CN112980973A - Preparation method and application of personalized micro-ecological bacterial fertilizer for improving grape continuous cropping obstacle - Google Patents
Preparation method and application of personalized micro-ecological bacterial fertilizer for improving grape continuous cropping obstacle Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a personalized micro-ecological bacterial fertilizer for improving grape continuous cropping obstacles, and belongs to the field of personalized customization of micro-ecological fertilizers. The invention is formed by scientifically formulating fermentation bacteria liquid of bacillus subtilis, bacillus mucilaginosus, photosynthetic bacteria and the like, organic fertilizer, organic material, inorganic material, decomposed microbial inoculum and the like. The active compound microecological bacterial fertilizer is constructed by analyzing biological information of original microbial flora of a soil sample, optimizing and formulating a targeted active compound microecological bacterial fertilizer formula, fermenting natural plants by using the microbial inoculum formula and constructing the active compound microecological bacterial fertilizer under an individualized formula. The personalized bacteria liquid has the functions of improving the root ecology of crops and optimizing the nutrient absorption environment of the crops, so that the continuous cropping crops can grow normally and the effects of increasing the production and income are achieved. The microbial ecological fertilizer has the effects of improving soil, preventing soil-borne diseases, decomposing toxic substances in soil, promoting plant growth and improving the stress resistance of plants, also solves the problems of the traditional single production process, further optimizes resources and makes virtuous circle, is fully and accurately applied in the field of modern agriculture, fills up the industrial blank, and is a microbial ecological fertilizer capable of effectively solving the continuous cropping obstacle of grapes.
Description
Technical Field
The invention belongs to the technical field of personalized customization of a micro-ecological organic fertilizer, and particularly relates to a method for formulating a dynamic formula of a microbial fertilizer.
Background
The grape cultivation history is long, the distribution area is large, large-area cultivation is carried out all over the world, the grape cultivation area and the yield increase speed in recent years of China are obviously higher than those of other fruit trees, the industrial layout is shifted to the dominant region, and the facility cultivation is developed quickly in the non-suitable region. However, with the increase of successive cropping years, the grape plants have weak growth and spread of diseases, and the development of the grape industry is severely restricted. Therefore, continuous cropping obstacles have become a widespread and serious-endangering productivity problem. One of the main reasons for the continuous cropping obstacle is that the microbial population and structure of rhizosphere soil are changed, the number of rhizosphere soil bacteria and actinomycetes after the continuous cropping of grapes is obviously reduced, and the number of fungi is obviously increased. However, at present, the continuous cropping obstacles are mainly solved by changing the planting mode, such as crop rotation or intercropping, and the problems are not solved fundamentally.
The preparation method and the application of the personalized microecological bacterial manure for improving the grape continuous cropping obstacle are guided and developed by combining the microbiology analysis and the traditional separation analysis, and the effect of the customized personalized microecological bacterial manure is evaluated by combining the previously analyzed result and the crop growth condition, so that the problem of the grape continuous cropping obstacle is solved. The micro ecological bacterial fertilizer has the effects of improving soil, preventing soil-borne diseases, decomposing toxic substances in soil, promoting plant growth and improving the stress resistance of plants, also solves the problem of the traditional single production process, further optimizes resources and makes virtuous circle, is fully and accurately applied in the field of modern agriculture, fills up the industrial blank, and is a micro ecological bacterial fertilizer capable of effectively solving continuous cropping obstacles of grapes.
Disclosure of Invention
1. Problems to be solved
The patent aims to research and develop a preparation method and application of a personalized micro-ecological bacterial fertilizer for improving grape continuous cropping obstacles, so that the grape continuous cropping obstacles are improved to a great extent, the grape planting soil is self-repaired, the application amount of the fertilizer is reduced, the quality of grapes is ensured, and the soil condition is improved.
2. Technical scheme
In order to solve the problems, the invention provides a preparation method and application of a personalized micro-ecological bacterial fertilizer for improving grape continuous cropping obstacles, which comprises the following steps:
collecting and processing a grape cultivation soil sample:
and measuring the target area, and sampling by adopting a five-point sampling method, wherein the soil layer is sampled by 25-35 cm. After the sample is roughly processed, extracting the microbial DNA in the sample by adopting a micro-bead breaking or ultrasonic method, and measuring the DNA concentration by adopting a fluorescence quantitative method. After purification after treatment, the V3-V4 hypervariable region of the 16SrRNA gene of the microbial DNA was amplified using 515F/806RPCR (515F-GTGCCAGCMGCCGCGGTAA, 806R-GGACTACHVGGGTWTCTAAT) primers. The concrete steps are detailed in a preparation method and accurate application (patent number: 2018103380602) of a patent microbial fertilizer dynamic microbial inoculum formula of the company, and a flow chart of a preparation method of the personalized micro-ecological microbial fertilizer is shown in a figure I;
determining the total amount of microorganisms and the proportion of microbial flora in the soil: extracting total DNA of microorganisms in target soil by using an MP Biomedicals FastDNA soil sample extraction kit, then carrying out absolute quantification by using a 515F/806R PCR primer by using 16SrRNA as a target sequence, and using escherichia coli as a standard substance; sequencing the amplified products by a high-throughput gene sequencing technology (the sequencing length can reach 500-600 bases at both ends and can completely comprise 16S gene fragments; and PCR amplified products of more than 20000 samples can be simultaneously determined), analyzing and evaluating the sequencing result, and determining the relative content and proportion of different active microbial floras in the soil sample.
Determining a microbial inoculum formula: and comparing the content of the measured microbial flora with the content distribution of the microbial flora in the self-established grape planting soil biological information database to determine the personalized microbial inoculum formula of the grape planting soil. If the relative content is more than 25 percent, the activity content of the microbial flora in the provided microbial inoculum formula is not less than 1 multiplied by 109cfu/g (mL); if the relative content is less than or equal to 25 percent, the activity content of the microbial flora in the provided microbial inoculum formula is not less than 5 multiplied by 109cfu/g(mL)。
Fermentation: the bacterial liquid fermentation substrate comprises fresh natural plant blocks, stems and fruits of different species (preferably Chinese cabbage, potato, pumpkin, pineapple, Korla pear, tomato, sugarcane and watermelon, each l0 kg), which are respectively cleaned, cut into pieces/crushed (the length is less than 1.5cm) and stored after impurities are removed; the proportion is 3: 10 (plant: water) and 5-10% of purified water and yeast extract. Diluting the natural microbial active floras in the personalized microbial inoculum formula determined in the step (3) according to the proportion of 1:500, mixing with a fermentation substrate for fermentation, and fermenting until the pH value of the fermentation substrate reaches or approaches 5.0. Cooling the fermentation liquid, mixing, filtering, separating, detecting, blending, and storing in a place without direct sunlight and cool and ventilated place. After fermentation is mature, organic fertilizer is used as a carrier to be mixed.
Furthermore, in the step (1), the microbial DNA in the sample is extracted by bead breaking or ultrasonic method, and the DNA concentration is determined by fluorescence quantitative method.
Further, the 515F/806RPCR primer used in step (1) is 515F: GTGCCAGCMGCCGCGGTAA 806R; 806R: GGACTACHVGGGTWTCTAAT are provided. The bacterial 16SrRNA gene shares nine hypervariable regions, V1, V2 to V9. The V3-V4 hypervariable region of the 16SrRNA gene was amplified by using a double-ended encoded 515F/806RPCR primer in the present invention. The amplified products were subjected to high throughput sequencing by Illumina or other sequencing platform. The 16SrRNA gene is a DNA sequence corresponding to coding rRNA on bacteria, exists in the genome of all bacteria, and has high conservation and specificity and a relatively complete reference library for bacterial typing analysis.
Furthermore, the microbial flora in the grape planting soil comprises various floras such as photosynthetic flora, lactic acid flora, yeast flora, gram-positive actinomycete flora, filamentous flora of fermentation system, bifidobacterium, bacillus, rhizobium, clostridium sporogenes, trichoderma, aspergillus niger, actinomycete, potassium bacteria, phosphorus bacteria, glomus sacculus on the earth surface, nitrogen-fixing bacteria and the like, and is prepared by dynamic compound strains according to the specific growth environment and detection result of the grapes.
Furthermore, a method for autonomously constructing a grape planting soil biological information database analysis and management platform by selecting the determination of microbial gene sequences of a plurality of strains 16SrRNA commonly seen in grape planting soil samples and referring to system classification names and DNA sequence information of strains in a public database (RDP, SILVA, GreenGene, UNIT).
Further, the fermentation substrate of the bacterial liquid comprises fresh natural plant blocks, stems and fruits of different species (preferably Chinese cabbage, potato, pumpkin, pineapple, Korla pear, tomato, sugarcane and watermelon each l0kg,) which are respectively cleaned, cut into pieces/crushed (length is less than 1.5cm) and stored after impurities are removed; the proportion is 3: 10 (plant: water) adding purified water and 8-13% of sucrose (preferably 8% of sucrose).
Further, diluting natural microbial active floras in the personalized microbial inoculum formula determined in the step (3) according to a ratio of 1:500, mixing the diluted natural microbial active floras with a fermentation substrate for fermentation, setting the temperature of a fermentation tank to be 28 ℃, completely sealing the fermentation tank in the first 1-3 days, ventilating a small amount in 4-7 days to fully amplify the active compound microorganisms, sealing the fermentation tank again after 7 days of fermentation when no large amount of gas is generated so as to kill pathogenic bacteria, worm eggs and the like, and keeping the period for fifteen to twenty days (preferably: 18 days) in order to improve the degree of harmless treatment; at this stage, the moisture is reduced due to the temperature rise. The fermentation process continues and gradually transforms into more stable microecological products. When the temperature is stabilized, the fermentation matures when the pH of the fermentation substrate reaches or approaches 5.0.
Further, the fermentation liquor is cooled to room temperature, and is fully and uniformly mixed with a carrier (decomposed organic fertilizer), dried and granulated to prepare the customized microecological bacterial fertilizer. The detection shows that the solid microecological product is brownish black and soft, has a pH of 5.0-8.5, and has a total content of various formula bacteria of more than or equal to 0.2 × 10 within 12 months9cfu/g, meeting the standard.
3. Advantageous effects
Compared with the prior art, the invention has the beneficial effects that:
(1) according to the method, biological information analysis is carried out on microbial floras of soil samples, a targeted active compound microecological bacterial fertilizer formula is optimized and formulated, natural plants are fermented by the microbial inoculum formula, an individualized active compound microecological bacterial fertilizer is constructed, crop root ecology is improved through the individualized bacterial liquids, and a crop nutrition absorption environment is optimized, so that continuous cropping crops can grow normally, and the effects of increasing production and income are achieved.
(2) The micro ecological bacterial fertilizer has the effects of improving soil, preventing soil-borne diseases, decomposing toxic substances in soil, promoting plant growth and improving the stress resistance of plants, also solves the problem of the traditional single production process, further optimizes resources and makes virtuous circle, is fully and accurately applied in the field of modern agriculture, fills up the industrial blank, and is a micro ecological bacterial fertilizer capable of effectively solving continuous cropping obstacles of grapes.
(3) The patent introduces the concept of accurate medical treatment into accurate agriculture by combining the most advanced biological information technology at present, so that resources are further optimized and virtuous cycle is realized, the industrial blank is filled, the national conditions of China are greatly met, and the application prospect is wide.
Drawings
FIG. 1 is a flow chart of a preparation method of a personalized microecological bacterial fertilizer
FIG. 2 is the information of the proportion of the distribution of the microbial composition in a grape planting soil sample
FIG. 3 shows the microbial groups with significant effect of different groups of microbes in grape planting soil samples
FIG. 4 shows the significant difference between the grape planting soil sample groups
FIG. 5 shows the significance of the average relative abundance and difference test of key species in the grape planting soil sample
Detailed Description
For further illustration of the invention, the following individual embodiments are listed. And meanwhile, the Zheng statement: these examples are not intended to limit the scope of the invention in any way, the invention is not limited to these examples, and certain changes and modifications within the scope of the claims are intended to be included therein.
Collecting culture soil of east foot grapes of Ningxia Helan mountain, and customizing a microbial fertilizer which meets the soil requirement and improves continuous cropping obstacles. Firstly, autonomously constructing a grape planting soil biological information database, obtaining the microorganism composition distribution proportion information in 500 soil samples in the area through high-throughput 16SrRNA gene sequencing, and then determining the formula of a biological fertilizer dynamic microbial inoculum according to the following method:
(1) collecting and processing a grape cultivation soil sample: estimating the planting area of the east foot grapes in the Helan mountain, and sampling by adopting a five-point sampling method in a cell, wherein the sampling soil layer is 25-35 cm, and the number of samples is 500.
(2) Determining the total amount of microorganisms and the proportion of microbial flora in the sample soil: extracting total DNA of microorganism in soil, amplifying V3-V4 hypervariable region of 16SrRNA gene of microorganism DNA by using 16SrRNA as target sequence and 515F/806RPCR (515F-GTGCCAGCMGCCGCGGTAA, 806R-GGACTACHVGGGTWTCTAAT) primer, and using colibacillus as standard substance and gradient of 1.0 × 106，5.0×106，1.0×107，5.0×107，1.0×108，5.0 ×108，1.0×109cfu/g; the total content of the microorganisms in the soil sample is determined to be 2.6 multiplied by 107cfu/g. The soil sample contains Bacillus 2.2 × 106cfu/g, actinomycetes 5.5X 106cfu/g, Rhizobium 7.3X 104cfu/g, photosynthetic bacteria 7.6X 104cfu/g, Clostridium sporogenes 2.8X 104cfu/g, Trichoderma 6.5X 104cfu/g, Aspergillus niger 2.7X 105cfu/g, 6.5X 10 actinomycetes5cfu/g, 3.3X 10 potassium bacteria4cfu/g, 2.7X 10 phosphate solubilizing bacteria4cfu/g, ground sacculus mildew 3.4 x 104cfu/g, 6.0 x 10 azotobacter5cfu/g, the specific strain distribution ratio and the microbial population with significant effect are shown in fig. 2 and 3.
(3) Determining a microbial inoculum formula: the content of bacillus in different soil samples in the area is less than or equal to 25 percent and is determined to be 2.2 multiplied by 106cfu/g (critical value of 25%, the same applies hereinafter), the rhizobium content of less than or equal to 25% is 7.3X 104cfu/g, the content of photosynthetic bacteria is less than or equal to 25 percent and is 7.6 multiplied by 104cfu/g, actinomycetes less than or equal to 25% is 5.5 multiplied by 106cfu/g。
(4) According to the result of biological information analysis, the proportion of partial strains is adjusted, and the total active content of the microbial flora in the microbial inoculum formula is optimized and determined to be 6.5 multiplied by 109cfu/g, 40-60% of bacillus subtilis, 10-30% of bacillus mucilaginosus, 5-10% of bifidobacterium, 10-30% of bacillus megatherium, 8-15% of photosynthetic bacteria and 15-25% of microzyme; preferably: subtilation40% of bacillus, 20% of bacillus mucilaginosus, 5% of bifidobacterium, 15% of bacillus megaterium, 12% of photosynthetic bacteria and 18% of microzyme.
The method is used for fermentation, drying and granulation, and after trial by customers, the trial group is obviously different from the control group, and the trial group is superior to the control group in the aspects of leaf size, glossiness, result rate and the like.
A grape cultivation soil sample in the Ningxia red temple area is collected, and a microbial fertilizer which meets the soil requirement and improves continuous cropping obstacles needs to be customized. Firstly, autonomously constructing a grape planting soil biological information database, obtaining the microorganism composition distribution proportion information in 500 soil samples in the area through high-throughput 16SrRNA gene sequencing,
then determining the formula of the dynamic microbial inoculum of the biological fertilizer according to the following method:
(1) collecting and processing a grape cultivation soil sample: estimating the planting area of the grapes at east foot of Helan mountain, sampling by a cell five-point sampling method, sampling 25-35 cm of soil layer and 500 parts of samples, and extracting and purifying microbial DNA in the samples by using an MPBiomecals FastDNA soil sample extraction kit after treatment.
(2) Determining the total amount of microorganisms and the proportion of microbial flora in the soil: extracting total DNA of microorganism in soil, performing absolute quantification with 16S rRNA as target sequence and 515F/806RPCR primer, and gradient of 1.0 × 10 using Escherichia coli as standard6，5.0×106，1.0×107，5.0×107，1.0×108，5.0×108，1.0×109cfu/g; the total microorganism content in the soil sample was 7.2X 107cfu/g. The soil sample contains bacillus 3.8X 106cfu/g, actinomycetes 1.6X 106cfu/g, Rhizobium 5.6X 106cfu/g, photosynthetic bacteria 5.8X 104cfu/g, Clostridium sporogenes 3.6X 104cfu/g, Trichoderma 7.8X 104cfu/g, Aspergillus niger 4.3X 105cfu/g, 7.7X 10 Potassium bacteria4cfu/g, 6.5X 10 phosphate solubilizing bacteria4cfu/g, 8.2 x 10 sacculus terrestris on ground5cfu/gAzotobacteria 7.6X 104cfu/g. Determining the content of Bacillus in the soil sample to be 3.8 × 106cfu/g, actinomycetes 1.6X 106cfu/g, nitrifying bacteria 2.4X 105cfu/g, photosynthetic bacteria 5.8X 104cfu/g, and the like.
The content of bacillus in the soil sample in the area is more than or equal to 25 percent and is 3.8 multiplied by 108cfu/g (critical value of 25%, the same applies below), actinomycete content of 1.6 x 10 when the content is more than or equal to 25%6cfu/g, the content of nitrobacteria is less than or equal to 25 percent and is 2.4 multiplied by 105cfu/g, 5.8 multiplied by 10 with the content of photosynthetic bacteria more than or equal to 25 percent4cfu/g. Wherein, the relative abundance histograms of the species in each group, the log2 value of the average relative abundance ratio of the same species in the two groups, and the significant difference are shown in fig. 4, and the species with the abundance of the top 10, the average relative abundance and the significance of the difference test are shown in fig. five.
(3) Determining a microbial inoculum formula: according to the result of biological information analysis, the proportion of partial strains is adjusted, and the total active content of the microbial flora in the microbial inoculum formula is optimized and determined to be 2.0 multiplied by 109cfu/g, 30-50% of bacillus subtilis, 10-30% of bacillus mucilaginosus, 5-10% of bifidobacterium, 15-30% of bacillus megatherium, 8-15% of photosynthetic bacteria and 15-25% of microzyme; preferably: 35% of bacillus subtilis, 15% of bacillus mucilaginosus, 8% of bifidobacterium, 18% of bacillus megaterium, 14% of photosynthetic bacteria and 20% of microzyme.
(4) The method is used for fermentation, drying and granulation, and after trial by customers, the trial group is obviously different from the control group, and the trial group is superior to the control group in the aspects of leaf size, glossiness, result rate and the like.
Claims (9)
1. A preparation method and application of a personalized micro-ecological bacterial fertilizer for improving grape continuous cropping obstacles comprise the following steps:
(1) collecting and processing a grape planting soil sample: and measuring the target area, and sampling by adopting a five-point sampling method, wherein the soil layer is sampled by 25-35 cm. After the sample is roughly processed, extracting the microbial DNA in the sample by adopting a micro-bead breaking or ultrasonic method, and measuring the DNA concentration by adopting a fluorescence quantitative method. After purification after treatment, the V3-V4 hypervariable region of the 16S rRNA gene of the microbial DNA was amplified using 515F/806R PCR primers. The concrete steps are detailed in the preparation method and the accurate application of the formula of the microorganism fertilizer dynamic microbial inoculum (patent number: 2018103380602)
(2) Determining the total amount of microorganisms and the proportion of microbial flora in the soil of the target area: the total DNA of microorganisms in target soil is determined by using an MP Biomedicals FastDNA soil sample extraction kit, then 16S rRNA is used as a target sequence, 515F/806R PCR primers are used for absolute quantification, and escherichia coli is used as a standard substance to determine the relative content and proportion of different active microbial floras in a soil sample.
(3) Preparing a personalized microbial inoculum formula: and comparing the content of the measured microbial flora with the content distribution of the microbial flora in the self-established grape planting soil biological information database to determine the personalized microbial inoculum formula of the grape planting soil. If the relative content is more than 25 percent, the activity content of the microbial flora in the provided microbial inoculum formula is not less than 1 multiplied by 109cfu/g (mL), if the relative content is less than or equal to 25 percent, the activity content of the microbial flora in the microbial inoculum formula is not less than 5 multiplied by 109cfu/g(mL)。
(4) Fermentation: diluting the natural microbial active floras in the personalized microbial inoculum formula determined in the step (3) according to the proportion of 1:500, mixing with a fermentation substrate for fermentation, and fermenting until the pH value of the fermentation substrate reaches or approaches 5.0.
2. The preparation method and the application of the personalized micro-ecological bacterial fertilizer for improving the grape continuous cropping obstacle as claimed in claim 1 are characterized in that: in the step (1), microbial DNA in a sample is extracted by adopting a method of breaking up microbeads or adopting ultrasound, and the DNA concentration is measured by adopting a fluorescence quantitative method.
3. The preparation method and the application of the personalized micro-ecological bacterial fertilizer for improving the grape continuous cropping obstacle as claimed in claim 1 or 2 are characterized in that: the 515F/806R PCR primers adopted in the step (1) are 515F-GTGCCAGCMGCCGCGGTAA and 806R-GGACTACHVGGGTWTCTAAT.
4. The preparation method and the application of the personalized micro-ecological bacterial fertilizer for improving the grape continuous cropping obstacle as claimed in claim 1 are characterized in that: the microbial flora in the grape planting soil comprises photosynthetic flora, lactic acid flora, yeast flora, gram-positive actinomycete flora, filamentous flora of a fermentation system, bifidobacteria, bacillus, rhizobium, clostridium sporogenes, trichoderma, aspergillus niger, actinomycete, potassium bacteria, phosphorus bacteria, glomus sacculus on the earth surface, nitrogen-fixing bacteria and other floras, and is prepared by dynamic compound bacteria according to the specific growth environment and detection result of grapes.
5. The preparation method and the application of the personalized micro-ecological bacterial fertilizer for improving the grape continuous cropping obstacle as claimed in claim 1 are characterized in that: the method is a method for autonomously constructing a grape planting soil biological information database analysis and management platform by selecting and measuring microbial gene sequences of 16S rRNA of various common strains in grape planting soil samples and referring to system classification names and DNA sequence information of strains in a public database (RDP, SILVA, GreenGene and UNITE).
6. The preparation method and the application of the personalized micro-ecological bacterial fertilizer for improving the grape continuous cropping obstacle as claimed in claim 1 are characterized in that: the bacterial liquid fermentation substrate comprises fresh natural plant blocks, stems and fruits of different species, and is respectively cleaned, cut into pieces/crushed (the length is less than 1.5cm) and stored after impurities are removed; the proportion is 3: 10 (plant: water) adding purified water.
7. The preparation method and the application of the personalized micro-ecological bacterial fertilizer for improving the grape continuous cropping obstacle as claimed in claim 1 are characterized in that: the bacterial liquid fermentation substrate comprises 70% of water and 8-13% of cane sugar (preferably 8%) by mass fraction.
8. The preparation method and the application of the personalized micro-ecological bacterial fertilizer for improving the grape continuous cropping obstacle as claimed in claim 6 or 7 are characterized in that: the fermentation process in the step (5) is as follows: after fermenting for 7 days at the temperature of 25-30 ℃, sealing the fermentation tank for at least 15-20 days when a large amount of gas is not generated; after the temperature is stabilized and the pH reaches or approaches 5.0, the fermentation is mature and cooled; and (5) after the fermentation is finished, filtering and separating to obtain the personalized formula bacteria liquid.
9. The preparation method and the application of the personalized microecological bacterial fertilizer for improving the grape continuous cropping obstacle, according to the claim 1, the personalized formula bacterial liquid and the carrier to be used are fully and uniformly mixed, dried and granulated to prepare the customized microecological bacterial fertilizer.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120015806A1 (en) * | 2009-03-25 | 2012-01-19 | Sitaram Prasad Paikray | Novel formulation of microbial consortium based bioinoculant for wide spread use in agriculture practices |
| CN108424184A (en) * | 2018-04-16 | 2018-08-21 | 银川尧玥生物科技有限公司 | A kind of formulating method of microbial manure dynamic microbial inoculum formula and accurate application |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120015806A1 (en) * | 2009-03-25 | 2012-01-19 | Sitaram Prasad Paikray | Novel formulation of microbial consortium based bioinoculant for wide spread use in agriculture practices |
| CN108424184A (en) * | 2018-04-16 | 2018-08-21 | 银川尧玥生物科技有限公司 | A kind of formulating method of microbial manure dynamic microbial inoculum formula and accurate application |
Non-Patent Citations (2)
| Title |
|---|
| 张仕颖等: "不同种植年限葡萄根际微生物区系及其与肥力因子的相关分析", 《云南农业大学学报(自然科学)》 * |
| 陈景蕊等: "《宁夏葡萄酒与防沙治沙职业学院校本系列教材 葡萄栽培技术》", 31 December 2016 * |
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