CN115851502B - Bacillus pseudomycoides, microbial inoculum and liquid bio-organic fertilizer and application thereof - Google Patents
Bacillus pseudomycoides, microbial inoculum and liquid bio-organic fertilizer and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of microorganisms and organic fertilizers, in particular to bacillus pseudomycoides, a microbial inoculum, a liquid biological organic fertilizer and application thereof. The bacillus sphamoids SH-1464 provided by the invention has the characteristics of high-efficiency phosphate and potassium dissolving, nitrogen fixation, IAA activity production and the like. The liquid organic fertilizer provided by the invention takes byproducts or wastes of production such as potassium fulvate and the like as main raw materials, has the advantages of improving soil biological indexes, improving soil microorganism diversity and enzyme activity and the like through the cooperation of the components, and is easy to form soil microecological conditions suitable for tea tree growth after application, thereby promoting tea tree plant growth and improving the content of tea polyphenol in tea. After the bacillus pseudomycoides provided by the invention is added into the liquid organic fertilizer, the effect is further improved.
Description
Technical Field
The invention relates to the technical field of microorganisms and organic fertilizers, in particular to bacillus pseudomycoides, a microbial inoculum, a liquid biological organic fertilizer and application thereof.
Background
Microbial fertilizers are also called bacterial fertilizers and biological fertilizers, contain living microorganisms, and can activate nutrients in soil through the vital activities of the microorganisms so as to promote the plants to absorb the nutrients; the microbial fertilizer is applied in the field, so that the agricultural ecological environment can be effectively improved, and the quality of agricultural products can be improved. Improper application of fertilizers and pesticides causes land force reduction and soil nutrient loss, prevents nutrient absorption of vegetable crops and limits the growth of the vegetable crops. Searching for clean, green, pollution-free and strong fertilizer efficiency fertilizer is of great importance to the development of the grain industry in China, and through continuous research and exploration, microbial fertilizer becomes the best choice for improving agricultural productivity and reducing environmental damage.
However, the strain commonly adopted in the existing microbial fertilizers is usually a strain subjected to mutation screening or directional modification, and although the strain can obtain better growth promotion parameter index data in a laboratory research stage, the strain is easy to be colonized in soil or plants in practical application, or is difficult to proliferate or difficult to exert a growth promotion effect due to the influence of environmental factors (such as complex planting environment changes, interactions of other microorganisms in the soil and the like). Therefore, the development of microbial fertilizers based on natural strains is becoming an ongoing research topic in the art. However, there are still few reports on microbial fertilizers for promoting growth of tea trees, especially microbial fertilizers for promoting growth of tea trees prepared based on natural microorganisms.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art and providing bacillus pseudomycosis, a microbial inoculum and a liquid bio-organic fertilizer and application thereof. The bacillus pseudomycoides provided by the invention is a natural plant rhizosphere bacterium, and has better soil and plant rhizosphere colonization capacity compared with genetically engineered bacteria. The research shows that the strain can effectively improve the quality of tea leaves and improve the soil condition after application.
In order to achieve the above object, in one aspect, the present invention provides a bacillus pseudomycoides (Bacillus paramycoides) having a preservation number of cctccc NO: m2022924.
In a second aspect, the invention provides a microbial agent comprising a bacillus pseudomycoides strain according to the first aspect and optionally adjuvants.
In a third aspect, the invention provides a bacillus pseudomycoides strain according to the first aspect and/or the use of a microbial inoculum according to the second aspect in the preparation of a bio-organic fertilizer.
The fourth aspect of the invention provides a liquid bio-organic fertilizer comprising cyanobacteria polysaccharide, an amino acid source, a fertilizer molding agent, potassium fulvate and optionally bacillus pseudomycoides;
wherein, in the liquid bio-organic fertilizer, the weight ratio of blue algae polysaccharide, amino acid source, fertilizer molding agent and potassium fulvate is 3-5:5-10:3-5:77-84.
In a fifth aspect, the invention provides the use of a bacillus pseudomycoides as defined in the first aspect, a microbial inoculum as defined in the second aspect, or a liquid bio-organic fertilizer as defined in the fourth aspect for soil nutrient improvement and/or for promoting plant growth.
In a sixth aspect the present invention provides a method of promoting the growth of tea trees, the method comprising applying to the soil a bacillus pseudomycoides as defined in the first aspect, a microbial inoculum as defined in the second aspect or a liquid bio-organic fertilizer as defined in the fourth aspect.
Through the technical scheme, the invention at least has the following beneficial effects:
(1) The bacillus pseudomycoides provided by the invention is a natural strain separated from plant rhizosphere soil, and has better soil colonization capability compared with an induced mutant strain or a genetically engineered strain which is commonly used for microbial fertilizer preparation in the field. The strain or the microbial fertilizer prepared based on the strain is more beneficial to exerting the growth promoting property of the strain, so that the excellent growth promoting effect can be obtained.
(2) The liquid biological organic fertilizer provided by the invention has good effect of promoting the growth of tea trees, can improve the content of tea polyphenol in tea, and can effectively solve the problems that the existing liquid biological organic fertilizer has poor effect of promoting the growth of tea trees or can not realize continuous promotion of the growth. After the bacillus pseudomycoides provided by the invention is added into the liquid bio-organic fertilizer, the growth promoting effect of tea trees is further improved.
(3) The liquid biological organic fertilizer provided by the invention has the characteristics of wide raw material sources, low cost, environment friendliness and the like. Meanwhile, the liquid biological organic fertilizer has the advantages of simple preparation method, high storage stability and the like. In addition, through selection of the types and the amounts of raw materials in the liquid bio-organic fertilizer, the viable count in the fertilizer can reach a higher level when the bacillus pseudomycoides provided by the invention is added into the liquid bio-organic fertilizer.
(4) The method for promoting the growth of the tea trees is simple and easy to implement, does not have adverse effect on environmental safety, and can promote sustainable development of agricultural production.
Drawings
FIG. 1 is a phylogenetic tree of Bacillus pseudomycoides SH-1464 constructed in example 1.
FIG. 2 is a graph showing the results of the phosphorus dissolution test of Bacillus muscarinic SH-1464 in example 1.
FIG. 3 is a graph showing the results of nitrogen fixation test of Bacillus muscarinic SH-1464 in example 1.
FIG. 4 is a graph showing the results of qualitative IAA production test of Bacillus muscarinic SH-1464 in example 1.
Preservation of organisms
The bacillus pseudomycoides (Bacillus paramycoides SH-1464) provided by the invention is preserved in China center for type culture collection (China) for 6 months and 20 days, and has the address of eight paths 299 of Wuchang district of Wuhan, hubei province, and the preservation number of CCTCC NO: m2022924.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
In the present invention, the bacillus myxoides SH-1464 and the bacillus myxoides CCTCC NO: m2022924 is the same strain, both of which are synonymous, and their names (numbers) are used interchangeably.
In the present invention, "optional" means that the component belongs to an optional component, and a person skilled in the art can choose whether to add the component according to the actual situation. In the technical scheme provided by the invention, the purpose of the invention (such as promoting the growth of tea trees, improving the content of tea polyphenol in tea leaves and the like) can be realized without adding an optional component, but the effect can be further improved if the optional component is added.
The inventor of the present invention isolated a strain of bacillus muscari (Bacillus paramycoides), named SH-1464, deposited in the China center for type culture collection (CCTCC NO) at 2022, 6 and 20 days during the course of research: m2022924. Through further research, the inventor skillfully discovers that when the bacillus pseudomycoides is applied to the soil for planting tea trees, the growth of the tea trees can be effectively promoted, and the quality of the tea trees can be improved.
Based on the above findings, the present invention provides, in one aspect, a strain of bacillus myxoides (Bacillus paramycoides) having a preservation number of cctccc NO: m2022924.
In a second aspect, the invention provides a microbial inoculum, which contains the bacillus pseudomycoides (CCTCC NO: M2022924) and optional auxiliary materials according to the first aspect.
In the present invention, the specific formulation of the microbial agent is not particularly limited, and any type of microbial agent commonly used in the art may be applied to the present invention. For example, the bacterial agent may be a solid bacterial agent, a liquid bacterial agent, or a semisolid (concentrated) bacterial agent.
Preferably, the content of the bacillus pseudomycoides in the microbial inoculum is not less than 1×10 10 cfu/g (solid microbial agent) or 1X 10 10 cfu/mL (liquid or concentrated). For example, it may be 1X 10 10 -1×10 11 cfu/g or 1X 10 10 -1×10 11 cfu/mL。
In the invention, the auxiliary materials and the content thereof selected in the microbial inoculum are not particularly limited, so long as the auxiliary materials and the content thereof are used for preparing the bacillus mycoides CCTCC NO: the effect of M2022924 is not adversely affected or is not substantially adversely affected (for example, the effect of the strain after the addition of the auxiliary material is not less than 80% of that when the strain alone is used). Adjuvants commonly used in the art for the preparation of bacterial agents are suitable for use in the present invention, such as protective agents, excipients, binders, fillers, and the like.
The inventor of the present invention found in the research that the bacillus pseudomycoides provided by the present invention can effectively promote the growth of crops and improve the crop yield and the product quality when being applied to the soil alone or together with certain organic fertilizer materials. Especially has better growth promoting effect on tea trees, not only can promote the tea trees to grow faster, but also can improve the content of active ingredients such as tea polyphenol in the tea, and the like, thereby obtaining the tea with higher quality.
Based on this, a third aspect of the invention provides a bacillus pseudomycoides as defined in the first aspect and/or the use of a microbial inoculum as defined in the second aspect in the preparation of a bio-organic fertilizer.
The inventor of the invention also discovers in the research that the liquid biological organic fertilizer prepared by mixing blue algae polysaccharide with a proper amount of amino acid source, fertilizer molding agent, potassium fulvate and the like is applied to soil, so that the content of effective nitrogen, phosphorus and potassium in the soil can be effectively improved, the soil fertility is improved, the conversion of nitrogen, phosphorus and potassium in plant rhizosphere and the absorption of nitrogen, phosphorus and potassium are promoted, and the usage amount of chemical fertilizer is reduced. In addition, the liquid organic fertilizer can also improve the biological index of soil, regulate the diversity of soil microorganisms and enzyme activity, and promote the formation of soil microecological conditions for healthy growth of tea trees, thereby promoting the growth of tea trees, improving the content of tea polyphenol in tea, and improving the yield of high-quality tea. Through further research, the inventor also discovers that the bacillus pseudomycoides CCTCC NO provided by the invention is added on the basis of the liquid bio-organic fertilizer prepared by the components: m2022924 (or a microbial inoculum prepared based on the strain) can further improve the effect of improving the quality of the tea yield.
Based on the above findings, the fourth aspect of the present invention provides a liquid bio-organic fertilizer comprising cyanobacteria polysaccharide, an amino acid source, a fertilizer molding agent, potassium fulvate and optionally bacillus pseudomycoides;
wherein, in the liquid bio-organic fertilizer, the weight ratio of blue algae polysaccharide, amino acid source, fertilizer molding agent and potassium fulvate is 1:1-5:0.5-1.5:10-30.
That is, the liquid bio-organic fertilizer provided by the invention has the formula of any one of the following:
(1) Blue algae polysaccharide, an amino acid source, a fertilizer molding agent and potassium fulvate in a weight ratio of 1:1-5:0.5-1.5:10-30;
(2) Blue algae polysaccharide, amino acid source, fertilizer molding agent, potassium fulvate and bacillus pseudomycoides (microbial inoculum) with the weight ratio of 1:1-5:0.5-1.5:10-30.
Preferably, in the liquid bio-organic fertilizer, the weight ratio of blue algae polysaccharide, amino acid source, fertilizer molding agent and potassium fulvate is 1:1.5-4.5:0.8-1.2:15-28. More preferably 1:2-4:0.8-1.2:15-20.
In the invention, the raw materials (such as blue algae polysaccharide, amino acid, fertilizer molding agent, potassium fulvate and the like) used as the liquid fertilizer are not particularly limited, and can be any related products which can be used for preparing the liquid fertilizer in the field, and can be related products obtained commercially or prepared by self according to the prior art.
According to a preferred embodiment of the present invention, the cyanobacteria polysaccharide has a moisture content of 30 to 40 wt%. Preferably 30-32% by weight. The invention is not particularly limited in the specific components and sources of the blue algae polysaccharide selected, and any blue algae polysaccharide having the above characteristics can be applied to the invention. It is either directly commercially available or is a related product prepared by itself according to the prior art.
Preferably, the cyanobacteria polysaccharide has a sugar content of 20-40 wt%, a soluble colloid content of 10-20 wt%, a mineral content of 5-12 wt% and a crude protein content of 3-10 wt% based on dry matter.
More preferably, the cyanobacteria polysaccharide has a sugar content of 25-35 wt%, a soluble colloid content of 12-18 wt%, a mineral content of 5-10 wt%, and a crude protein content of 3-6 wt%, on a dry matter basis.
According to a preferred embodiment of the invention, the free amino acid content of the amino acid source is 20-40% by weight on a dry matter basis. Preferably 25 to 35% by weight.
In the present invention, the specific type and source of the amino acid source are not particularly limited, and any reagent capable of providing a liquid bio-organic fertilizer with free amino acid can be used as the amino acid source in the present invention. Preferably, the amino acid source may be selected from amino acids (solutions) prepared using plant-or animal-derived proteins. Preferably, the plant-derived protein may be provided by a fermentation product of at least one of soybean, soybean meal, corn bran, peanut bran, or by at least one of bean products, monosodium glutamate, wood processing offcuts, and the like. The animal-derived protein may be provided by at least one of animal hair (e.g., feathers, pig hair, etc.), animal blood, viscera, skin bones, low-fat fish meal, silkworm chrysalis, slaughterhouse waste, etc. The amino acid source can be related products with the characteristics obtained by direct purchase, or related products prepared by self by adopting the raw materials according to the prior art.
The inventor of the invention discovers that when polyglutamic acid is used as a fertilizer synergist to be applied to the liquid biological organic fertilizer provided by the invention, not only the fertilizer efficiency can be further improved, but also stress resistance of crops, especially tea trees, and tea quality can be improved. In addition, in the liquid organic fertilizer containing the bacillus mycoides, polyglutamic acid is adopted as a fertilizer synergist, so that the number of viable bacteria in the organic fertilizer can be further increased.
According to a preferred embodiment of the present invention, wherein the fertilizer synergist is selected from polyglutamic acid.
Preferably, the polyglutamic acid has a weight average molecular weight of 1 ten thousand to 1.5 ten thousand.
More preferably, the polyglutamic acid has a weight average molecular weight of 1 ten thousand to 1.3 ten thousand.
According to a preferred embodiment of the present invention, in the potassium fulvate, the content of fulvic acid is 40-60 wt%, the content of humic acid is 10-20 wt%, the content of total nitrogen is 2-5 wt%, the content of total phosphorus is 0.1-0.5 wt%, and the content of total potassium is 5-15 wt%, on a dry matter basis.
Preferably, in the potassium fulvate, the content of the fulvic acid is 45-55 wt%, the content of the humic acid is 15-20 wt%, the content of the total nitrogen is 2-4 wt%, the content of the total phosphorus is 0.2-0.3 wt%, and the content of the total potassium is 5-10 wt%, based on dry matter.
According to a preferred embodiment of the present invention, wherein the bacillus pseudomycoides is selected from the group consisting of the species having the accession number cctccc NO: m2022924 or, alternatively, provided by the microbial agent of the second aspect.
Preferably, the amount of the bacillus mycoides agent is 0.1-1 wt% based on the total weight of the liquid bio-organic fertilizer. Preferably 0.3 to 0.8% by weight. More preferably 0.5 to 0.7% by weight.
More preferably, the amount of the Bacillus pseudomycoides or the microbial inoculum is such that the content of the Bacillus pseudomycoides in the liquid bio-organic fertilizer is not less than 1×10 6 cfu/mL. Preferably 1X 10 6 -5×10 7 cfu/mL. More preferably 1X 10 7 -5×10 7 cfu/mL. Further preferably 1X 10 7 -2×10 7 cfu/mL. When the bacillus pseudomycoides is added into the liquid bio-organic fertilizer in the form of a solid microbial inoculum, the content of the bacillus pseudomycoides in the liquid bio-organic fertilizer after the liquid bio-organic fertilizer is added according to the proportion of 1mL of the finally obtained liquid bio-organic fertilizer being 1 g.
According to a preferred embodiment of the present invention, the liquid bio-organic fertilizer further comprises an auxiliary material, preferably at least one selected from the group consisting of nitrogen fertilizer, phosphate fertilizer and potassium fertilizer.
In the present invention, there is no particular limitation on the nitrogen fertilizer, the phosphate fertilizer and the potash fertilizer selected as the auxiliary materials, and any of the agents commonly used in the art as the above-mentioned fertilizers may be applied to the present invention. For example, urea, superphosphate, potassium sulfate and the like can be selected as auxiliary materials in the liquid bio-organic fertilizer provided by the invention.
The specific amounts and proportions of the nitrogen fertilizer, the phosphate fertilizer and the potash fertilizer used as auxiliary materials are not particularly limited in the invention, and can be adjusted correspondingly according to actual conditions (such as the condition of soil in a planting field, the growth condition and the growth characteristics of crops, and the like). According to a preferred embodiment of the invention, the auxiliary material is selected from the group consisting of mixtures of nitrogen fertilizer, phosphate fertilizer and potassium fertilizer in such an amount that the weight ratio of N, P to K in the auxiliary material is 1:1.5-2.5:3-4.
Preferably, the total content of the auxiliary materials is 0.1-3 wt% based on the total weight of the liquid bio-organic fertilizer. Preferably 0.5 to 2% by weight. More preferably 0.5 to 1% by weight. For example, the amount may be 0.5 wt%, 0.55 wt%, 0.6 wt%, 0.65 wt%, 0.7 wt%, 0.75 wt%, 0.8 wt%, 0.9 wt%, 1 wt%, or any intermediate value between any two of the above values.
In a fifth aspect, the invention provides the use of a bacillus pseudomycoides as defined in the first aspect, a microbial inoculum as defined in the second aspect, or a liquid bio-organic fertilizer as defined in the fourth aspect for soil nutrient improvement and/or for promoting plant growth.
According to a preferred embodiment of the present invention, wherein the plant is a camellia plant. Tea tree is preferred.
In the present invention, promoting plant growth means increasing the growth rate of plants (e.g., plant height increase, leaf growth, etc. over a period of time) and improving the quality of crops (e.g., increasing the content of a beneficial active ingredient in crops, etc.).
In the present invention, soil nutrient improvement means to increase the content of effective nutrients in soil (i.e., nutrients that can be directly absorbed and utilized by plants, such as N, P, K, etc.), to increase biological indexes of soil (e.g., to increase microbial diversity of soil, to increase enzymatic activity in soil, etc.), to improve the microenvironment of soil so as to make it more favorable for crop growth, etc.
In a sixth aspect the present invention provides a method of promoting the growth of tea trees, the method comprising applying to the soil a bacillus pseudomycoides as defined in the first aspect, a microbial inoculum as defined in the second aspect or a liquid bio-organic fertilizer as defined in the fourth aspect.
That is, the above method may include the following several ways:
(1) The bacillus or bacillus-mimetic agent is applied directly to the soil (which may typically be applied in the form of a culture);
(2) Applying a liquid bio-organic fertilizer free of bacillus mycoides to the soil;
(3) A liquid bioorganic fertilizer containing bacillus myxomatosis is applied to the soil.
In the present invention, the specific application mode of the microbial inoculum and/or the liquid bio-organic fertilizer is not particularly limited, and any mode commonly used in the art for applying fertilizer in soil can be applied to the present invention. For example, the microbial inoculum and/or the liquid bio-organic fertilizer can be used for root irrigation and soil covering after ditching soil around the plants, or can be directly sprayed on the rhizosphere of the plants.
The specific characteristics of the bacillus myxoides, the microbial inoculum and the liquid bio-organic fertilizer adopted in the method provided by the invention are as described above and are not described in detail herein.
In the invention, the specific dosage of the bacillus pseudomycoides, the microbial inoculum and the liquid bio-organic fertilizer is not particularly limited, so long as the growth of tea plants can be promoted.
According to a preferred embodiment of the present invention, wherein, for the above-mentioned modes (1) and (3), the amount of the Bacillus pseudomycoides, the microbial inoculum or the liquid bio-organic fertilizer to be applied is such that the amount of the Bacillus pseudomycoides is not less than 1X 10 8 cfu/strain/time. Preferably 1X 10 9 -1×10 13 cfu/strain/time. More preferably 3X 10 9 -3×10 11 cfu/strain/time. For example, it may be 3X 10 9 cfu/strain/time, 4X 10 9 cfu/strain/time, 5X 10 9 cfu/strain/time, 6X 10 9 cfu/strain/time, 8×10 9 cfu/strain/time, 1×10 10 cfu/strain/time, 5X 10 10 cfu/strain/time, 8×10 10 cfu/strain/time, 1×10 11 cfu/strain/time, 2X 10 11 cfu/strain/time, 3×10 11 cfu/strain/time, or any intermediate value between any two values mentioned above. The dosage of the bacillus pseudomycoides is calculated according to the application dosage of the microbial inoculum or the liquid organic fertilizer and the quantity of the bacillus pseudomycoides contained in the bacillus pseudomycoides, wherein the liquid microbial inoculum and the liquid organic fertilizer are converted according to 1 g/mL.
The bio-organic fertilizer and the bacillus pseudomycoides provided by the invention have a synergistic effect when being used together, so that the dosage of the bacillus pseudomycoides in the mode (1) can be appropriately higher than that in the mode (3) in order to obtain a better effect.
According to a preferred embodiment of the present invention, wherein, for the above-mentioned modes (2) and (3), the liquid bio-organic fertilizer may be applied to the soil in an amount of 200 to 500 mL/plant/time. Preferably 300-400 mL/strain/time.
Preferably, the rate of application of the bacillus myxoides, microbial agents or liquid bio-organic fertilizer is 2-3 times per year.
The present invention will be described in detail by examples. It should be understood that the following examples are provided for further explanation and illustration of the present invention and are not intended to limit the present invention.
Example 1
This example is useful in illustrating the isolation, purification, identification and preservation of Bacillus pseudomycoides SH-1464 provided herein.
(1) Strain isolation and purification
In the research process, the inventor adopts a dilution coating flat plate method to separate and obtain a strain with the number of SH-1464 from healthy tobacco rhizosphere soil which is rotation-produced for a long time in Lijiang city of Yunnan province.
(2) Identification of strains
Strain SH-1464 was identified for morphological and physiological-biochemical characteristics by reference to the "berkovich system classification handbook" and the "common bacterial systems identification handbook". The results were as follows: bacterial strain SH-1464 is gram positive bacteria, bacterial colony is white, flat in shape, positive in contact enzyme, starch hydrolysis, nitric acid reduction reaction, indole reaction, citric acid reaction, arginine double hydrolysis, sucrose fermentation reaction, glucose fermentation reaction, nitrogen fixation reaction, gelatin reaction and the like, and negative in MR reaction and urease reaction.
The strain SH-1464 was identified by molecular biology using the following method: inoculating strain SH-1464 into purified liquid culture medium, shaking and culturing at 37deg.C in shaking table at 180r/min overnight, sampling under aseptic condition, and reading culture bacterial liquid OD under ultraviolet spectrophotometer 600 Value, when OD 600 The value is approximately 1 (about 1X 10) 9 cfu/mL) and stopping the culture. The strain genomic DNA was extracted using TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit ver.3.0 kit. PCR amplification was performed using bacterial 16S rDNA universal primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5 '-TACGGY TACCTTGTTACGACTT-3'). PCR reaction procedure: pre-denaturation at 94℃for 5min; denaturation at 94℃for 60s; annealing at 53 DEG C60s; extending at 72 ℃ for 2min;35 cycles; finally, the mixture is extended for 7min at 72 ℃ and stored at 4 ℃. After the reaction was completed, 5. Mu.L of the reaction product was taken and subjected to 1% agarose gel electrophoresis, and observed in a gel imaging system. The amplified product was purified and recovered by agarose gel electrophoresis and sent to Beijing qingke biosciences, inc. for sequencing. After BLAST search (https:// BLAST. Ncbi. Lm. Nih. Gov/BLAST. Cgi), the sequencing result is compared and analyzed with the gene sequences of related species in a GenBank database, a mode strain sequence with higher homology is selected as a reference object, clustal X1.8 software is used for carrying out multi-sequence comparison, and the similarity between the tested strain and the reference strain sequence is calculated. The phylogenetic analysis was performed by excluding the base deletion site, and constructing a phylogenetic tree between the test strain and the reference strain using MEGA 7.0 by the Neighbor-joining analysis method. Wherein, the Bootstrap value is set to be 1000, and the rest are default values.
The molecular biology identification results are as follows: the 16SrDNA sequence of SH-1464 was 100% similar to that of Bacillus muscarinic MAOI01000012 as analyzed by NCBI Blast, and a phylogenetic tree constructed by the neighbor-Joining method (neighbor-Joining) is shown in FIG. 1.
The strain SH-1464 is bacillus pseudomycoides (Bacillus paramycoides) with the combination of morphological and biochemical characteristics and molecular biological identification results
(3) Strain characterization study
And (3) detecting a phosphate dissolving effect: the purified strain SH-1464 is inoculated onto inorganic phosphate-decomposing bacteria selection medium (10.0 g/L of glucose, 0.5g/L of ammonium sulfate, 0.5g/L of yeast extract powder, 0.3g/L of sodium chloride, 0.3g/L of potassium chloride, 0.3g/L of magnesium sulfate, 0.03g/L of ferrous sulfate, 0.03g/L of manganese sulfate, 5.00g/L of calcium phosphate and pH 7-7.5), and the mixture is placed in a 30 ℃ incubator to be cultured for 72 hours, and the production condition of phosphate solubilizing rings is observed and recorded.
The results are detailed in FIG. 2. From the figure, it can be seen that the strain SH-1464 has better phosphate dissolving capacity, and the average diameter of the phosphate dissolving ring is 16.11mm.
Nitrogen fixation activity detection: purified strain SH-1464 was inoculated into nitrogen-fixing bacteria selection medium (KH 2 PO 4 0.2g/L、MgSO 4 0.2g/L、NaCl 0.2g/L、CaCO 3 5.0g/L mannitol10.0g/L、CaSO 4 0.1g/L, 18.0g/L of agar and pH of 6.8-7), and culturing in a 30 ℃ incubator for 72 hours, and observing the occurrence of transparent rings of the record.
The results are detailed in FIG. 3. From the figure, the strain SH-1464 can generate transparent rings on the nitrogen-fixing bacteria selection medium, and the diameter of the transparent rings is larger, so that the strain SH-1464 has better nitrogen-fixing activity.
Indoleacetic acid (IAA) production assay: (1) qualitative detection: SH-1464 single colony is selected and inoculated into KB culture medium, shake culture is carried out for 24 hours at 180rpm/min at 30 ℃, 1mL of fermentation liquor is absorbed in a centrifuge tube under aseptic condition, and quick and 4mL of Sackowcki's color reagent is added (preparation method comprises the steps of slowly adding 150mL of concentrated sulfuric acid into 250mL of deionized water while stirring, adding 7.5mL of FeCl with the concentration of 0.5mol/L after the solution is cooled 3 ·6H 2 O solution), standing at normal temperature in dark place for color development for 40min, and observing and recording color change.
(2) Quantitative detection: weighing 10mg IAA standard substance, dissolving in small amount of ethanol, adding distilled water to volume of 100mL (concentration of 100 μg/mL), gradient diluting to 0 (distilled water only), 4 μg/mL, 8 μg/mL, 12 μg/mL, 16 μg/mL, 20 μg/mL, 24 μg/mL, 1mL each, adding 4mL of color development agent, mixing, standing at 40deg.C in dark for 40min, and measuring OD 535 Values, and plotting a standard curve. Centrifuging the bacterial suspension and blank for 10min at 10000r/min, collecting supernatant 4mL, adding 4mL colorimetric solution, mixing, standing in the dark for 40min, and measuring OD with spectrophotometer 535 The values are repeated for 3 times, the blank colorimetric solution is zeroed, the drawn standard curve is compared, the color change at different fermentation time is continuously measured for 7 days, and the IAA yield of the strain at each time period is calculated to obtain the optimal fermentation time and yield of the IAA produced by the strain.
The results are shown in detail in FIG. 4. As can be seen from the graph, the strain can react with Sackowcki's color reagent in a color development way, and IAA yield is increased along with the extension of fermentation time (the color of the culture solution at the rightmost side of the graph is slightly darker than that of the culture solution at the left side of the graph for 24 h), and the IAA secretion amount of the strain SH-1464 is calculated to be about 21.56-24.88 mug/mL through quantitative detection.
(4) Preservation of strains
The inventor reserves the bacillus pseudomycoides SH-1464 separated by the method in China Center for Type Culture Collection (CCTCC) in the 6 th and 20 th 2022, wherein the preservation number is CCTCC NO: m2022924.
Example 2
Liquid bio-organic fertilizer was formulated according to the formulation in table 2. The specific preparation method comprises the following steps: the raw materials are weighed according to the proportion shown in Table 2, 1L/kg of water is added, and the liquid bio-organic fertilizer is obtained after uniform mixing.
The blue algae polysaccharide is purchased from Yunnan Yuxi light chemical industry Co., ltd, polyglutamic acid is purchased from Henan Ai Pusheng agricultural technology Co., ltd (weight average molecular weight is 1.2 ten thousand), and potassium fulvate is purchased from Henan Ai Pusheng agricultural technology Co. The microbial inoculum is a solid microbial inoculum prepared by amplifying and drying the bacillus pseudomycoides SH-1464 obtained in the example 1, wherein the content of the bacillus pseudomycoides is about 2 multiplied by 10 10 cfu/g. The auxiliary materials are prepared by urea, calcium superphosphate and potassium sulfate, wherein the weight ratio of N, P, K is 8:16:26. The amino acid source is a plant source amino acid concentrate from Sichuan blue Biotechnology Co., ltd, wherein the content of free amino acid is 30+ -0.5 wt%.
The sugar content in blue algae polysaccharide is measured by a sulfuric acid phenol method, the soluble colloid content is measured by a 3, 5-dimethylphenol colorimetric method, the mineral content is measured by an atomic absorption spectrophotometry method, and the crude protein content is measured by a Kaplan nitrogen determination method. The content of fulvic acid in the potassium fulvate is measured by adopting a potassium dichromate oxidation colorimetry, the content of humic acid is measured by adopting a sodium pyrophosphate alkaline liquor extraction method, the content of total nitrogen is measured by adopting a Kjeldahl digestion method, the content of total phosphorus is measured by adopting a molybdenum-antimony colorimetry, the content of total potassium is measured by adopting a flame photometer method, and the content of the potassium fulvate is calculated according to the content of other substances.
The results of the measurements are shown in Table 1 (wherein the water content is calculated based on the total weight of the material and the remaining components are calculated based on the dry matter in the material).
TABLE 1 liquid bioorganic fertilizer material compositions
Table 2 liquid bioorganic fertilizer formulation
Example 3
The formulation of the liquid bio-organic fertilizer B1 in Table 2 is adopted, and the potassium fulvate is replaced by the biochemical potassium fulvate with the same weight (the components are shown in Table 3 in detail). Obtaining the liquid bio-organic fertilizer B6.
TABLE 3 substitution of Biochemical potassium Fulvate Components
Test example 1
A batch of potted tea seedlings with basically consistent initial heights (the difference among the plants is not more than 1 cm) are selected, and 1 plant is planted in each pot. The liquid bio-organic fertilizer obtained in the above example was applied to the rhizosphere soil of tea seedlings in the amounts shown in table 4, and then simultaneously cultured under other conditions kept uniform. The specific fertilization mode comprises the following steps: digging pits of potted tea seedlings before fertilization, namely digging soil deeply along the periphery of the tea seedlings by using a soil digging shovel by 3-5cm and 10-13cm away from the ground, and carrying out root irrigation treatment on the periphery of the tea trees by using liquid biological organic fertilizer, and then covering soil to finish fertilization.
The growth of tea trees and the biological index of the rhizosphere soil of tea trees were investigated two months after fertilization. Specifically, the method comprises the steps of measuring the average plant height of each group of tea trees; the method in national standard of the people's republic of China (detection method of tea polyphenol and catechin content in tea) (GB/T8313-2008) is adopted to measure the total amount of tea polyphenol in tea; the rhizosphere soil of each group of tea trees is collected, the activity of the polyphenol oxidase (PPO) is measured by using a polyphenol oxidase (PPO) activity detection kit purchased from Beijing Soy Bao technology Co., ltd.), and the diversity index of the soil bacteria Shannon is measured by using High throughput sequencing (High-throughput sequencing) (specific test method refers to the diversity characteristic of the anaerobic microbial community of rhizosphere of High carbon reserves farmland paddy in Er-sea river basin). The results are detailed in Table 4.
As can be seen from the data in table 4, in the case of applying the liquid bio-organic fertilizer provided by the present invention, the growth condition of tea trees and the content of tea polyphenol in tea leaves are both superior to those of the control group tea seedlings, and when the applied liquid bio-organic fertilizer adopts the preferred components of the present invention and the content ratio of each component is within the preferred range of the present invention, the effect is further improved. In addition, from the measurement result of the biological index of the rhizosphere soil of the tea tree, the liquid biological organic fertilizer provided by the invention has a good improvement effect on the activity of the soil after being applied, and the diversity of soil microorganisms is greatly improved.
TABLE 4 test conditions and results for liquid bioorganic fertilizer to tea plant fruit set
Note that: the data in Table 4 are the average of the measured data of 10 tea seedlings in the group, "control" refers to the control group to which no fertilizer was applied, "fertilizer" refers to the control group to which fertilizer was applied, wherein the fertilizer used was a mixture of commercially available urea, potassium sulfate and calcium magnesium phosphorus compound fertilizer (weight ratio of 12.5:4:2.5).
Test example 2
Culturing Bacillus pseudomycosis SH-1464 obtained in example 1 with NB medium (powder medium purchased from Haibo technology Co., ltd., preparation method comprises weighing 18g of culture medium powder with balance, adding 1000mL of deionized water for dissolving, packaging, sterilizing with 121 deg.C high temperature steam for 15 min), diluting the obtained culture solution with appropriate amount of fresh NB medium to obtain viable count of about 3×10 8 cfu/mL of the liquid bacterial agent of the bacillus mycoides.
Potted tea seedlings were treated as follows, and then investigated for tea tree growth and tea tree rhizosphere soil biological index as in test example 1.
Experimental group I: the culture solution of the bacillus muscari was poured onto the rhizosphere of tea plants (10 plants per test group), and the growth condition of tea plants and the biological index of the rhizosphere soil of tea plants were investigated two months after application (specific detection indexes and detection methods are the same as in test example 1, and the following are the same).
Experimental group II: the preparation method comprises drying a Bacillus pseudomycosis (solid preparation obtained by drying liquid preparation, and the number of viable bacteria is about 2×10) 8 cfu/g) was applied to the rhizosphere soil of tea plant (10 plants per test group) according to the method in test example 1, and the growth of tea plant and the biological index of the rhizosphere soil of tea plant were investigated two months after application.
Experimental group III: liquid bio-organic fertilizers A2 and B1 in Table 2 were poured onto the rhizosphere of tea plant (10 plants per test group) respectively, and the growth condition of tea plant and the biological index of the soil of the rhizosphere of tea plant were investigated two months after application.
Control group: neither the mycosphaerella mycoides nor any fertiliser is applied.
The growth promoting effect and soil biological index of each group of tea trees were measured according to the calculation method in test example 1, and the results are shown in Table 5.
As can be seen from the results of experimental groups I and II in Table 5, the bacillus pseudomycoides provided by the invention has a good growth promoting effect on tea trees, can effectively promote the growth of tea trees regardless of the application of liquid microbial agents or solid microbial agents, improves the content of tea polyphenol in the tea, has a good improvement effect on the activity of soil, and particularly improves the activity of polyphenol oxidase and the diversity of microorganisms in the soil. As can be seen from comparison of the results of experiment group III in Table 5 and A2 and B1 in Table 4, the liquid bio-organic fertilizer provided by the invention has little difference in growth promotion effect and soil activity improvement effect on tea trees when applied in different modes, which indicates that the liquid bio-organic fertilizer provided by the invention can adapt to different planting and management modes and has better universality in production practice.
TABLE 5 test conditions and results of the test of the application of Bacillus pseudomycoides and liquid organic fertilizer to tea plant fruit growth promotion effect
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Claims (20)
1. The bacillus pseudomycoides is characterized in that the preservation number of the bacillus pseudomycoides is CCTCC NO: m2022924.
2. A microbial agent comprising the bacillus pseudomycoides of claim 1 and optionally an adjuvant.
3. The use of the bacillus pseudomycoides of claim 1 and/or the microbial inoculum of claim 2 in the preparation of a bio-organic fertilizer.
4. A liquid bio-organic fertilizer, which is characterized by comprising blue algae polysaccharide, an amino acid source, a fertilizer molding agent, potassium fulvate and optionally bacillus pseudomycoides;
wherein, in the liquid biological organic fertilizer, the weight ratio of blue algae polysaccharide, amino acid source, fertilizer molding agent and potassium fulvate is 1:1-5:0.5-1.5:10-30;
the bacillus pseudomycoides is selected from the group consisting of the bacillus pseudomycoides with a preservation number of CCTCC NO: m2022924, or alternatively, provided by the microbial agent of claim 2.
5. The liquid bio-organic fertilizer according to claim 4, wherein the moisture content of the cyanobacteria polysaccharide is 30-40 wt%;
the free amino acid content of the amino acid source is 20-40 wt% based on dry matter;
the fertilizer plastic effect agent is polyglutamic acid;
in the potassium fulvate, the content of the fulvic acid is 40-60 wt%, the content of the humic acid is 10-20 wt%, the content of the total nitrogen is 2-5 wt%, the content of the total phosphorus is 0.1-0.5 wt%, and the content of the total potassium is 5-15 wt% based on dry matter.
6. The liquid bio-organic fertilizer according to claim 5, wherein the cyanobacteria polysaccharide has a sugar content of 20-40 wt%, a soluble colloid content of 10-20 wt%, a mineral content of 5-12 wt%, and a crude protein content of 3-10 wt%, on a dry matter basis;
the weight average molecular weight of the polyglutamic acid is 1 ten thousand to 1.5 ten thousand.
7. The liquid bioorganic fertilizer of claim 4, wherein said bacillus pseudomycosis is used in an amount of 0.1-1% by weight based on the total weight of said liquid bioorganic fertilizer.
8. The liquid bio-organic fertilizer according to claim 4, wherein the content of the bacillus pseudomycoides in the liquid bio-organic fertilizer is not less than 1 x 10 6 cfu/mL。
9. The liquid bio-organic fertilizer according to any one of claims 4 to 8, wherein the liquid bio-organic fertilizer further comprises an auxiliary material.
10. The liquid bio-organic fertilizer according to claim 9, wherein the auxiliary material is selected from at least one of a nitrogen fertilizer, a phosphorus fertilizer and a potassium fertilizer.
11. The liquid bio-organic fertilizer according to claim 10, wherein the total content of the auxiliary material is 0.1 to 3% by weight based on the total weight of the liquid bio-organic fertilizer.
12. The liquid bio-organic fertilizer according to claim 11, wherein the total content of the auxiliary material is 0.5 to 2% by weight based on the total weight of the liquid bio-organic fertilizer.
13. Use of a bacillus pseudomycoides as defined in claim 1, a microbial inoculant as defined in claim 2, or a liquid bioorganic fertilizer as defined in any one of claims 4-12 for soil nutrient improvement and/or for promoting plant growth.
14. The use according to claim 13, wherein the plant is a camellia plant.
15. The use according to claim 14, wherein the plant is tea tree.
16. A method of promoting tea tree growth comprising applying the muscarinic stem of claim 1, the microbial inoculant of claim 2, or the liquid bioorganic fertilizer of any one of claims 4-12 to soil.
17. The method according to claim 16, wherein the amount of the Bacillus pseudomycoides, the microbial inoculum or the liquid bio-organic fertilizer is applied such that the amount of the Bacillus pseudomycoides is not less than 1X 10 8 cfu/strain/time;
and/or the liquid bio-organic fertilizer is applied to the soil in an amount of 200-500 mL/plant/time.
18. The method according to claim 17, wherein the amount of the bacillus pseudomycoides, the microbial inoculum or the liquid bio-organic fertilizer applied is such that the amount of the bacillus pseudomycoides is 1 x 10 9 -1×10 13 cfu/strain/time;
and/or the liquid bio-organic fertilizer is applied to the soil in an amount of 300-400 mL/plant/time.
19. The method according to claim 18, wherein the amount of the bacillus pseudomycoides, the microbial inoculum or the liquid bio-organic fertilizer applied is such that the amount of the bacillus pseudomycoides is 3 x 10 9 -3×10 11 cfu/strain/time.
20. The method of any one of claims 16-19, wherein the rate of administration of the bacillus pseudomycoides, microbial inoculum, or liquid bio-organic fertilizer is 2-3 times per year.
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| WO2012085647A1 (en) * | 2010-12-22 | 2012-06-28 | Zero Waste Agro Organics Pvt Ltd | Organic fertilizer and a method for preparing the same |
| CN108503466A (en) * | 2018-06-29 | 2018-09-07 | 贵州大学 | A kind of tea tree special bio bacterial manure and the preparation method and application thereof |
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| EP3892716A1 (en) * | 2020-04-07 | 2021-10-13 | Biofor System d.o.o. | Novel plant growth promoting bacterial strains, an improved method for encapsulation of bacteria and a biofertilizer comprising encapsulated bacteria prepared by the said method |
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