CN108841761B - Method for promoting growth of clover and/or increasing yield of clover and microbial inoculum used by method - Google Patents
Method for promoting growth of clover and/or increasing yield of clover and microbial inoculum used by method Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
Abstract
The invention discloses a method for promoting the growth of clover and/or improving the yield of clover and a microbial inoculum used by the method. The method for promoting the growth of the clovers and/or improving the yield of the clovers comprises the steps of uniformly mixing clover seeds with a microbial inoculum and then sowing the mixture into soil; the microbial inoculum contains rhizobium meliloti or/and metabolites of rhizobium meliloti; the strain number of the rhizobium meliloti is 92-29, and the preservation number of the rhizobium meliloti in the common microorganism center of China Committee for culture preservation and management of microorganisms is CGMCC No. 15533. The rhizobium meliloti 92-29 of the invention can obviously promote the growth of the clovers and obviously improve the yield of the clovers under the acid soil condition: the plant height, nodulation rate, root nodule number and/or root nodule weight of the clover are improved; increasing the fresh weight and dry weight of the plant of the clover, and increasing the fresh grass yield and the dry grass yield of the clover. The invention has wide application prospect in the clover planting industry.
Description
Technical Field
The invention relates to a method for promoting the growth of clover and/or improving the yield of clover in the field of agricultural microorganisms and a microbial inoculum used by the method.
Background
Trifolium (Trifolium) is the most widely distributed genus of the Leguminosae family and is a short-lived perennial legume. Clover species are distributed throughout the world in about 360 varieties, of which about 25 varieties have higher agronomic value. The three-leaf clover is widely planted in China, mainly distributed in temperate to tropical regions, distributed in south to Yunnan and north to Heilongjiang in wild species and cultivated species, and mostly cultivated in China are red three leaves, white three leaves and miscellaneous three leaves. The clover is excellent forage grass with nitrogen fixation capacity, contains rich protein and mineral substances, has strong cold resistance, long green period, high heat resistance, low requirement on soil, adaptability to acid and alkaline soil, high regeneration capacity, high harmful gas pollution resistance and high disease and pest resistance, can improve soil fertility, maintain soil temperature, control weed growth, increase ground coverage and reduce soil erosion. Clover can be used as excellent feed for livestock, good first crop of crops, green manure, good water and soil conservation plants, garden greening plants, honey source and medicinal materials.
The nitrogen is one of the most important nutrient elements in plant growth, the rhizobia is a bacterium which can infect roots (a few stems) of leguminous plants to form root nodules for biological nitrogen fixation, a symbiotic system of the rhizobia and the leguminous plants is a system with the strongest effect in biological nitrogen fixation, nitrogen-fixing microorganisms in soil convert nitrogen in air into ammonia which can be absorbed by plants, and the fixed nitrogen is about 65 percent of the total amount of biological nitrogen fixation. Currently, growing legumes in degraded soil is of increasing interest because degraded soil is poor in nitrogen nutrients, soil fertility can be improved through symbiotic nitrogen fixation of legumes and rhizobia, and inoculation of rhizobia can improve yield and nitrogen storage of legumes. If the planted leguminous plants do not have corresponding high-efficiency rhizobia strains to symbiotically nodulate with the leguminous plants and fix nitrogen in the air, the leguminous plants are completely dependent on combined nitrogen in soil, and the nitrogen in the soil can not be supplemented but consumed, so that the soil fertility is reduced. Therefore, in order to fully exert the symbiotic nitrogen fixation function of rhizobia and leguminous plants and maintain the nitrogen balance of soil, efficient strains need to be screened and artificially inoculated.
Disclosure of Invention
The invention aims to solve the technical problem of how to obviously promote the growth of clovers and/or improve the yield of the clovers in acid soil.
In order to solve the technical problems, the invention provides a microbial inoculum for promoting the growth of clovers and/or improving the yield of the clovers.
The microbial inoculum for promoting the growth of the clovers and/or improving the yield of the clovers, which is provided by the invention, contains Rhizobium japonicum (Rhizobium trifolii) or/and metabolites of the Rhizobium japonicum (Rhizobium trifolii);
the strain number of the Rhizobium meliloti (Rhizobium trifolii) is 92-29, and the preservation number of the Rhizobium meliloti in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 15533.
The rhizobium meliloti 92-29 belongs to gram stain negative, is short and rod-shaped, has no spore, has terminal single flagellum and can move. The cell size was 0.7X 2.2. mu.m. The bacterial colony grows on a yeast juice mannitol agar culture medium plate, the bacterial colony is in a circular micro-bulge shape, the edge is neat, the surface is smooth and moist, the bacterial colony is light milky white, and the bacterial lawn is sticky. Rhizobium meliloti 92-29 has the 16S rDNA sequence of sequence 1 in the sequence table.
The seed coating agent of the rhizoctonia trifoliata containing the microbial inoculum or the biological fertilizer containing the microbial inoculum also belongs to the protection scope of the invention.
The clover seeds coated with the microbial inoculum also belong to the protection scope of the invention.
As mentioned above, the active ingredient of the above fungicide can be Rhizobium japonicum 92-29 or/and metabolites of Rhizobium japonicum 92-29, the active ingredient of the fungicide can also contain other biological ingredients or non-biological ingredients, and the other active ingredients of the fungicide can be determined by those skilled in the art according to the promoting effect of the fungicide on the plant weight and/or plant height of the clovers.
The microbial inoculum may also comprise a carrier, as hereinbefore described. The carrier may be a solid carrier or a liquid carrier. The solid carrier is a mineral material or a biological material; the mineral material may be at least one of grass peat, clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the biological material is at least one of straws, pine shells, rice straws, peanut shells, corn flour, bean flour, starch, grass peat and animal manure of various crops; the liquid carrier can be water; in the microbial inoculum, rhizobium meliloti 92-29 or/and metabolites of rhizobium meliloti 92-29 can exist in the form of cultured living cells, fermentation liquor of the living cells, filtrate of cell culture or mixture of the cells and the filtrate. The preparation formulation of the microbial inoculum can be various preparation formulations, such as liquid, emulsion, suspending agent, powder, granules, wettable powder or water dispersible granules.
In the above, according to needs, a surfactant (such as tween 20, tween 80, and the like), a binder, a stabilizer (such as an antioxidant), a pH regulator, and the like may also be added to the microbial inoculum.
To solve the above technical problems, the present invention provides a method for promoting the growth and/or increasing the yield of clovers.
The method for promoting the growth of the clovers and/or improving the yield of the clovers comprises the step of uniformly mixing clover seeds with the microbial inoculum and then sowing the mixture into soil.
As used herein, promoting clover growth may be increasing clover plant height, nodulation rate, number of nodules, and/or weight of nodules.
As used herein, the promoting the growth of clover can be promoting the growth of clover in neutral medium or acidic medium.
As used herein, the increase in yield of clover can be an increase in the weight per plant (fresh and/or dry) or the yield per unit area of clover.
As used herein, the increased yield of clover can be increased in neutral medium or acidic medium.
In the above context, the medium may be soil.
As used herein, the pH of the acidic medium can be greater than or equal to 4.2 and less than 7, such as from 4.8 to 6.2.
As used herein, the clover may be white clover.
The application of the microbial inoculum, the clover rhizoma bacterium seed coating agent and/or the pelleted clover seeds in promoting the growth of clovers and/or improving the yield of the clovers also belongs to the protection scope of the invention.
Experiments prove that the rhizobium pratense 92-29 of the invention can obviously promote the growth of clovers and obviously improve the yield of the clovers under the acidic (pH value is 4.8-6.2) soil condition: the plant height, nodulation rate, root nodule number and/or root nodule weight of the clover are improved; increasing the fresh weight and dry weight of the plant of the clover, and increasing the fresh grass yield and the dry grass yield of the clover. The invention has wide application prospect in the clover planting industry.
Deposit description
The strain name is as follows: root nodule bacteria of clover (Rhizobium trifolii)
The strain number is as follows: 92-29
The preservation organization: china general microbiological culture Collection center
The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)
Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district
The preservation date is as follows: 3, month and 30 days 2018
Registration number of the preservation center: CGMCC No.15533
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The preparation method of the solid medium in the following examples is as follows: 5ml of glycerol, 5g of mannitol and 1g of yeast extract powder, K2HPO40.5g, anhydrous MgSO4 0.2g，CaSO4·2H2O 0.2g，NaCl 0.1g，1％(NH4)6Mo7O24·4H2O1ml、1％H3BO31ml of agar and 20g of agar, adding water to a constant volume of 1000ml, adjusting the pH value to 6.8-7.0, and sterilizing at 121 ℃ for 30 min.
The liquid medium in the following examples was prepared as follows: 5ml of glycerol, 5g of mannitol and 1g of yeast extract powder, K2HPO40.5g, anhydrous MgSO4 0.2g，CaSO4·2H2O 0.2g，NaCl 0.1g，1％(NH4)6Mo7O24·4H2O1ml、1％H3BO31ml, adding water to a constant volume of 1000ml, adjusting pH to 6.8-7.0, and sterilizing at 121 deg.C for 30 min.
The Rhizobium trifolii (Rhizobium trifolii) ACCC18019 in the following examples was collected at 19.12.1981 in the agricultural microorganism center of China Committee for culture Collection of microorganisms (abbreviated as ACCC, address: southern street 12 of Guancun, Haizu province, Beijing, China academy of agricultural sciences, institute of agricultural resources and agricultural divisions, postal code 100081), and was publicly available from the agricultural microorganism center of the Committee for culture Collection of microorganisms in China on the day of collection. Rhizobium trifolii (Rhizobium trifolii) ACCC18019 is hereinafter abbreviated as Rhizobium trifolii ACCC 18019.
Example 1 isolation and characterization of Rhizobium trifolium (Rhizobium trifolii)92-29CGMCC No.15533
1. Isolation of the Strain
Collecting root nodules of wild white clover from the area of Yunnan Trijing, and streaking and separating by using a flat plate (taking 5g of mannitol, 5g of glycerol, 1g of yeast extract powder, 0.5g of dipotassium hydrogen phosphate, 0.2g of calcium sulfate, 0.2g of magnesium sulfate, 0.1g of sodium chloride, 1ml of ammonium molybdate aqueous solution with the mass content of 1 percent, 1ml of boric acid aqueous solution with the mass content of 1 percent, 1ml of 0.5 percent Congo red, 20g of agar, 1L of distilled water and the pH value of 6.8-7.0) to obtain the strain 92-29.
2. Identification of strains
2.1 morphological identification
And (3) observing the state of a single colony of the strain 92-29 which is in a logarithmic growth phase and has stable colony size and is obtained by separating and purifying the strain in the step 1, wherein the observation mainly comprises the size, color, transparency, wettability, colony surface state and colony edge state of the colony. On the other hand, the strain 92-29 in the logarithmic growth phase was stained by smear and then the form of the cells was observed by an optical microscope.
The result shows that the strain 92-29 belongs to gram-negative stain, is short and rod-shaped, has no spore, has terminal single flagellum and can move. The cell size was 0.7X 2.2. mu.m. The bacterial colony grows on a yeast juice mannitol agar culture medium plate, the bacterial colony is in a circular micro-bulge shape, the edge is neat, the surface is smooth and moist, the texture is uniform, the bacterial colony is light milky white, and the bacterial lawn is sticky.
2.2, 16S rDNA sequence homology analysis
The 16S rDNA fragment of the strain 92-29 obtained in step 1 was amplified by colony PCR, and the reagents were provided by Takara Shuzo Co. The results of amplifying and cloning sequencing the 16S rDNA gene fragment show that the 16S rDNA of the strain 92-29 has a nucleotide sequence of a sequence 1 in a sequence table. The similarity of the 16S rDNA of strains 92-29 to the 16S rRNA gene (Sequence ID: MRDL01000029) of Rhizobium trifolii strain USDA 2370(T) was up to 99.93%.
2.3, physiological and biochemical characterization
The physiological and biochemical characteristics of strains 92-29 were determined with reference to "handbook of identification of common bacteria systems" (Dongxu bead, Chuia Miaoying. handbook of identification of common bacteria systems. Beijing: scientific Press, 2011) and "microbiology experiment" (Shenping, Fangxiong capacity, Liguanwu. microbiology experiment (third edition); Beijing: advanced education Press, 1999.). The result shows that the strain 92-29 is chemoheterotrophic and grows aerobically, and can grow by utilizing glucose, lactose, D-ribose, D-cellobiose, D-arabinose, mannitol, xylose, D-galactose, fructose, dulcitol and inositol; ammonium salt, nitrate and most amino acids are used as nitrogen sources, Congo red color absorption reaction micro color absorption, B.T.B reaction produces acid, gelatin is positive in liquefaction, starch cannot be hydrolyzed, cellulose cannot be utilized, casein cannot be hydrolyzed, 3-ketolactose cannot be utilized, citrate cannot be utilized, and the beef extract-peptone medium is not grown with abundant extracellular mucus when the beef extract-peptone medium grows on the sugar-containing medium.
In view of the above results of morphological analysis, physiological and biochemical characteristic analysis and 16S rDNA sequence homology analysis, the strain 92-29 isolated and purified in step 1 was identified as Rhizobium trifolii (Rhizobium trifolii). The Rhizobium trifolii (Rhizobium trifolii)92-29 is preserved in China general microbiological culture Collection center (CGMCC, address: No. 3 of Xilu No.1 of Beijing area on the morning sun) in 2018, 3 months and 30 days, and the preservation number is CGMCC No.15533, which is hereinafter referred to as Rhizobium trifolii 92-29.
Example 2 preparation of Rhizobium meliloti 92-29 bacterial preparation
1. Slant culture of Rhizobium meliloti 92-29
The Rhizobium bifidum 92-29 of example 1 was selected and inoculated into a solid medium for slant culture, and cultured at 28 ℃ for 56 hours to obtain the slant-cultured Rhizobium bifidum 92-29.
2. Activation of bacterial species
And (3) selecting the rhizobium meliloti 92-29 cultured on the slant in the step (1), inoculating the rhizobium meliloti 92-29 into 200mL of liquid culture medium, and carrying out shaking culture at 28 ℃ and 150rpm for 48 hours to obtain a rhizobium meliloti 92-29 bacterial liquid.
3. Seeding tank culture
Taking 3L of the rhizobium meliloti 92-29 bacterial liquid obtained in the step 2, inoculating the bacterial liquid into a 100L seed tank (containing 60L of liquid culture medium) for seed culture, and ventilating and stirring at 28 ℃ and 150rpm for culturing for 48 hours to obtain rhizobium meliloti 92-29 seed liquid.
4. Cultivation in fermenter
Inoculating the Rhizobium meliloti 92-29 seed solution obtained in step 3 into a 1000L fermentation tank (containing 600L of liquid culture medium) according to a proportion (volume ratio) of 10%, fermenting and culturing, ventilating and stirring at 28 ℃ and 150rpm for 48 hours to obtain Rhizobium meliloti 92-29 fermentation broth, wherein the Rhizobium meliloti 92-29 fermentation brothThe content of bacteria 92-29 is 4 × 109cfu/mL。
5. Preparation of rhizobium meliloti 92-29 microbial inoculum
Naturally drying the grass peat, then crushing, sieving with a 100-mesh sieve, then adjusting the pH value to 7.0 with lime water, and sterilizing at 121 ℃ for 60min to obtain the sterile grass peat.
And (4) uniformly mixing the rhizobium meliloti 92-29 fermentation liquor obtained in the step (4) with the sterile grass carbon, and performing propagation culture for 50h at the temperature of 28 ℃ to obtain the rhizobium meliloti 92-29 microbial inoculum. The finished product is qualified through detection, and the content of the rhizobium meliloti 92-29 in the rhizobium meliloti 92-29 microbial inoculum is 3.0 multiplied by 108cfu/g.
Example 3 comparison of the growth of Rhizobium trefoil 92-29 on acidic Medium at different pH (tolerance test)
After the liquid culture medium is sterilized, the pH value of the culture medium is adjusted to 4.2, 4.4, 4.5, 4.6, 4.8, 5.0, 5.5, 6.0 and 7.0 by using a sterilized 1% NaOH solution and a sterilized 1% HCl solution to obtain liquid culture media with different pH values, each treatment is repeated for 3 times, rhizobium meliloti 92-29 and rhizobium meliloti ACCC18019 strains are respectively inoculated into the liquid culture media with different pH values, and after the liquid culture media are subjected to constant temperature shaking culture at 150rpm and 28 ℃ for 48 hours, the OD is measured by using the liquid culture media subjected to each treatment of non-inoculated bacteria as a blank control600The value is obtained. As shown in Table 1, it was revealed that Rhizobium meliloti 92-29 grew at pH 4.2-7.0, that Rhizobium meliloti ACCC18019 grew only at pH 4.8-7.0, and that the growth of Rhizobium meliloti 92-29 was superior to that of Rhizobium meliloti ACCC18019 at pH 4.2-7.0. The acid resistance of the rhizobium meliloti 92-29 is better than that of the rhizobium meliloti ACCC18019, and the rhizobium meliloti is an acid-resistant rhizobium.
TABLE 1 OD of Rhizobium trefoil cultured in liquid medium at various pH values for 48 hours600Value of
Example 3 Rhizobium meliloti 92-29 promotes clover growth and increases clover yield
Preparation of root nodule bacteria ACCC18019 bacterial preparation of clover
1. Slant culture of Rhizobium meliloti ACCC18019
Selecting rhizobium meliloti ACCC18019, inoculating to solid culture medium, slant culturing at 28 deg.C for 56 hr to obtain slant cultured rhizobium meliloti ACCC 18019.
2. Activation of bacterial species
And (3) selecting the rhizobium meliloti ACCC18019 cultured on the slant in the step (1), inoculating the rhizobium meliloti ACCC18019 into 200mL of liquid culture medium, and performing shaking culture at 28 ℃ and 150rpm for 48 hours to obtain a rhizobium meliloti ACCC18019 bacterial liquid.
3. Seeding tank culture
Taking 3L of the rhizobium meliloti ACCC18019 bacterial liquid obtained in the step 2, inoculating the bacterial liquid into a 100L seed tank (containing 60L of liquid culture medium) for seed culture, and ventilating and stirring at 28 ℃ and 150rpm for culturing for 48 hours to obtain the rhizobium meliloti ACCC18019 seed liquid.
4. Cultivation in fermenter
Inoculating the rhizobium meliloti ACCC18019 seed solution obtained in the step 3 into a 1000L fermentation tank (containing 600L of liquid culture medium) according to a proportion (volume ratio) of 10 percent for fermentation culture, and performing aeration-stirring culture at 28 ℃ and 150rpm for 48 hours to obtain rhizobium meliloti ACCC18019 fermentation liquor, wherein the content of the rhizobium meliloti ACCC18019 in the rhizobium meliloti ACCC18019 fermentation liquor is 4 multiplied by 109cfu/mL。
5. Preparation of rhizobium meliloti ACCC18019 microbial inoculum
Naturally drying the grass peat, then crushing, sieving with a 100-mesh sieve, then adjusting the pH value to 7.0 with lime water, and sterilizing at 121 ℃ for 60min to obtain the sterile grass peat.
And (4) uniformly mixing the rhizobium meliloti ACCC18019 fermentation liquor obtained in the step (4) with the sterile grass carbon, and performing proliferation culture at the temperature of 28 ℃ for 50h to obtain the rhizobium meliloti ACCC18019 microbial inoculum. The finished product is qualified through detection, and the content of the rhizobium meliloti ACCC18019 in the rhizobium meliloti ACCC18019 microbial inoculum is 3.0 multiplied by 108cfu/g.
Secondly, the rhizobium meliloti 92-29 promotes the growth of the clovers and improves the yield of the clovers in the soil with the pH value of 6.15
The test site is in Qiyang county of Yongzhou city of Hunan province. The test adopts a random block design, three treatment areas are randomly arranged, and each treatment area is provided with three cell repetitions. The three treatment areas are respectively a non-inoculation contrast treatment area, a 92-29 microbial inoculum treatment area and an ACCC18019 microbial inoculum treatment area. Each small area is 4 multiplied by 4 square meters, the soil pH value is 6.15, and the average number of rhizobium abortus is 2.0 multiplied by 102cfu/g dry soil. According to the sowing quantity of the clover being 2kg/hm2Seeds are prepared.
Selecting Trifolium repens seeds with uniform size, soaking for 1 minute by concentrated sulfuric acid, and washing with sterile water for several times to remove acid to obtain treated seeds. The treated seeds were subjected to the following three treatments:
mixing the rhizobium meliloti 92-29 microbial inoculum prepared in the example 2 and the seeds of the clover leaves according to the proportion of 1: 10, and the obtained seeds are called 92-29-trifoliate repens seeds.
And (3) mixing the rhizobium meliloti ACCC18019 microbial inoculum prepared in the step one and the seeds of the clover leaves according to the ratio of 1: 10, and the obtained seeds are called ACCC 18019-trifoliate repens seeds.
Mixing the sterile grass peat and the white clover seeds according to the proportion of 1: 10, and the obtained seeds are called sterile grass carbon-white clover seeds.
The seeds are uniformly sown in the soil according to the cell design, the sowing method is drill sowing, and the row spacing is 10 cm. The conditions in the above treatment zones are identical except for the different seeds to be sown. Wherein the seeds sowed in the non-inoculation control treatment area are aseptic grass peat-Trifolium repens seeds; the seeds sowed in the 92-29 microbial inoculum treatment area are 92-29-trifoliate repens; the seeds sowed in the ACCC18019 microbial inoculum treatment area are ACCC 18019-Trifolium repens seeds.
Recording the number of nodules of the underground part of each plant, the fresh weight of the nodules of the underground part of each plant, the nodule rate of the underground part of each plant, the height of the plant of the overground part of each plant, the fresh weight of the plant of the overground part of each plant and the dry weight of the plant of the overground part of each plant under the same condition after 100 days of sowing, and calculating the average value of each group to respectively obtain the average nodule rate, the average number of nodules, the average nodule fresh weight, the average height of the plant of the overground part of each plant, the average fresh weight of the plant of the overground part of each plant, the weight of the fresh grass of the overground part of each plant and the average dry weight of the plant of the overground part of each plant.
TABLE 2 test results of the respective treatments
The results are shown in table 2 and indicate:
1. compared with the white clover in the non-inoculated control treatment area, the white clover in the 92-29 microbial inoculum treatment area has the advantages that the nodulation rate is increased by 1125 percent, the nodulation number is increased by 2800 percent, the root nodule weight is increased by 143.75 percent, the average height of each plant is increased by 96.15 percent, the average fresh weight of each plant is increased by 213.33 percent, the fresh grass weight is increased by 108.91 percent, and the dry grass weight is increased by 87.67 percent.
2. Compared with the white clover in the ACCC18019 microbial inoculum treatment area, the white clover in the 92-29 microbial inoculum treatment area has the advantages that the nodulation rate is increased by 30.67 percent, the nodulation number is increased by 112.20 percent, the root nodule weight is increased by 85.71 percent, the average height of each plant is increased by 37.84 percent, the average fresh weight of each plant is increased by 51.61 percent, the fresh grass weight is increased by 36.61 percent, and the hay weight is increased by 48.91 percent.
The results show that the rhizobium meliloti 92-29 has more remarkable growth promoting and yield increasing effects on the rhizobium meliloti in neutral subacid soil than the rhizobium meliloti ACCC 18019.
Thirdly, the root nodule bacteria 92-29 of clover promotes the growth of clover and improves the yield of the clover in the soil with pH4.8
Three treatments of no inoculation contrast treatment, 92-29 microbial inoculum treatment and ACCC18019 microbial inoculum treatment are set in the test.
Selecting Trifolium repens seeds with uniform size, soaking for 1 minute by concentrated sulfuric acid, and washing with sterile water for several times to remove acid to obtain treated seeds. The treated seeds were subjected to the following three treatments:
mixing the rhizobium meliloti 92-29 microbial inoculum prepared in the example 2 and the seeds of the clover leaves according to the proportion of 1: 10, and the obtained seeds are called 92-29-trifoliate repens seeds.
And (3) mixing the rhizobium meliloti ACCC18019 microbial inoculum prepared in the step one and the seeds of the clover leaves according to the ratio of 1: 10, and the obtained seeds are called ACCC 18019-trifoliate repens seeds.
Mixing the sterile grass peat and the white clover seeds according to the proportion of 1: 10, and the obtained seeds are called sterile grass carbon-white clover seeds.
The above-mentioned sterile grass peat-Trifolium repens seeds, 92-29-Trifolium repens seeds and ACCC 18019-Trifolium repens seeds were sown respectively in pots filled with the same soil (1.5 kg of soil in a 15 cm. times.18 cm pot, pH 4.80, from Qiyang county, Yongzhou city, Hunan province), and 30 seeds were sown per pot. The conditions were identical except for the different seeds sowed in each treatment. Wherein the seed sowed by the non-inoculation control treatment is aseptic grass carbon-white clover seed; the seeds sowed by the 92-29 microbial inoculum are 92-29-Trifolium repens seeds; the seeds sowed by the ACCC18019 microbial inoculum are ACCC 18019-Trifolium repens seeds. The experiment was repeated four times, each time with 4 pots of treatment. After sowing, placing the seeds in a greenhouse to be cultured under the same conditions until flowering, recording the underground part nodulation rate (short for nodulation rate), the height of the overground part of the plant, the fresh weight of the overground part (fresh weight of the plant) and the dry weight of the overground part (dry weight of the plant) of each treatment, calculating the average value of each group, and respectively obtaining the nodulation rate, the average height of the overground part of the plant (short for average height of the plant), the average fresh weight of the overground part of the plant (short for average fresh weight of the plant) and the average dry weight of the overground part of the plant (short for average dry weight of the plant).
TABLE 3 test results of the respective treatments
The results are shown in table 3, indicating that:
1. compared with the trifolium pratense treated by the non-inoculated control, the nodulation rate of the trifolium pratense treated by the 92-29 microbial inoculum is increased by 1550%, the average height of each plant is increased by 66.67%, the average fresh weight of each plant is increased by 121.43%, and the average dry weight of each plant is increased by 107.14%.
2. Compared with the white clover treated by ACCC18019, the white clover treated by 92-29 microbial inoculum has the advantages that the nodulation rate is increased by 88.57%, the average height of each plant is increased by 40.00%, the average fresh weight of each plant is increased by 55.00%, and the average dry weight of each plant is increased by 51.63%.
The results show that the rhizobium meliloti 92-29 has more remarkable growth promoting and yield increasing effects on the white clover than the rhizobium meliloti ACCC18019 in acid soil.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
<110> institute of agricultural resources and agricultural regionalism of Chinese academy of agricultural sciences
<120> method for promoting growth and/or increasing yield of clover and fungicide used therein
<130> GNCFH181205
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1341
<212> DNA
<213> Rhizobium trifolii (Rhizobium trifolii)
<400> 1
tgcctccttg cggttagcgc actaccttcg ggtaaaacca actcccatgg tgtgacgggc 60
ggtgtgtaca aggcccggga acgtattcac cgcggcatgc tgatccgcga ttactagcga 120
ttccaacttc atgcactcga gttgcagagt gcaatccgaa ctgagatggc ttttggagat 180
tagctcacac tcgcgtgctc gctgcccact gtcaccacca ttgtagcacg tgtgtagccc 240
agcccgtaag ggccatgagg acttgacgtc atccccacct tcctctcggc ttatcaccgg 300
cagtcccctt agagtgccca actgaatgct ggcaactaag ggcgagggtt gcgctcgttg 360
cgggacttaa cccaacatct cacgacacga gctgacgaca gccatgcagc acctgtgtcc 420
cggtccccga agggaacctt gcatctctgc aagtagccgg gcatgtcaag ggctggtaag 480
gttctgcgcg ttgcttcgaa ttaaaccaca tgctccaccg cttgtgcggg cccccgtcaa 540
ttcctttgag ttttaatctt gcgaccgtac tccccaggcg gaatgtttaa tgcgttagct 600
gcgccaccga acagtatact gcccgacggc taacattcat cgtttacggc gtggactacc 660
agggtatcta atcctgtttg ctccccacgc tttcgcacct cagcgtcagt aatggaccag 720
tgagccgcct tcgccactgg tgttcctccg aatatctacg aatttcacct ctacactcgg 780
aattccactc acctcttcca tactccagat cgacagtatc aaaggcagtt ccagggttga 840
gccctgggat ttcacccctg actgatcgat ccgcctacgt gcgctttacg cccagtaatt 900
ccgaacaacg ctagccccct tcgtattacc gcggctgctg gcacgaagtt agccggggct 960
tcttctccgg ataccgtcat tatcttctcc ggtgaaagag ctttacaacc ctagggcctt 1020
catcactcac gcggcatggc tggatcaggc ttgcgcccat tgtccaatat tccccactgc 1080
tgcctcccgt aggagtttgg gccgtgtctc agtcccaatg tggctgatca tcctctcaga 1140
ccagctatgg atcgtcgcct tggtaggcct ttaccccacc aactagctaa tccaacgcgg 1200
gctcatcctt gaccgataaa tctttctccc gaaggacaca tacggtatta gcacaagttt 1260
ccctgcgtta ttccgtagtc aagggtagat tcccacgcgt tactcacccg tctgccgctc 1320
cccttgcggg gcgctcgact g 1341
Claims (2)
1. A method for promoting the growth of clover in soil and/or increasing the yield of clover in soil includes such steps as mixing clover seeds with bacterial preparation, sowing them in soil, where the pH value of soil is 4.8, and the bacterial preparation contains rhizobium (A)Rhizobium trifolii) Or/and said Rhizobium meliloti: (Rhizobium trifolii) A metabolite of (a); root nodule bacteria of clover (A), (B)Rhizobium trifolii) The strain number of (1) is 92-29, and the preservation number of the strain in the China general microbiological culture Collection center is CGMCC No. 15533.
2. The method of claim 1, wherein: the promotion of the growth of the clovers is to improve the plant height, nodulation rate, the number of root nodules and/or the weight of the root nodules of the clovers, and the improvement of the yield of the clovers is to improve the yield of the single plant weight or unit area of the clovers.
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