing and screening again to obtain the microbial strain with the best antagonistic effect on pathogenic bacteria of pestalotiopsis.
The colony morphology of the strain is shown in figure 3, and the strain has strong temperature resistance and salt tolerance. And the plate antagonism rate of the strain to the pestalotiopsis clavuligerus reaches 80%, and the antagonism effect is obvious, as shown in figure 4.
Example 2 identification of Bacillus amyloliquefaciens
Example 2 the microbial strain obtained in example 1 was further identified as bacillus amyloliquefaciens. The method comprises the following steps: the sequence of the microbial strain was obtained by 16S rRNA sequencing, and phylogenetic tree analysis was performed using MEGA7.0, as shown in FIG. 5 (the strain is abbreviated as J4 in FIG. 5). The results indicated that the isolated strain was bacillus amyloliquefaciens.
The DNA sequence of the strain for coding 16S rRNA is as follows:
ACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAGATGGGAGCT TGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGC CTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTG TTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACA GATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGG CGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAG ACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATG GACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGAT CGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGC ACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGC CGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAG GGCTCGCAGG(SEQ ID NO:2)
the strain is identified as bacillus amyloliquefaciens, and is preserved with the preservation number of CGMCC No.17844, the preservation unit is China general microbiological culture Collection center, and the preservation address is the institute of microbiology of China academy of sciences No. 3, North Xilu No.1, North Chen Yang district, Beijing.
Example 3 disease-resistant and growth-promoting effects of Bacillus amyloliquefaciens (accession number CGMCC No.17844)
1. Antagonistic effect of plate
Preparing a PDA culture medium by adopting a plate confronting method, punching a fungus cake with the diameter of 5mm at the edge of the pestalotiopsis of strawberry root rot pathogenic bacteria by using a puncher, transplanting the fungus cake in the center of a plate, inoculating a strain of the bacillus amyloliquefaciens CGMCC No.17844 around the plate by using toothpicks, comparing the antagonistic effect of the strain with that of purchased bacillus amyloliquefaciens, bacillus subtilis and bacillus mucilaginosus, culturing the plate at a constant temperature of 25 ℃ after inoculation, and measuring the antagonistic rate.
Wherein the antagonism rate is obtained by the following calculation method:
according to the formula: antagonism (control colony diameter-treated colony diameter)/control colony diameter 100
The measurement results are shown in table 1 below:
TABLE 1 antagonistic Effect of the plates
Experimental results show that compared with externally purchased bacillus amyloliquefaciens or bacillus subtilis and bacillus mucilaginosus, the antagonistic rate of the bacillus amyloliquefaciens on the plate of the pestalotiopsis is improved by 20-28%.
2. Disease-resistant effect of pot culture test
The disease prevention effect of different microbial strains is verified by the following method, which comprises the following steps:
(1) preparation of a suspension of pathogenic spores. Inoculating two kinds of pathogenic bacteria on PDA plate, culturing at 25 deg.C for 5-7d, washing off spores with sterile water after the strain grows over the plate, centrifuging, collecting pathogenic bacteria spores, and using hemocytometerCounting plates under microscope, diluting spore suspension to 106One/ml for standby.
(2) Preparing an antagonistic bacteria suspension. Inoculating the bacillus amyloliquefaciens to a 100ml LB liquid shake flask from a plate, fermenting at the temperature of 30 ℃, the pH value of 6.8 and the rotation speed of 140rpm, and culturing for 24 hours. Adjusting the concentration of the bacterial suspension to 10 after 24 hours8cfu/ml for use.
(3) And (5) disease resistance test. The test is carried out in a test greenhouse of the research and development center limited company of the Linyi city of Shandong province, the tests are divided into a control group 1, a control group 2 and an experimental group 1-7, each group comprises 20 pots, each pot is planted with a healthy strawberry plant with consistent growth and no leaf spots, the pot is 30cm multiplied by 20cm multiplied by 30cm (the diameter of the pot mouth is multiplied by the diameter of the pot bottom is multiplied by the height of the pot), and 3.5kg of sterilized soil is filled in the pot.
Wherein different bacterial suspensions of the experiment groups 1 to 7 are respectively inoculated into root soil of each pot plant after 15 days after seedling revival, and pathogenic bacteria spore suspension (Pestalotiopsis pseudopezii with the concentration of 10) is inoculated after 48 hours6cfu/ml) test period is 30 days (calculated after 15 days of seedling recovery), the watering amount per day of each treatment is the same, and the treatment is unified. Wherein the experimental group 1 is a bacillus amyloliquefaciens group (with the number of CGMCC No.17844), an external collection amylolytic bacteria agent group 1, a group 2 and a group 3, a bacillus mucilaginosus group W4, a bacillus subtilis group W5 and a bacillus subtilis group W6, and the control group 1 is not treated at all; control group 2 was inoculated with two pathogen spore suspensions at a concentration of 106cfu/ml; experimental group 1 administration of the antagonist bacterial suspension of step (2); experimental group 2 to experimental group 7 applied the antibiotic-inoculated suspension prepared as described above. The incidence was calculated with reference to the following methods, respectively.
The strawberry root rot disease incidence rate is calculated by the following method, and the incidence rate is calculated by the same method in the following examples:
the disease degree of the strawberry root rot is divided into 6 grades: 0 level is that the root system is not attacked; grade 1 is that the incidence of root system is less than or equal to 30 percent and the leaves are normal; grade 2 is 30%, the incidence rate of roots is less than or equal to 60%, and the leaves are normal; grade 3 is 60%, the incidence rate of roots is less than or equal to 80%, and leaves turn yellow; grade 4 is that the incidence rate of root systems is more than 80 percent, and leaves wither; grade 5 indicates death of the whole plant and dry leaves.
Disease incidence (%) (∑ (disease grade × number of plants at that grade)/(total number of plants × highest grade value of disease) × 100
Wherein fresh weight (g/plant) represents the weight of strawberry plants; the number of results (number/plant) represents the number of strawberries knotted per plant; yield (g/plant) represents the weight of strawberry fruit; sweetness (%) was obtained by detection with a brix meter.
In the following examples, the fresh weight, the number of results, the yield, the sweetness and the like have the same meanings and the same calculation methods as those of the examples.
The results are shown in Table 2.
TABLE 2 growth promotion test results of strawberry potting
From the results shown in table 2, it can be seen that strawberries obtained by applying the bacillus amyloliquefaciens provided by the invention have improved fresh weight and number of results, yield and sweetness compared with those of bacillus amyloliquefaciens with long market, bacillus subtilis, bacillus mucilaginosus and the like, especially fresh weight and number of results. Moreover, compared with other treatment groups, the incidence rate of strawberries treated by the bacillus amyloliquefaciens provided by the invention is obviously reduced.
Example 4 preparation of Bacillus amyloliquefaciens (CGMCC No.17844) bacterial agent
Example 4 provides a method for preparing a microbial agent by fermentation with a liquid fermentation medium and by fermentation with a solid fermentation medium, wherein the following two methods are used:
1. fermentation with the aid of a liquid fermentation Medium
Inoculating bacillus amyloliquefaciens CGMCC NO.17844 into a 1000mL LB liquid shake flask for culture, preparing a seed solution, transferring the seed solution into a 10L liquid fermentation tank containing a liquid fermentation medium A, fermenting for 2 days at 30 ℃ at an inoculum size of 9 percent and a pH value of 7.0 at a rotating speed of 160rpm, and spray-drying a fermentation product to obtain a dry powder corresponding bactericide. The effective viable count of bacillus amyloliquefaciens in each gram of the microbial inoculum is 8 multiplied by 1011。
Wherein the formula of the liquid fermentation medium A is as follows: 10g of peptone, 2.5g of ammonium sulfate, 3g of beef extract powder, 5g of sodium chloride, 2g of dipotassium hydrogen phosphate, 5g of polyvinyl alcohol and 1000mL of distilled water, wherein the pH value is 7.2.
2. Fermentation with the aid of a solid fermentation Medium
Inoculating Bacillus amyloliquefaciens CGMCC NO.17844 into a 100ml LB liquid shake flask for independent fermentation at 30 deg.C, pH7.0, and rotation speed of 160rpm, and culturing for 20 hr. Then transferring the strain to a solid fermentation culture medium B with the inoculum size of 8 percent, and fermenting for 4 days at 30 ℃ to obtain a solid fermentation product.
Wherein the formula of the solid fermentation medium B comprises the following components in parts by weight: bean pulp: magnesium sulfate: the weight ratio of sodium chloride is 4:1:0.1:0.1, and the water content is 55%.
Drying and crushing the solid fermentation product to obtain the corresponding microbial inoculum in a dry powder state. The effective viable count of bacillus amyloliquefaciens in each gram of the microbial inoculum is 9 multiplied by 1011。
Example 5 application of Bacillus amyloliquefaciens (CGMCC No.17844) in preparation of composite microbial inoculum
The embodiment provides the compound microbial inoculum, and the compound microbial inoculum is obtained by mixing different microbial inocula according to a certain proportion. The bacillus amyloliquefaciens, the bacillus mucilaginosus, the trichoderma viride and the aspergillus terreus can be mixed according to the proportion of 1-3: 1-3: 1-3: 1-3, preferably, the mixture can be prepared by mixing bacillus amyloliquefaciens, bacillus mucilaginosus, trichoderma viride and aspergillus terreus according to the proportion of 1-1.5: 1-1.5: 1-1.5: 1 to 1.5, at least one of them is blended. The method comprises the following steps of preparing different microbial agents, and compounding the different microbial agents, wherein the method comprises the following steps:
1. preparing the bacillus amyloliquefaciens CGMCC NO.17844 microbial inoculum. Referring to the first method of example 4, fermentation was carried out in a liquid fermentation medium, and the number of viable bacteria of Bacillus amyloliquefaciens per gram of the microbial inoculum was 8X 1011。
2. Preparing the bacillus mucilaginosus agent. Bacillus mucilaginosus was fermented by a method similar to the first method in example 4 except that fermentation was carried out using Bacillus mucilaginosus. The obtained bacterial agent contains 8 multiplied by 10 effective viable count of bacillus mucilaginosus per gram10。
3. Preparing aspergillus terreus and trichoderma viride. Respectively inoculating aspergillus terreus and trichoderma viride to a solid fermentation culture medium, wherein the inoculation amount is 8%, fermenting at 30 ℃, culturing in the dark for 24 hours after illumination for 24 hours, continuously fermenting for 9 days to respectively obtain fermentation products of the aspergillus terreus and the trichoderma viride, drying and crushing to obtain dry powder-shaped aspergillus terreus microbial inoculum and trichoderma viride agent, wherein each gram of the aspergillus terreus microbial inoculum contains 5 multiplied by 10 effective viable bacteria of the aspergillus terreus10The effective viable count of trichoderma viride in each gram of trichoderma viride is 4 multiplied by 1010。
4. And compounding the prepared bacillus amyloliquefaciens agent, the prepared bacillus mucilaginosus agent, the prepared trichoderma viride agent and the prepared aspergillus terreus agent according to the mass ratio listed in the table 3 to obtain the composite microbial agent.
TABLE 3 examples of complex microbial agents
The prepared compound microbial inoculum is verified for the field growth promotion and disease resistance effect, and is tested in the town of the Taurus county in Linyi city, Shandong province, specifically as follows:
selecting 1 strawberry greenhouse for 2 mu, and respectively arranging a blank control group and a control group, wherein the blank control is only subjected to conventional fertilization without adding a microbial inoculum, the control group uses a market purchased microbial inoculum, other treatment groups use the compound microbial inoculum prepared in the table 3, and other field management is the same. The experimental results are shown in the following table 4:
table 4 strawberry greenhouse test results
From the results given in table 4, it can be seen that by using the complex microbial inoculum provided by the invention, compared with a blank control group or a control group, the physiological indexes of strawberry plants are improved, fruits are plump, malformed fruits are few, the taste is sweet, the mouthfeel is good, and the field morbidity of strawberry root rot is remarkably reduced.
Example 6 application of Bacillus amyloliquefaciens (CGMCC No.17844) in preparation of microbial fertilizer
Embodiment 6 provides a fertilizer, which includes a bio-organic fertilizer and a complex microbial inoculum, and the preparation method thereof can adopt the following steps:
1. the complex microbial inoculum is prepared according to the method of the embodiment 5, and comprises complex microbial inoculum 1-22.
2. Weighing different materials according to the content of 40% of fermented cow dung, 30% of carbon-based fertilizer, 20% of small molecular organic material and 10% of calcium silicate, and mixing to obtain the corresponding biological organic fertilizer. And uniformly adding the microbial inoculum which accounts for 1-1.5 per mill of the content of the biological organic fertilizer and is obtained in the step 1 to obtain the corresponding fertilizer. Examples of fertilizers prepared using different bioorganic fertilizers are listed in table 5 below, and the fertilizers prepared in this example are named with the bioorganic fertilizer number for the purpose of distinguishing from the fertilizers prepared in examples 7 and 8 below.
The compound microbial agents used are respectively 1-21, so that the prepared fertilizer respectively serves as 1-21.
Table 5 bio-organic fertilizer examples
The prepared fertilizer is subjected to field growth promotion and disease resistance verification, and the test is carried out in the road town of Junan county in Linyi City, Shandong province, and the method comprises the following steps:
selecting 1 strawberry greenhouse for 2 mu, and respectively arranging a blank control group and a control group, wherein the fertilizer used in the blank control group does not contain microbial inoculum compared with each experimental group, and the control group uses the market purchased microbial inoculum to replace the compound microbial inoculum to prepare the microbial fertilizer. Each experimental group used each fertilizer prepared above, and other field management was the same.
TABLE 6 strawberry greenhouse test results
From the results listed in the table 6, compared with the control, the fresh weight of the strawberry plant is improved by 46%, the number of the results is improved by 50%, and the yield is improved by 42% by using the biological organic fertilizer provided by the invention; the fruits are full, the malformed fruits are few, the sugar degree is improved by 17%, and the taste is better; meanwhile, the incidence rate of strawberry root rot is reduced by 37%, and the disease resistance of plants is remarkably improved.
Example 7
Example 7 provides a fertilizer prepared by adding the complex microbial inoculum to an organic-inorganic compound fertilizer 15-40 (16-12-12/S) (wherein 15-40 refers to the ratio of organic fertilizer to inorganic fertilizer, and 16-12-12/S refers to the ratio of nitrogen, phosphorus and potassium). After the organic and inorganic fertilizers are granulated, the compound microbial inoculum and the anti-caking agent are uniformly mixed and added in an outer wrapping mode in a coating working section to obtain the granular fertilizer.
The compound microbial agents 1 to 22 prepared in example 5 were used, and the proportions of the added amount of the compound microbial agent to the amount of the inorganic-inorganic compound fertilizer are shown in table 7 below, thereby obtaining corresponding fertilizers. Meanwhile, for the sake of easy distinction, the prepared fertilizers are named with corresponding base fertilizers.
TABLE 7 examples of organic-inorganic Compound fertilizers
The prepared fertilizer is verified to have the effects of field resistance, growth promotion and disease resistance, and the experiment is carried out in a test area of strawberry sheds at the level of the Shandong Junan county, and comprises the following steps:
compared with the experimental group, the blank control group uses organic-inorganic compound fertilizer without adding complex microbial inoculum, and the control group 1 uses organic-inorganic compound fertilizer with adding microbial inoculum purchased in the market. The other test groups applied the organic-inorganic compound fertilizer prepared above. 3 treatments are arranged in the shed, each treatment is carried out in 3 cells, each cell is 66.6 square meters, and the application amount of the organic fertilizer is 120 Kg/mu. The results of the experiment are shown in table 8 below:
TABLE 8 strawberry greenhouse test results
From the results given in table 8, it can be seen that the physiological indexes of strawberry plants are significantly improved and the field incidence of strawberry root rot is significantly reduced by using the fertilizer provided by the present invention compared with the control group.
Example 8
Embodiment 8 provides a fertilizer, which is prepared by adding a complex microbial inoculum to 15-15-15% of an inorganic compound fertilizer (the contents of nitrogen, phosphorus and potassium are 15%, 15% and 15%, respectively). After the inorganic compound fertilizer is granulated, the compound microbial inoculum and the anti-caking agent are uniformly mixed and added outside a coating working section in a wrapping mode to obtain the granular fertilizer.
The compound microbial agents used in the method are compound microbial agents 1 to 22 prepared in example 5, and the proportions of the compound microbial agents in the inorganic compound fertilizer are respectively shown in table 9 below, thereby obtaining corresponding fertilizers. Meanwhile, for the convenience of distinguishing, the prepared fertilizers are named as corresponding basic fertilizers.
TABLE 9 Fertilizer examples
The prepared fertilizer is verified to have the effects of field resistance, growth promotion and disease resistance, and the experiment is carried out in a test area of strawberry sheds at the level of the Shandong Junan county, and comprises the following steps:
compared with the experimental group, the blank control group uses inorganic compound fertilizer without adding complex microbial inoculum, and the control group uses inorganic compound fertilizer with adding commercial microbial inoculum. The other test groups applied the inorganic fertilizer prepared above. 3 treatments are arranged in the shed, each treatment is carried out in 3 cells, each cell is 66.6 square meters, and the application amount of the organic fertilizer is 120 Kg/mu.
TABLE 10 strawberry greenhouse experimental results
Compared with a control, the physiological indexes of strawberry plants are obviously improved, and the field morbidity of the root rot of the strawberries is obviously reduced by using the fertilizer.
The terms "first", "second" and "first" are used herein for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, schematic representations of the above-described terms are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
SEQUENCE LISTING
<110> research and development center of Zhonghua agriculture (Linyi)
<120> Bacillus amyloliquefaciens CGMCC number 17844 and application thereof
<130> PIDC3194246
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 468
<212> DNA
<213> Artificial Sequence
<220>
<223> Pestalotiopsis clavuligerus
<400> 1
taccttttgt tgcctcggca gaagttatag gtcttcttat agctgctgcc ggtggaccat 60
taaactcttg ttattttatg taatctgagc gtcttatttt aataagtcaa aactttcaac 120
aacggatctc ttggttctgg catcgatgaa gaacgcagcg aaatgcgata agtaatgtga 180
attgcagaat tcagtgaatc atcgaatctt tgaacgcaca ttgcgcccat tagtattcta 240
gtgggcatgc ctgttcgagc gtcatttcaa cccttaagcc tagcttagtg ttgggaatct 300
acttctttta ttagttgtag ttcctgaaat acaacggcgg atttgtagta tcctctgagc 360
gtagtaattt tttttctcgc ttttgttagg tgctataact cccagccgct aaacccccaa 420
ttttttgtgg ttgacctcgg atcaggtagg aatacccgct gaacttaa 468
<210> 2
<211> 560
<212> DNA
<213> Artificial Sequence
<220>
<223> Bacillus amyloliquefaciens
<400> 2
acgctggcgg cgtgcctaat acatgcaagt cgagcggaca gatgggagct tgctccctga 60
tgttagcggc ggacgggtga gtaacacgtg ggtaacctgc ctgtaagact gggataactc 120
cgggaaaccg gggctaatac cggatggttg tttgaaccgc atggttcaga cataaaaggt 180
ggcttcggct accacttaca gatggacccg cggcgcatta gctagttggt gaggtaacgg 240
ctcaccaagg cgacgatgcg tagccgacct gagagggtga tcggccacac tgggactgag 300
acacggccca gactcctacg ggaggcagca gtagggaatc ttccgcaatg gacgaaagtc 360
tgacggagca acgccgcgtg agtgatgaag gttttcggat cgtaaagctc tgttgttagg 420
gaagaacaag tgccgttcaa atagggcggc accttgacgg tacctaacca gaaagccacg 480
gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc cggaattatt 540
gggcgtaaag ggctcgcagg 560
Claims (13)
1. A Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is preserved in China general microbiological culture collection center with the preservation number of CGMCC No. 17844.
2. A microbial agent, comprising the bacillus amyloliquefaciens of claim 1.
3. The microbial inoculant according to claim 2, wherein the microbial inoculant is in the form of a dry powder and comprises at least 8 x 10 viable bacteria per gram of Bacillus amyloliquefaciens bacteria10CFU。
4. The microbial agent according to claim 2, wherein the microbial agent is in a dry powder form, and the effective viable count of the bacillus amyloliquefaciens is 1 x 10 per gram of the microbial agent11CFU。
5. A complex microbial inoculant comprising a first microbial inoculant and a second microbial inoculant different from the first microbial inoculant, wherein the first microbial inoculant is the microbial inoculant of any one of claims 2 to 4;
the second microbial agent comprises at least one of a bacillus mucilaginosus agent, a trichoderma viride agent and an aspergillus terreus agent.
6. The complex microbial inoculant according to claim 5, wherein the complex microbial inoculant comprises 1-3 parts by weight of the first microbial inoculant and at least one of the following components:
1-3 parts by weight of a bacillus mucilaginosus microbial agent;
1-3 parts by weight of a trichoderma viride agent;
1-3 parts of an aspergillus terreus microbial inoculum.
7. The complex microbial inoculant according to claim 5, wherein the complex microbial inoculant comprises 1-1.5 parts by weight of the first microbial inoculant, and at least one of the following microbial inoculant:
1-1.5 parts by weight of a Bacillus mucilaginosus microbial agent;
1-1.5 parts by weight of a trichoderma viride agent;
1-1.5 parts by weight of an aspergillus terreus microbial inoculum.
8. A fertilizer, characterized in that the fertilizer comprises the microbial agent of any one of claims 2 to 4 or the complex microbial agent of any one of claims 5 to 7.
9. The fertilizer according to claim 8, further comprising a base fertilizer, wherein the base fertilizer comprises one selected from a bio-organic fertilizer, an organic compound fertilizer, an inorganic compound fertilizer or an organic-inorganic compound fertilizer, and the content of the microbial agent or the compound microbial agent is 1-5% of the content of the base fertilizer.
10. The fertilizer according to claim 9, wherein the bio-organic fertilizer comprises 30-50 parts by weight of fermented cow dung, 20-40 parts by weight of carbon-based fertilizer, 10-30 parts by weight of small-molecule organic material and 5-15 parts by weight of calcium silicate.
11. The use of the bacillus amyloliquefaciens of claim 1 in the preparation of a microbial agent, a complex microbial agent or a fertilizer.
12. A method of controlling root rot, comprising:
applying an effective amount of bacillus amyloliquefaciens, a microbial agent, a complex microbial agent or a fertilizer to a plant, wherein the bacillus amyloliquefaciens is the bacillus amyloliquefaciens of claim 1, the microbial agent is the microbial agent of any one of claims 2 to 4, the complex microbial agent is the complex microbial agent of any one of claims 5 to 7, and the fertilizer is the fertilizer of any one of claims 8 to 10; the root rot disease is caused by pestalotiopsis.
13. The method of claim 12, wherein the plant is a strawberry.
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