CN106967652B - Rhizobium for promoting growth of common vetch and application of rhizobium - Google Patents
Rhizobium for promoting growth of common vetch and application of rhizobium Download PDFInfo
- Publication number
- CN106967652B CN106967652B CN201710354470.1A CN201710354470A CN106967652B CN 106967652 B CN106967652 B CN 106967652B CN 201710354470 A CN201710354470 A CN 201710354470A CN 106967652 B CN106967652 B CN 106967652B
- Authority
- CN
- China
- Prior art keywords
- rhizobium
- rhizobium leguminosarum
- common vetch
- qinghai
- leguminosarum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/41—Rhizobium
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- 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
-
- A—HUMAN NECESSITIES
- 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
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/20—Bacteria; Culture media therefor
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- General Engineering & Computer Science (AREA)
- Environmental Sciences (AREA)
- Agronomy & Crop Science (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Pest Control & Pesticides (AREA)
- Biomedical Technology (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses rhizobium for promoting growth of common vetch and application thereof. The Rhizobium leguminosarum is Rhizobium leguminosarum (Rhizobium leguminosarum), the strain number of the Rhizobium leguminosarum is JW1401, and the preservation number of the Rhizobium leguminosarum in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 11876. Experiments prove that the Rhizobium victoriae (Rhizobium leguminosarum) JW1401 CGMCC11876 and common vetch inoculation combination has a remarkable yield increasing effect on common vetch, has high practicability and popularization, and is expected to be developed into a new excellent combination technology in the field of common vetch Rhizobium application. The common vetch planting method has wide application prospect in common vetch planting industry.
Description
Technical Field
The invention relates to a rhizobium for promoting growth of vicia sativa in the field of agricultural microorganisms and application of the rhizobium.
Background
Common vetch (Vicia sativa L.) also known as big vetch, spring vetch, common vetch, wild pea, salvaged wild pea, etc.) is an annual or perennial bean family herbaceous plant. The common vetch is suitable for sandy soil and clay which are dry in climate, cold-resistant, warm and cool, and good in drainage, is suitable for the pH value of soil to be 6.5-8.5, and has wide adaptability. Is slightly worse than the sweet potato. Early hairFast growing, early maturing, high and stable seed yield, N0.64% and P as nutrients2O50.1 percent and K0.59 percent. Common vetch is green manure and good forage grass. It is distributed in grassland and mountain land in Jiangsu, Jiangxi, Taiwan, Shaanxi, Yunnan, Qinghai, Gansu province (region) of China, and it is grown in barren mountains with elevation of 50-3000 m, Tianbian grass clumps and forests.
Nitrogen is an essential element of all life, the conversion of inorganic nitrogen into organic nitride is one of the most important nutrient elements in plant growth, and in three major groups in the biological world, plants cannot directly utilize molecular nitrogen, while some microorganisms, namely nitrogen-fixing microorganisms, can utilize molecular nitrogen, and the action that living organisms convert molecular nitrogen in the atmosphere into ammonia is called biological nitrogen-fixing action. Biological nitrogen fixation in nature has two main forms, autogenous nitrogen fixation and symbiotic nitrogen fixation. Symbiotic nitrogen-fixing microorganisms must be matched with another organism under natural conditions to ensure nitrogen-fixing life. Biological nitrogen fixation is an important link of nitrogen element cycle conversion in geochemistry, and plays an important role in the nitrogen cycle process in the nature. Rhizobia is a kind of bacteria capable of infecting leguminous plant roots (few stems) to form root nodules for biological nitrogen fixation, and a symbiotic system of rhizobia and leguminous plants is a system with the strongest effect in biological nitrogen fixation. The nitrogen fixing capacity of the self-growing nitrogen fixing bacteria is much lower than that of the nodule bacteria for symbiotic nitrogen fixing, and is only one tenth of that of common nodule bacteria. The nitrogen fixing microorganisms in the soil convert nitrogen in the air into ammonia which can be absorbed by plants, and the fixed nitrogen is about 65 percent of the total biological nitrogen fixing amount. The soil fertility can be improved through the symbiotic nitrogen fixation effect of leguminous plants and rhizobia, and the inoculation of rhizobia can improve the yield and the nitrogen storage capacity of leguminous plants. 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. In the biological nitrogen fixation process, according to the specificity of rhizobia to host plants, plants suitable for nodulation and nitrogen fixation of the same rhizobia are classified as a group, and the rhizobia can be switched among the plants in the group, so that the method is called as an inter-inoculation ethnicity. The vetch and the pea are of an inter-inoculation ethnic group in the application of rhizobia, the pea rhizobia can be used for inoculating different vetch and pea varieties, and the difference of the inoculation effects is large due to the difference of the varieties.
The common vetch has many and early nodules, so rhizobia in seedling stage has certain nitrogen fixation capacity, and the nitrogen fixation activity is continuously improved along with the acceleration of vegetative growth. Nodules are generally formed when 2-3 true leaves exist, and most nodules are single nodules during seedling development; the change of the quantity and the quality of the common vetch root nodule generally forms positive correlation with the nitrogen fixation activity of the root nodule; the nitrogen fixation amount in the vegetative growth stage accounts for more than 95% of the whole growth period. The nitrogen fixation activity of the root nodule in the striking period of autumn sowing in the south and the stretching period of spring sowing in the north. After the flowering period, the root nodules of most common vetch varieties die naturally, and the nitrogen fixation activity is weak. Common vetch as a green manure crop is also a leguminous crop, and the research and application of legume rhizobia have been in the world for over one hundred years, and rhizobia inoculation to leguminous crops is promoted almost all over the world. At present, inoculation of common vetch rhizobia is still an important technical measure for improving the yield of fresh grass. Nearly ten years, the common vetch planting area of china constantly enlarges, and the research and the application of the common vetch inoculation rhizobium technique that attention is turned up in all places to the cultivation and the utilization of common vetch are also constantly improved in all places.
Disclosure of Invention
The invention aims to solve the technical problem of how to obviously promote the growth of common vetch.
In order to solve the technical problems, the invention provides a rhizobium strain.
The Rhizobium strain for inoculating common vetch provided by the invention is (Rhizobium leguminosarum), the strain number of the Rhizobium strain is JW1401, the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms and management (CCM) 12-14 th month 2015, the preservation number is CGMCC No.11876, and the strain is called as pea Rhizobium JW1401 for short.
Rhizobium leguminosarum (Rhizobium leguminatum) JW1401 CGMCC11876 belongs to gram-negative stain, small short rod shape, no spore, terminal flagella or periphytic flagella, and can move. The size of the cells is (0.5-0.9) × (1.0-3.0) microns, and the forms of the cells growing in different environments are different. Under the culture conditions, the bacteria are in small short rod shape, the bacterial body is not uniformly stained, a ring body with colored and non-colored parts is formed, and the lipid content of the non-colored parts is high. The bacterial colony grows on the plane of a yeast juice mannitol agar culture medium, is round, has neat edges, is slightly protuberant, is colorless and semitransparent or light milky white, and has thick bacterial lawn.
In order to solve the technical problems, the invention further provides a microbial inoculum for promoting the growth of common vetch.
The microbial inoculum for promoting the growth of common vetch provided by the invention contains metabolites of rhizobium japonicum JW1401 or/and rhizobium japonicum JW 1401.
Among the above-mentioned microbial inoculum, the microbial inoculum is the microbial inoculum that improves common vetch output and/or improve common vetch plant height.
The active ingredients of the microbial inoculum can be rhizobium japonicum JW1401 or/and metabolites of rhizobium japonicum JW1401, the active ingredients of the microbial inoculum can also contain other biological ingredients or non-biological ingredients, and other active ingredients of the microbial inoculum can be determined by a person skilled in the art according to the promoting effect of the microbial inoculum on the output and/or plant height of common vetch.
The microbial inoculum may also include a carrier. 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, the rhizobium leguminosarum JW1401 or/and the metabolites of rhizobium leguminosarum JW1401 can be present in the form of cultured living cells, a fermentation broth of the living cells, a filtrate of a cell culture or a 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.
Wherein the metabolite of the rhizobium leguminosarum JW1401 is obtained by culturing the rhizobium leguminosarum JW1401 in a microbial liquid fermentation culture medium.
According to the requirement, the microbial inoculum can also be added with 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.
The biological organic fertilizer containing the microbial inoculum also belongs to the protection scope of the invention.
The application of the rhizobium peas JW1401 or the microbial agent in preparing a product (such as a biological fertilizer) for promoting the growth of common vetch and the application of the rhizobium peas JW1401 or the microbial agent or the biological organic fertilizer in promoting the growth of common vetch also belong to the protection scope of the invention.
In above-mentioned application, promote vicia sativa to grow for improving vicia sativa output and/or improving vicia sativa plant height.
In the above, the variety of common vetch may be Qinghai white arrow or Qinghai grey arrow.
In order to solve the technical problems, the invention also provides a method for culturing the rhizobium leguminosarum JW 1401.
The method for culturing the rhizobium leguminosarum JW1401 provided by the invention comprises the step of culturing the rhizobium leguminosarum JW1401 in a culture medium for culturing rhizobium.
The culture medium for culturing rhizobia can be prepared according to the following method: 3-5ml of glycerol, 2-5g of mannitol, 0.8-1g of yeast extract powder and K2HPO40.5-1g, anhydrous MgSO4 0.1-0.2g，CaSO4·2H2O 0.1-0.2g，NaCl 0.1-0.2g，1％(NH4)6Mo7O24·4H2O 1-1.5ml、1％H3BO31 to 1.5ml of agar and 20g of agar, adding water to a constant volume of 1000ml, adjusting the pH value to 6.8 to 7.0, and sterilizing to obtain the culture medium for culturing rhizobia.
The invention provides a Rhizobium leguminosum JW1401 CGMCC11876 for promoting growth of common vetch, and a microbial inoculum taking the Rhizobium leguminosum as an active ingredient of the Rhizobium jW1401 CGMCC 11876. The water culture test of the common vetch inoculation combined effect proves that after the common vetch is inoculated with the strain, the height, the fresh weight and the dry weight of the common vetch are respectively increased by 8.62%, 32.59% and 56.67% on average compared with a control plant without the strain; in a plot field test, compared with the green sea white arrow which is not inoculated with the rhizobium leguminosarum JW1401 microbial inoculum and is treated by contrast, the plant height of the green sea white arrow is increased by 6.26%, and the plot yield is increased by 62.62%; compared with the green gram gray arrow which is not inoculated with the rhizobium leguminosarum JW1401 microbial inoculum and treated, the plant height of the green gram gray arrow is increased by 10.11 percent, and the yield of a cell is increased by 66.77 percent. The tests prove that the Rhizobium japonicum (Rhizobium leguminosarum) JW1401 CGMCC11876 and common vetch inoculation combination has a remarkable yield increasing effect on common vetch, has high practicability and popularization, and is expected to be developed into a new excellent combination technology in the common vetch Rhizobium application field. The common vetch planting method has wide application prospect in common vetch planting industry.
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 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.
Pea rhizobia (Rhizobium leguminosarum) ACCC16505 in the following examples was collected at the agricultural microbial center of the china microbial cultures collection management committee (abbreviated as ACCC, address: southern avenue 12 of guancun in the hail lake area of beijing, institute of agricultural resources and agricultural compartmentalization, japan, zip code 100081) at 4.1.1990, and the strain was publicly available from the agricultural microbial centers of the chinese microbial cultures collection management committee since the date of collection. Rhizobium leguminosarum ACCC16505 is hereinafter referred to as Rhizobium leguminosarum ACCC 16505.
The preparation method of the solid medium in the following examples is as follows: glycerol 5ml, mannitol5g, yeast extract powder 1g, K2HPO40.5g, anhydrous MgSO4 0.2g，CaSO4·2H2O 0.2g，NaCl 0.1g，1％(NH4)6Mo7O24·4H2O 1ml、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)6Mo7O24·4H2O 1ml、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.
Example 1 isolation and characterization of Rhizobium leguminosarum JW1401 CGMCC11876
1. Isolation of the Strain
A wild common vetch root nodule sample is collected from the autonomous county and the safety area of the soil family of the mutual aid city of Shandong, Hailai, Qinghai, and is subjected to streak separation by using a flat plate (10 g of mannitol, 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 and 20g of agar, wherein the water volume is fixed to 1L, and the pH value is 6.8-7.0) to obtain a strain JW 1401.
2. Identification of strains
2.1 morphological identification
And (3) performing single colony state description on the strain JW1401 which is in a logarithmic growth phase and has stable colony size and is separated and purified in the step 1, wherein the single colony state description mainly comprises the size, color, transparency, wettability, colony surface state and colony edge state of a colony. On the other hand, strain JW1401 at the logarithmic growth phase was stained by smear, and the form of the cells was observed by an optical microscope.
The results showed that the strain JW1401 is gram-negative, small, short, rod-shaped, spore-free, possessing terminal or perigenic flagella, and capable of locomotion. The size of the cells is (0.5-0.9) × (1.0-3.0) microns, and the forms of the cells growing in different environments are different. Under the culture conditions, the bacteria are in small short rod shape, the bacterial body is not uniformly stained, a ring body with colored and non-colored parts is formed, and the lipid content of the non-colored parts is high. The bacterial colony grows on the plane of a yeast juice mannitol agar culture medium, is round, has neat edges, is slightly protuberant, is colorless and semitransparent or light milky white, and has thick bacterial lawn.
2.2, 16S rDNA sequence homology analysis
The 16S rDNA fragment of the strain JW1401 obtained in step 1 was amplified by colony PCR, and the reagents were provided by the same company as the whole gold company. The results of amplifying and cloning and sequencing the 16S rRNA gene fragment show that the 16SrDNA of the strain JW1401 has a nucleotide sequence of a sequence 1 in a sequence table. The 16S rDNA of strain JW1401 has a similarity of up to 100% to the 16S ribosomal RNA gene partial sequence (sequence ID: DQ835299.2) of Rhizobium leguminosarum (Rhizobium leguminatum bv. vitamin) CCBAU 75042.
2.3, physiological and biochemical characterization
The physiological and biochemical characteristics of strain JW1401 were determined with reference to "Manual of identification of common bacterial systems" (Dongxu bead, Chuia Miaoying. Manual of identification of common bacterial systems.: Beijing: scientific Press, 2011.) and "microbiological experiments" (Shen, Fangxiong, Liguanwu. microbiological experiments (third edition); Beijing: advanced education Press, 1999.). The results show that the strain JW1401 is chemoheterotrophic, can utilize various carbohydrates and salts of organic acids as carbon sources, such as glucose, lactose, D-ribose, D-cellobiose, D-arabinose, mannitol, xylose, D-galactose, fructose, dulcitol and inositol; ammonium salts, nitrates and most amino acids can be used as nitrogen sources; slightly absorbing color by congo red color absorption reaction; the litmus milk does not coagulate and produce acid in reaction; cellulose and starch cannot be utilized; the casein cannot be hydrolyzed; 3-ketolactose is not utilized; producing acid from mannitol; growth on sugar-containing media is often accompanied by abundant extracellular mucus; no hydrogen sulfide is produced; no precipitation was produced in calcium glycerophosphate medium.
In view of the above results of morphological analysis, physiological and biochemical characteristics analysis and 16s rDNA sequence homology analysis, the strain JW1401 isolated and purified in step 1 was identified as Rhizobium leguminosarum. The Rhizobium leguminosarum JW1401 has been preserved in the general microorganism center of China Committee for culture Collection of microorganisms (CGMCC, address: No. 3 of No.1 Hospital of Xilu north Chen of the Korean district in Beijing) within 12 months and 14 days of 2015, and the preservation number is CGMCC No.11876, which is hereinafter referred to as Rhizobium leguminosarum JW 1401.
Example 2 preparation of Rhizobium leguminosarum JW1401 microbial inoculum
1. Slant culture of Rhizobium leguminosarum JW1401
The Rhizobium leguminosarum JW1401 obtained in example 1 was inoculated into a solid medium and subjected to slant culture at 28 ℃ for 56 hours to obtain the Rhizobium leguminosarum JW 1401.
2. Activation of bacterial species
And (3) selecting the rhizobium leguminosarum JW1401 cultured on the slant in the step (1), inoculating the rhizobium leguminosarum JW1401 into 500mL of liquid culture medium, and culturing for 48 hours at 28 ℃ to obtain a rhizobium leguminosarum JW1401 bacterial liquid.
3. Seeding tank culture
Taking 3L of the rhizobium leguminosarum JW1401 bacterial solution obtained in the step 2, inoculating the rhizobium leguminosarum JW1401 bacterial solution into a 100L seed tank (containing 60L of liquid culture medium) for seed culture, and performing shaking culture at 30 ℃ and 120rpm for 56 hours to obtain the rhizobium leguminosarum JW1401 seed solution.
4. Cultivation in fermenter
Inoculating the rhizobium leguminosarum JW1401 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% for fermentation culture, and performing shaking culture at 28 ℃ and 150rpm for 56 hours to obtain rhizobium leguminosarum JW1401 fermentation liquor, wherein the content of rhizobium leguminosarum JW1401 in the rhizobium leguminosarum JW1401 fermentation liquor is 30 hundred million cfu/mL.
5. Preparation of rhizobium leguminosarum JW1401 microbial inoculum
Naturally drying the grass peat, then crushing, sieving with a 100-mesh sieve, then adjusting the pH value to 6.8 with lime water, and sterilizing at 121 ℃ for 60min to obtain the sterile grass peat.
Mixing the rhizobium leguminosarum JW1401 fermentation liquor obtained in the step 4 with the sterile grass peatUniformly mixing, carrying out propagation culture for 48h at 28 ℃ to obtain the rhizobium leguminosarum JW1401 microbial inoculum, and subpackaging and warehousing. The finished product is qualified through detection, and the content of the rhizobium leguminosarum JW1401 in the rhizobium leguminosarum JW1401 microbial inoculum is 2.0 multiplied by 108cfu/g.
Comparative example 1 isolation and identification of Rhizobium leguminosarum (Rhizobium leguminatum) H10 CGMCC No.11878
1. Isolation of the Strain
Wild pea nodules are collected from Lo city county, Qinghai province, and are streaked and separated by using a flat plate (10 g of mannitol, 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 congo red with the mass content of 0.5 percent and 20g of agar, the volume is determined to be 1L by water, and the pH value is 6.8-7.0) to obtain a strain H10.
2. Identification of strains
2.1 morphological identification
And (3) performing single colony state description on the strain H10 which is in a logarithmic growth phase and has stable colony size and is separated and purified in the step 1, wherein the single colony state description mainly comprises the size, color, transparency, wettability, colony surface state and colony edge state of the colony. On the other hand, the strain H10 in the logarithmic growth phase was stained by smear, and the morphology of the cells was observed by an optical microscope.
The result shows that the strain H10 belongs to gram-negative stain, is in a small short rod shape, has the thallus size of 0.5-0.9 micrometer multiplied by 1.2-6.0 micrometers, and has different thallus forms growing in different environments. Typically containing granules of poly-beta-hydroxybutyrate. Has no spore, terminal flagellum or perigenic flagellum, and is aerobic. The optimum growth temperature is 25-30 ℃, and the optimum pH value is 6.0-7.0. The bacterial colony is round, neat in edge, slightly convex, colorless, translucent or light milky white, and viscous. Growing on a yeast juice mannitol inorganic salt agar culture medium plate for 3-6 days, and then, the diameter is 2-4 mm. Growths on sugar-containing media are often accompanied by abundant extracellular mucus.
2.2, 16S rDNA sequence homology analysis
The 16S rDNA fragment of the strain H10 obtained in step 1 was amplified by colony PCR, and the reagents were provided by Takara Shuzo Co. The results of amplifying and cloning and sequencing the 16S rRNA gene fragment show that the 16S rDNA of the strain H10 has a nucleotide sequence of a sequence 2 in a sequence table. The 16S rRNA of strain H10 has a similarity of up to 99% to the 16S rRNA gene of Rhizobium leguminosum strain INTA D156 (Sequence ID: KX 066064.1).
2.3, physiological and biochemical characterization
The physiological and biochemical characteristics of strain H10 were determined with reference to "Manual of identification of common bacterial systems" (Dongxu bead, Chuia Miaoying. Manual of identification of common bacterial systems. Beijing: scientific Press, 2011) and "microbiological experiments" (Shenping, Fangxiong, Liguanwu. microbiological experiments (third edition); Beijing: advanced education Press, 1999.). The result shows that the strain H10 is chemoheterotrophic, can utilize various carbohydrates and salts of organic acids as carbon sources, such as glucose, lactose, D-ribose, D-cellobiose, D-arabinose, mannitol, xylose, D-galactose, fructose, dulcitol and inositol; ammonium salt, nitrate and most amino acids can be used as nitrogen sources; slightly absorbing color by congo red color absorption reaction; the litmus milk does not coagulate and produce acid in reaction; cellulose and starch cannot be utilized; the casein cannot be hydrolyzed; 3-ketolactose is not utilized; producing acid from mannitol; growth on sugar-containing media is often accompanied by abundant extracellular mucus; no hydrogen sulfide is produced; no precipitation was produced in calcium glycerophosphate medium.
The strain H10 isolated and purified in step 1 was identified as Rhizobium leguminosarum (Rhizobium leguminatum) in view of the above results of morphological, physiological and biochemical characteristic analyses and 16s rDNA sequence homology analyses. The Rhizobium leguminosarum H10 has been deposited in the general microorganism center of China Committee for culture Collection of microorganisms (CGMCC, address: No. 3 of Xilu No.1 of Beijing area north Chenyang) within 12 months and 14 days of 2015, and the deposit number is CGMCC No.11878, hereinafter referred to as Rhizobium leguminosarum H10. Rhizobium leguminosarum H10 was filed by the applicant of the present application as a chinese patent application at 28/11/2016, application No. 201611072530.2.
Comparative example 2 preparation of Rhizobium leguminosarum H10 microbial inoculum
1. Slant culture of Rhizobium leguminosarum H10
The rhizobium leguminosarum H10 of comparative example 1 was picked up and inoculated into a solid medium for slant culture, and cultured at 28 ℃ for 56 hours to obtain slant-cultured rhizobium leguminosarum H10.
2. Activation of bacterial species
And (3) selecting the pea rhizobia H10 cultured on the slant in the step (1), inoculating the pea rhizobia H10 into 500mL of liquid culture medium, and culturing for 72 hours at 28 ℃ to obtain a pea rhizobia H10 bacterial liquid.
3. Seeding tank culture
Taking 3L of the rhizobium leguminosarum H10 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 performing shaking culture at 30 ℃ and 120rpm for 60 hours to obtain a rhizobium leguminosarum H10 seed liquid.
4. Cultivation in fermenter
Inoculating the rhizobium leguminosarum H10 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% for fermentation culture, and performing shaking culture at 28 ℃ and 150rpm for 60 hours to obtain rhizobium leguminosarum H10 fermentation liquor, wherein the content of rhizobium leguminosarum H10 in the rhizobium leguminosarum H10 fermentation liquor is 30 hundred million cfu/mL.
5. Preparation of rhizobium leguminosarum H10 microbial inoculum
Naturally drying the grass peat, then crushing, sieving with a 100-mesh sieve, then adjusting the pH value to 6.8 with lime water, and sterilizing at 121 ℃ for 60min to obtain the sterile grass peat.
And (4) uniformly mixing the pea rhizobium H10 fermentation liquor obtained in the step (4) with the sterile grass peat, performing propagation culture for 48 hours at the temperature of 28 ℃ to obtain a pea rhizobium H10 microbial inoculum, and subpackaging and warehousing. The finished product is qualified through detection, and the content of the rhizobium leguminosarum H10 in the rhizobium leguminosarum H10 microbial inoculum is 2.0 multiplied by 108cfu/g.
Comparative example 3 preparation of Rhizobium leguminosarum ACCC16505 microbial inoculum
1. Slant culture of Rhizobium leguminosarum ACCC16505
And (3) selecting the rhizobium leguminosarum ACCC16505 to inoculate into a solid culture medium for slant culture, and culturing for 56 hours at 28 ℃ to obtain the rhizobium leguminosarum ACCC 16505.
2. Activation of bacterial species
And (3) selecting the rhizobium leguminosarum ACCC16505 cultured by the slant in the step (1), inoculating the rhizobium leguminosarum ACCC16505 into 500mL of liquid culture medium, and culturing for 72 hours at 28 ℃ to obtain a rhizobium leguminosarum ACCC16505 bacterial liquid.
3. Seeding tank culture
And (3) taking 3L of the rhizobium leguminosarum ACCC16505 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 performing shaking culture at 30 ℃ and 120rpm for 60 hours to obtain a rhizobium leguminosarum ACCC16505 seed liquid.
4. Cultivation in fermenter
And (3) inoculating the rhizobium leguminosarum ACCC16505 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% for fermentation culture, and performing shaking culture at 28 ℃ and 150rpm for 60 hours to obtain a rhizobium leguminosarum ACCC16505 fermentation liquid, wherein the content of the rhizobium leguminosarum ACCC16505 in the rhizobium leguminosarum ACCC16505 fermentation liquid is 30 hundred million cfu/mL.
5. Preparation of rhizobium leguminosarum ACCC16505 microbial inoculum
Naturally drying the grass peat, then crushing, sieving with a 100-mesh sieve, then adjusting the pH value to 6.8 with lime water, and sterilizing at 121 ℃ for 60min to obtain the sterile grass peat.
And (4) uniformly mixing the rhizobium leguminosarum ACCC16505 fermentation liquor obtained in the step (4) with the sterile grass carbon, performing propagation culture at the temperature of 28 ℃ for 48 hours to obtain a rhizobium leguminosarum ACCC16505 microbial inoculum, and subpackaging and warehousing. The finished product is qualified through detection, and the content of the rhizobium leguminosarum ACCC16505 in the rhizobium leguminosarum ACCC16505 microbial inoculum is 2.0 multiplied by 108cfu/g.
Comparative example 4 isolation and identification of Rhizobium leguminosarum (Rhizobium leguminatum) m1-10-3 CGMCC11877
1. Isolation of the Strain
Collecting wild vetch root nodules from the safety area of Shandong city in Haidong, Qinghai, and carrying out streak separation by using a flat plate (taking 10g of mannitol, 1g of yeast extract powder, 0.5g of dipotassium 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 and 20g of agar, and using water to fix the volume to 1L, wherein the pH value is 6.8-7.0) to obtain a strain m 1-10-3.
2. Identification of strains
2.1 morphological identification
And (3) performing single colony state description on the strain m1-10-3 which is in a logarithmic growth phase and has stable colony size and is separated and purified in the step 1, wherein the single colony state description mainly comprises the size, color, transparency, wettability, colony surface state and colony edge state of the colony. On the other hand, the strain m1-10-3 in the logarithmic growth phase was stained by smear and then the morphology of the cells was observed by an optical microscope.
The results show that the strain m1-10-3 belongs to gram-negative stain, short and short rod-shaped strain, the bacterial stain is not uniform, a ring body of colored and non-colored parts is formed, and the lipid content of the non-colored part is higher. Has no spore, terminal flagella or perigenic flagella, and can move. The cell size is (0.5-0.8) × (1.1-2.9) μm. The bacterial colony grows on the plane of a yeast juice mannitol agar culture medium, is round and convex, has neat edges, smooth moist surface, uniform texture, light milky white color and thick lawn.
2.2, 16S rDNA sequence homology analysis
The 16S rDNA fragment of the strain m1-10-3 obtained in step 1 was amplified by colony PCR, and the reagents were supplied by Takara Shuzo Co. The results of amplifying and cloning sequencing the 16S rRNA gene fragment show that the 16SrDNA of the strain m1-10-3 has a nucleotide sequence of a sequence 2 in a sequence table. The similarity of the 16S rRNA gene fragment of strain m1-10-3 to the partial Sequence (Sequence ID: EU256422.1) of the 16S rRNA gene of Rhizobium leguminosarum strain CCBAU 85022 was up to 99%.
2.3, physiological and biochemical characterization
The physiological and biochemical characteristics of the strain m1-10-3 were determined with reference to "handbook of identification of common bacterial System (Dongxu bead, Chuia Miaoying. handbook of identification of common bacterial System", Beijing: scientific Press, 2011 ") and" experiments in microbiology "(Shenping, Fangxiu, Liguanwu. microbiological experiments (third edition); Beijing: advanced education Press, 1999.). The result shows that the strain m1-10-3 is chemoheterotrophic, can utilize various carbohydrates and salts of organic acids as carbon sources, such as glucose, lactose, D-ribose, D-cellobiose, D-arabinose, mannitol, xylose, D-galactose, fructose, dulcitol and inositol; ammonium salts, nitrates and most amino acids can be used as nitrogen sources; slightly absorbing color by congo red color absorption reaction; the litmus milk does not coagulate and produce acid in reaction; cellulose and starch cannot be utilized; the casein cannot be hydrolyzed; 3-ketolactose is not utilized; producing acid from mannitol; growth on sugar-containing media is often accompanied by abundant extracellular mucus; no hydrogen sulfide is produced; no precipitation was produced in calcium glycerophosphate medium.
In view of the above results of morphological analysis, physiological and biochemical characteristic analysis and 16s rDNA sequence homology analysis, the strain m1-10-3 isolated and purified in step 1 was identified as Rhizobium leguminosarum. The Rhizobium leguminosarum (Rhizobium leguminatum) m1-10-3 has been deposited in the general microorganism center of China Committee for culture Collection of microorganisms (CGMCC, address: No. 3 of Xilu 1. North Chen of the south-facing area of Beijing city) within 12 months and 14 days of 2015, and the preservation number is CGMCC No.11877, which is hereinafter referred to as "Rhizobium leguminatum" m 1-10-3.
Comparative example 5 preparation of Rhizobium leguminosarum m1-10-3 microbial inoculum
1. Slant culture of pea rhizobium m1-10-3
The rhizobium leguminosarum m1-10-3 of example 1 was selected and inoculated into a solid medium for slant culture, and cultured at 28 ℃ for 56 hours to obtain slant-cultured rhizobium leguminosarum m 1-10-3.
2. Activation of bacterial species
And (3) selecting the pea rhizobia m1-10-3 cultured on the slant in the step (1), inoculating the pea rhizobia m1-10-3 into 500mL of liquid culture medium, and culturing for 48 hours at 28 ℃ to obtain a pea rhizobia m1-10-3 bacterial liquid.
3. Seeding tank culture
Taking 3L of rhizobium leguminosarum m1-10-3 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 carrying out shake culture at 30 ℃ and 120rpm for 56 hours to obtain the rhizobium leguminosarum m1-10-3 seed liquid.
4. Cultivation in fermenter
Inoculating the rhizobium leguminosarum m1-10-3 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% for fermentation culture, and performing shaking culture at 28 ℃ and 150rpm for 56 hours to obtain rhizobium leguminosarum m1-10-3 fermentation liquor, wherein the content of rhizobium leguminosarum m1-10-3 in the rhizobium leguminosarum m1-10-3 fermentation liquor is 30 hundred million cfu/mL.
5. Preparation of pea rhizobium m1-10-3 microbial inoculum
Naturally drying the grass peat, then crushing, sieving with a 100-mesh sieve, then adjusting the pH value to 6.8 with lime water, and sterilizing at 121 ℃ for 60min to obtain the sterile grass peat.
And (4) uniformly mixing the rhizobium leguminosarum m1-10-3 fermentation liquor obtained in the step (4) with the sterile grass carbon, performing propagation culture at the temperature of 28 ℃ for 48 hours to obtain a rhizobium leguminosarum m1-10-3 microbial inoculum, and subpackaging and warehousing. The finished product is qualified through detection, and the content of the rhizobium leguminosarum m1-10-3 in the rhizobium leguminosarum m1-10-3 microbial inoculum is 2.0 multiplied by 108cfu/g.
Example 3 hydroponic test of inoculation of Rhizobium Pisi with JW1401 inoculum
The common vetch adopts a filter paper bridge test tube hydroponic method. Cutting the filter paper into strips, wherein the widths of the filter paper are 1.6cm respectively, making the filter paper into an M shape, making a V-shaped small hole in the middle concave part according to the size of a seedling, and subtracting 4cm from the length of a test tube by the height of the M-shaped filter paper. The test tube used was 2.0 cm. times.20 cm.
Respectively filling the prepared nitrogen-free nutrient solution into test tubes with filter paper supports, wherein the height of the nitrogen-free nutrient solution is positioned in the concave part of the M-shaped filter paper, repeating 4 test tubes for each treatment, arranging 4 reference tubes, sealing the test tubes with a high-temperature and high-pressure resistant plastic film after filling, and sterilizing the test tubes for 1 hour at 121 ℃ for later use.
Selecting seeds of Qinghai Ash arrow of the variety common vetch with uniform size, soaking in 95% ethanol for 5min, and then soaking in 0.1% HgCl2Sterilizing the surface for 5min, and washing with sterile water for 10 times.
And (3) placing the sterilized seeds in an incubator at 28-30 ℃ for heat preservation and germination acceleration. The illumination is required to be irradiated from the top, the root is protected from light, and the greenhouse or the illumination box can be placed, wherein the illumination intensity is about 7000lux to 8000 lux; temperature: 20 to 30 ℃; the germination length is 0.8-1.5 cm.
The experimental set was an inoculation-free control treatment and a rhizobium leguminosarum JW1401 treatment. The experiment is designed to be repeated for three times, and the experimental method of each repetition is as follows:
rhizobium leguminosarum JW1401 treatment: 10 seeds successfully germinated were placed in bacterial suspension (the fermentation broth of Rhizobium leguminosarum JW1401 from step 4 of example 2 was diluted with a liquid medium to a content of 2.0X 10 of Rhizobium leguminosarum JW14017cfu/mL to obtain bacterial suspension), carefully clamping out the seeds with sterile forceps, placing the seeds in a filter paper bridge of a water culture solution test tube, fixing, sowing, and evenly sucking the bacterial suspension into the test tube. After sealing, culturing in an illumination box by illumination, wherein the illumination is performed from the top for 14h/10h, the roots are protected from light, and the illumination intensity is 7000 lux-8000 lux; temperature: 25-30 ℃.
Inoculating-free control treatment: the only difference from the treatment with Rhizobium leguminosarum JW1401 was that the bacterial suspension was replaced with liquid medium and the other operations were exactly the same.
TABLE 1 hydroponic test results of vetch inoculation with Rhizobium JW1401
The results are shown in table 1, after vetch seeds are inoculated with vetch rhizobia (Rhizobium leguminosa) JW1401 CGMCC11876, the plant height, the plant fresh weight and the plant dry weight are respectively increased by 8.62%, 32.59% and 56.67% on average compared with the control plant without inoculation, which shows that the vetch rhizobia (Rhizobium leguminosa) JW1401 CGMCC11876 disclosed by the invention has a remarkable yield increase effect on vetch.
Example 4 comparative test of Effect of Rhizobium Pisi JW1401 inoculation with Rhizobium Pisi H10, Rhizobium Pisi m1-10-3, and Rhizobium Pisi ACCC16505 with variety of common vetch
Two common vetch varieties of the common vetch variety of the Qinghai white arrow and the common vetch variety of the Qinghai grey arrow are selected to be subjected to growth promotion experiments.
The test adopts a random block design, 10 treatment areas are randomly arranged, and each treatment area is provided with three cell repetitions. The 10 processing areas are two pea rhizobia JW1401 microbial inoculum processing areas, namely a JW 1401-Qinghai white arrow processing area and a JW 1401-Qinghai grey arrow processing area, two pea rhizobia m1-10-3 microbial inoculum processing areas, namely an m 1-10-3-Qinghai white arrow processing area and an m 1-10-3-Qinghai grey arrow processing area, two pea rhizobia H10 microbial inoculum processing areas, namely an H10-Qinghai white arrow processing area and an H10-Qinghai grey arrow processing area, two pea rhizobia ACCC16505 microbial inoculum processing areas, namely an ACCC 16505-Qinghai white arrow processing area and an ACCC 16505-Qinghai grey arrow processing area, and two non-inoculation contrast processing areas, namely a contrast-Qinghai white arrow processing area and a contrast-Qinghai grey arrow processing area.
Each small area is 4 square meters, the soil pH8.3, the average number of nodule bacteria in soil is 2.0X 103cfu/g dry soil. According to the sowing quantity of the vetch seeds of 7.5kg/hm2Seeds are prepared.
The JW 1401-Qinghai white arrow processing area and the JW 1401-Qinghai grey arrow processing area are respectively sown with Qinghai white arrow seeds and Qinghai grey arrow seeds (seeds obtained by respectively mixing and uniformly stirring the pea rhizobium JW1401 microbial inoculum of the embodiment 2 with the Qinghai white arrow seeds and the Qinghai grey arrow seeds according to the mass ratio of 1: 10 are respectively called as JW 1401-Qinghai white arrow seeds and JW 1401-Qinghai grey arrow seeds.
m 1-10-3-Qinghai white arrow processing area and m 1-10-3-Qinghai grey arrow processing area are respectively sown with Qinghai white arrow seeds and Qinghai grey arrow seeds (seeds obtained by respectively mixing and uniformly stirring the pea rhizobia m1-10-3 microbial inoculum of the comparative example 5, the Qinghai white arrow seeds and the Qinghai grey arrow seeds according to the mass ratio of 1: 10 are respectively called m 1-10-3-Qinghai white arrow seeds and m 1-10-3-Qinghai grey arrow seeds.
H10-Qinghai white arrow processing area and H10-Qinghai grey arrow processing area are respectively sown with Qinghai white arrow seeds and Qinghai grey arrow seeds (seeds obtained by respectively mixing and uniformly stirring the pea rhizobium H10 microbial inoculum of the comparative example 2 with the Qinghai white arrow seeds and the Qinghai grey arrow seeds according to the mass ratio of 1: 10 are respectively called H10-Qinghai white arrow seeds and H10-Qinghai grey arrow seeds.
The ACCC 16505-Qinghai white arrow processing area and the ACCC 16505-Qinghai grey arrow processing area are respectively sown with Qinghai white arrow seeds and Qinghai grey arrow seeds (seeds obtained by respectively mixing and uniformly stirring the pea rhizobium ACCC16505 microbial inoculum of the comparative example 3 with the Qinghai white arrow seeds and the Qinghai grey arrow seeds according to the mass ratio of 1: 10 are respectively called ACCC 16505-Qinghai white arrow seeds and ACCC 16505-Qinghai grey arrow seeds.
The control-Qinghai white arrow processing area and the control-Qinghai grey arrow processing area are respectively sown with control-Qinghai white arrow seeds and control-Qinghai grey arrow seeds (the seeds obtained by respectively mixing and uniformly stirring the sterile grass carbon in the example 2 with the Qinghai white arrow seeds and the Qinghai grey arrow seeds according to the mass ratio of 1: 10 are respectively called as control-Qinghai white arrow seeds and control-Qinghai grey arrow seeds).
The seeds are uniformly sown into 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. And culturing under the same conditions to the flowering period after sowing, recording the height (plant height) of the overground part of the plant, the number of the nodules and the dry weight of the overground part of each cell, and calculating the average value of each treatment area to respectively obtain the average height of the plant, the average number of the nodules of the plant and the yield of the cell.
TABLE 2 Combined field plot effect test data for vetch inoculation
Note: cell yield is on a dry weight basis.
Table 3 shows the growth promotion effect of the rhizobium leguminosarum JW1401 microbial inoculum, the rhizobium leguminosarum m1-10-3 microbial inoculum, the rhizobium leguminosarum H10 microbial inoculum and the rhizobium leguminosarum ACCC16505 microbial inoculum
Note: cell yield is on a dry weight basis. Note: JW1401 is a rhizobium leguminosarum JW1401 microbial inoculum treatment area, m1-10-3 is a rhizobium leguminosarum m1-10-3 microbial inoculum treatment area, H10 is a rhizobium leguminosarum H10 microbial inoculum treatment area, ACCC16505 is a rhizobium leguminosarum ACCC16505 microbial inoculum treatment area, and the control is a non-inoculated bacteria control treatment area.
The results are shown in tables 2 and 3, indicating that:
1. compared with the green sea white arrow which is not inoculated with the rhizobium leguminosarum JW1401 microbial inoculum and treated, the plant height of the green sea white arrow is increased by 6.26 percent, and the cell yield is increased by 62.62 percent; compared with the green gram gray arrow which is not inoculated with the rhizobium leguminosarum JW1401 microbial inoculum and treated, the plant height of the green gram gray arrow is increased by 10.11 percent, and the yield of a cell is increased by 66.77 percent;
2. compared with the green sea white arrow treated by the rhizobium leguminosarum JW1401 microbial inoculum, the plant height of the green sea white arrow treated by the rhizobium leguminosarum JW1401 microbial inoculum is increased by 6.33%, and the cell yield is increased by 71.08%; compared with the green sea ash arrow treated by the rhizobium leguminosarum JW1401 microbial inoculum, the plant height of the green sea ash arrow treated by the rhizobium leguminosarum JW1401 microbial inoculum is increased by 20.64%, and the cell yield is increased by 80.59%;
3. compared with the green sea white arrow treated by the pea rhizobia JW1401 microbial inoculum, the plant height of the green sea white arrow treated by the pea rhizobia JW1401 microbial inoculum is increased by 18.61%, and the cell yield is increased by 60.39%;
4. compared with the green sea white arrow treated by the pea rhizobia JW1401 microbial inoculum, the plant height of the green sea white arrow treated by the pea rhizobia JW1401 microbial inoculum is increased by 4.32%, and the cell yield is increased by 58.09%; compared with the Qinghai grey arrow treated by the pea rhizobium JW1401 microbial inoculum, the Qinghai grey arrow treated by the pea rhizobium JW1401 microbial inoculum has the advantages that the plant height is increased by-1.34%, and the cell yield is increased by 56.43%.
The results show that the rhizobium peasant JW1401 disclosed by the invention has more remarkable growth promoting and yield increasing effects on common vetch than the rhizobium peasant m1-10-3, the rhizobium peasant H10 and the rhizobium peasant ACCC 16505.
<110> institute of agricultural resources and agricultural regionalism of Chinese academy of agricultural sciences
<120> rhizobium for promoting growth of common vetch and application thereof
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1384
<212> DNA
<213> Rhizobium leguminosarum (Rhizobium leguminosarum)
<400> 1
tctgctcgca gcttacacat gcaagtcgag cgcgtagcaa tacgagcggc agacgggtga 60
gtaacgcgtg ggaatctacc cttgactacg gaataacgca gggaaacttg tgctaatacc 120
gtatgtgtcc ttcgggagaa agatttatcg gtcaaggatg agcccgcgtt ggattagcta 180
gttggtgggg taaaggccta ccaaggcgac gatccatagc tggtctgaga ggatgatcag 240
ccacattggg actgagacac ggcccaaact cctacgggag gcagcagtgg ggaatattgg 300
acaatgggcg caagcctgat ccagccatgc cgcgtgagtg atgaaggccc tagggttgta 360
aagctctttc accggagaag ataatgacgg tatccggaga agaagccccg gctaacttcg 420
tgccagcagc cgcggtaata cgaagggggc tagcgttgtt cggaattact gggcgtaaag 480
cgcacgtagg cggatcgatc agtcaggggt gaaatcccag ggctcaaccc tggaactgcc 540
tttgatactg tcgatctgga gtatggaaga ggtgagtgga attccgagtg tagaggtgaa 600
attcgtagat attcggagga acaccagtgg cgaaggcggc tcactggtcc attactgacg 660
ctgaggtgcg aaagcgtggg gagcaaacag gattagatac cctggtagtc cacgccgtaa 720
acgatgaatg ttagccgtcg ggcagtatac tgttcggtgg cgcagctaac gcattaaaca 780
ttccgcctgg ggagtacggt cgcaagatta aaactcaaag gaattgacgg gggcccgcac 840
aagcggtgga gcatgtggtt taattcgaag caacgcgcag aaccttacca gcccttgaca 900
tgcccggcta cttgcagaga tgcaaggttc ccttcgggga ccgggacaca ggtgctgcat 960
ggctgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctc 1020
gcccttagtt gccagcattg agttgggcac tctaagggga ctgccggtga taagccgaga 1080
ggaaggtggg gatgacgtca agtcctcatg gcccttacgg gctgggctac acacgtgcta 1140
caatggtggt gacagtgggc agcgagcacg cgagtgtgag ctaatctcca aaagccatct 1200
cagttcggat tgcactctgc aactcgagtg catgaagttg gaatcgctag taatcgcgga 1260
tcagcatgcc gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccatggg 1320
agttggtttt acccgaaggt agtgcgctaa ccgcaaggag gcagctaacc acgagacgcc 1380
gaaa 1384
<210> 2
<211> 1364
<212> DNA
<213> Rhizobium leguminosarum (Rhizobium leguminosarum)
<400> 2
tgcgctacca tgcaagtcga gcgcgtagca atacgagcgg cagacgggtg agtaacgcgt 60
gggaatctac ccttgactac ggaataacgc agggaaactt gtgctaatac cgtatgtgtc 120
cttcgggaga aagatttatc ggtcaaggat gagcccgcgt tggattagct agttggtggg 180
gtaaaggcct accaaggcga cgatccatag ctggtctgag aggatgatca gccacattgg 240
gactgagaca cggcccaaac tcctacggga ggcagcagtg gggaatattg gacaatgggc 300
gcaagcctga tccagccatg ccgcgtgagt gatgaaggcc ctagggttgt aaagctcttt 360
caccggagaa gataatgacg gtatccggag aagaagcccc ggctaacttc gtgccagcag 420
ccgcggtaat acgaaggggg ctagcgttgt tcggaattac tgggcgtaaa gcgcacgtag 480
gcggatcgat cagtcagggg tgaaatccca gggctcaacc ctggaactgc ctttgatact 540
gtcgatctgg agtatggaag aggtgagtgg aattccgagt gtagaggtga aattcgtaga 600
tattcggagg aacaccagtg gcgaaggcgg ctcactggtc cattactgac gctgaggtgc 660
gaaagcgtgg ggagcaaaca ggattagata ccctggtagt ccacgccgta aacgatgaat 720
gttagccgtc gggcagtata ctgttcggtg gcgcagctaa cgcattaaac attccgcctg 780
gggagtacgg tcgcaagatt aaaactcaaa ggaattgacg ggggcccgca caagcggtgg 840
agcatgtggt ttaattcgaa gcaacgcgca gaaccttacc agcccttgac atgcccggct 900
acttgcagag atgcaaggtt cccttcgggg accgggacac aggtgctgca tggctgtcgt 960
cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct cgcccttagt 1020
tgccagcatt gagttgggca ctctaagggg actgccggtg ataagccgag aggaaggtgg 1080
ggatgacgtc aagtcctcat ggcccttacg ggctgggcta cacacgtgct acaatggtgg 1140
tgacagtggg cagcgagcac gcgagtgtga gctaatctcc aaaagccatc tcagttcgga 1200
ttgcactctg caactcgagt gcatgaagtt ggaatcgcta gtaatcgcgg atcagcatgc 1260
cgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccatgg gagttggttt 1320
tacccgaagg tagtgcgcta accgcaagga ggcagcaacc gcga 1364
Claims (7)
1. Rhizobium leguminosarum (Rhizobium leguminosum) with the strain number of JW1401 has the preservation number of CGMCC No.11876 in the China general microbiological culture Collection center.
2. Promote the microbial inoculum that vicia sativa grows, its characterized in that: the microbial agent contains the pea rhizobium as claimed in claim 1, is a microbial agent for improving the yield and the plant height of common vetch, and is common vetch which is Qinghai white arrow or Qinghai grey arrow.
3. Use of Rhizobium leguminosum (Rhizobium leguminatum) according to claim 1 or microbial agent according to claim 2 in preparation of a product for promoting growth of vicia sativa, which is used for improving yield and plant height of vicia sativa, and is a common vetch which is an celosia angustifolia or celosia angustifolia.
4. A bio-organic fertilizer containing the microbial inoculum of claim 2.
5. Use of Rhizobium leguminosum (Rhizobium leguminatum) as claimed in claim 1 or the microbial agent as claimed in claim 2 or the bio-organic fertilizer as claimed in claim 4 for promoting the growth of vicia sativa; promote common vetch to grow for improving common vetch output and improving common vetch plant height, common vetch is blue and white arrow of Qinghai or blue and white arrow of Qinghai.
6. A method for culturing Rhizobium leguminosarum (Rhizobium leguminium) as claimed in claim 1, comprising the step of culturing the Rhizobium leguminium (Rhizobium leguminium) as claimed in claim 1 in a medium for culturing Rhizobium.
7. A method for producing the microbial agent according to claim 2, comprising a step of using the Rhizobium leguminosarum (Rhizobium leguminosarum) and/or the metabolite of the Rhizobium leguminosarum (Rhizobium leguminosarum) as an active ingredient of the microbial agent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710354470.1A CN106967652B (en) | 2017-05-09 | 2017-05-09 | Rhizobium for promoting growth of common vetch and application of rhizobium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710354470.1A CN106967652B (en) | 2017-05-09 | 2017-05-09 | Rhizobium for promoting growth of common vetch and application of rhizobium |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106967652A CN106967652A (en) | 2017-07-21 |
| CN106967652B true CN106967652B (en) | 2019-12-31 |
Family
ID=59325899
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710354470.1A Active CN106967652B (en) | 2017-05-09 | 2017-05-09 | Rhizobium for promoting growth of common vetch and application of rhizobium |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106967652B (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108124566A (en) * | 2017-12-26 | 2018-06-08 | 四川农业大学 | Subtract the method for applying synergy in arrow Kuo peas in cigarette the crop rotation of Rhizobium Inoculation |
| CN107904194B (en) * | 2017-12-26 | 2020-06-02 | 四川农业大学 | Common vetch rhizobium strain VS13-1 and application thereof |
| CN108034603B (en) * | 2017-12-26 | 2020-06-02 | 四川农业大学 | Common vetch rhizobium strain VS21-2 and application thereof |
| CN107955799B (en) * | 2017-12-26 | 2020-06-02 | 四川农业大学 | Common vetch rhizobium strain JSP1-3-1 and application thereof |
| CN108034606B (en) * | 2017-12-26 | 2020-06-02 | 四川农业大学 | Common vetch rhizobium strain VS6-3 and application thereof |
| CN107881134B (en) * | 2017-12-26 | 2020-06-02 | 四川农业大学 | Common vetch rhizobium strain VS5-1 and application thereof |
| CN114891658B (en) * | 2022-02-22 | 2024-02-23 | 河北大学 | Common vetch root nodule strain HBUJ010033 and application thereof |
| CN115927088B (en) * | 2022-10-28 | 2023-11-10 | 中国中医科学院中药研究所 | Ginseng rhizobium and application thereof |
| CN116286479A (en) * | 2023-02-01 | 2023-06-23 | 青海省农林科学院 | Rhizobium fava and application thereof in improving stress resistance or yield of leguminous crops |
-
2017
- 2017-05-09 CN CN201710354470.1A patent/CN106967652B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| Comparative investigations of the action of fungicides on Rhizobium leguminosarum Frank and its symbosis with Vicia sativa L.;KECSKES,M;《CAB Direct》;19701231;第505-514页 * |
| 根瘤菌对箭筈豌豆结瘤固氮的影响;付萍;《草业科学》;20160415;第33卷(第4期);第584-588页 * |
| 豌豆根瘤菌优良菌种筛选及应用的研究;宁国赞等;《中国草地》;19970330(第2期);第48-51页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106967652A (en) | 2017-07-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106967652B (en) | Rhizobium for promoting growth of common vetch and application of rhizobium | |
| CN110283751B (en) | Microbial inoculum for promoting growth of leguminous plants and strain and application thereof | |
| CN106987541B (en) | Efficient alfalfa rhizobium with stress resistance and growth promotion performance and application thereof | |
| CN107164261B (en) | One plant of rhizobium for promoting villose vetch to increase and its application | |
| CN103627662B (en) | A kind of Bradyrhizobium sp Arachis and uses thereof | |
| CN111484951B (en) | Bacillus for dissolving phosphorus and fixing nitrogen and application thereof in growth promotion | |
| CN102329756A (en) | Streptomyces albospinus strain BWL15-4 for preventing and treating banana vascular wilt and application thereof | |
| CN102329755A (en) | Nonabsorbent streptomyces ahygroscopicus strain BWL58 for preventing and treating banana vascular wilt and application thereof | |
| CN109055274B (en) | Caragana rhizobium and fermentation culture method and application thereof | |
| CN109182194B (en) | Rhizobium oridonii for promoting growth of corolla dentiger and culture method and application thereof | |
| CN103468591B (en) | Salt-tolerant trichoderma pleuroticola strain and application thereof | |
| CN107467075B (en) | Application of bacillus pumilus as rice growth promoter | |
| CN109735468A (en) | One plant with wide spectrum dross characteristic soybean slowly raw rhizobium and its application and with its preparation compound nitragin | |
| CN110791459B (en) | Bacillus subtilis for preventing and controlling continuous cropping lily soil-borne blight and application thereof | |
| CN108841761B (en) | Method for promoting growth of clover and/or increasing yield of clover and microbial inoculum used by method | |
| CN110218657B (en) | Trichoderma longibrachiatum MD30 and biological organic fertilizer developed by same | |
| CN109957535B (en) | Bacillus simplex, microbial agent prepared by using bacillus simplex, biological fertilizer and application | |
| CN105524874B (en) | Bacillus methylotrophicus strain NKG-1 and plant growth promoting method | |
| CN114350559B (en) | Salt-tolerant growth-promoting Liaoning slow rhizobium RY6 strain and application thereof | |
| CN107586748B (en) | A kind of China's sporangium and its application | |
| CN106520629B (en) | One Bacillus species QBJP-F4 and its application | |
| CN111808758A (en) | Crop growth-promoting endophytic fungus and application thereof | |
| CN108841762B (en) | Acid-resistant rhizobium meliloti and application thereof | |
| CN101255400A (en) | Biological activator for reinforcing nitrogen fixing capacity of nodule bacteria | |
| CN117645954B (en) | Paenibacillus mucilaginosus MSSW03 and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |