CN114686387B - Adhesive sword bacteria and application thereof in preparation of microbial fertilizer - Google Patents
Adhesive sword bacteria and application thereof in preparation of microbial fertilizer Download PDFInfo
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Classifications
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
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- Bioinformatics & Cheminformatics (AREA)
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- General Engineering & Computer Science (AREA)
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- Tropical Medicine & Parasitology (AREA)
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Abstract
The invention discloses an adhesive sword fungus, which is classified and named as adhesive sword fungusEnsiferadhaerens) The strain number is CN-02, and is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.21357; further discloses application of the adhesive sword bacteria in enhancing salt stress resistance of crops. The invention discloses a microbial agent with adhesive sword bacteria as active microorganisms; further discloses application of the microbial inoculum in enhancing salt stress resistance of crops. The adhesive sword bacteria can adhere to plant root systems, can enter plants to form symbiosis with the plants, can improve the absorption of the plants to external nutrient elements, can improve the self metabolic system, can maintain the water potential in the plants, and the like, so that the growth and development of the plants are promoted, the yield is improved, and meanwhile, the salt stress resistance of the plants is enhanced. Has extremely important significance and application value for fully exerting the ecological fertility of soil, keeping the balance of agricultural ecological environment and the like.
Description
Technical Field
The invention relates to the field of microbial agents, in particular to adhesive sword bacteria, a microbial agent and application thereof.
Background
Soil salinization and secondary salinization of soil caused by irrigation are a major environmental risk caused by natural or human activities, and generally occur in areas with drought weather, high soil evaporation intensity, high groundwater level and high content of soluble salts. It is counted that 20% of cultivated land and nearly half of irrigated land worldwide are threatened by varying degrees of salt damage, and this proportion is continually rising. The total area of the salinized lands in China is about 1 hundred million hectares, and the salinized lands are mainly distributed in 17 provinces including northwest, northeast, north China and coastal areas. When the salt concentration of the soil reaches a certain value, normal vital activity of plant cells is destroyed, so that the growth of plants is inhibited, the soil yield is low, the agricultural comprehensive productivity is seriously insufficient, and the ecological safety and the economic sustainable development of a region are directly affected.
Currently, how to improve salt and alkali resistance of crops has become a great challenge for scientific researchers. The promotion of plant growth in a salt-tolerant environment is critical to solving the problem, both from the perspective of soil improvement and from the perspective of improving crop salt tolerance and alkalinity. Plant growth-promoting rhizobacteria (PGPR) is a type of microorganism in Plant rhizosphere soil that can promote Plant growth, control diseases, and increase crop yield. The use of PGPR will help to mitigate the adverse effects of salinity on plants.
Disclosure of Invention
The invention aims to: in order to solve the problem of salt resistance of plants, the invention provides adhesive sword bacteria, and further provides a microbial agent taking the adhesive sword bacteria as active microorganisms and application of the microbial agent in enhancing the salt stress resistance of crops.
The technical scheme is as follows: the invention relates to an adhesive sword bacteria, which is classified and named as adhesive sword bacteria (Ensiferaddaerens), wherein the strain number is CN-02, the adhesive sword bacteria are preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms, the preservation time is 12 months and 11 days in 2020, the preservation number is CGMCC No.21357, and the preservation address is North Chen Xiyu No.1, 3 in the Beijing area, and the post code is 100101. The strain is a strain bred by the inventor from the soil of the paddy field in the Nanjing Shipu' er district Shang Quan in the year 5.
The adhesive sword bacteria CN-02 has the following properties:
1. colony morphology characterization
After culturing for 16-24h on LB solid medium, observing that the cells are single cells by microscope, the length of the thallus is 1.0-1.5 μm, and the width is 0.4-0.6 μm. Belongs to gram-negative bacteria and no spore. The bacterial colony is milky white, opaque, smooth, sticky, convex and regular in edge after being cultured for 12 hours at 30 ℃ in the culture medium.
2. 16SrDNA sequence analysis
The length of the 16SrDNA sequence is 1388bp, and the nucleotide sequence is shown in a sequence table SEQ.NO. 1. Comparing the 16SrDNA sequence with related species in a GeneBank database, and constructing a 16SrDNA full sequence-based phylogenetic tree. The results show that: strain CN-02 was 100% homologous to R.viscosus. The strain of the present invention was therefore considered to be Sword adhesion, and in particular Ensiferadhaerens CN-02.
The invention further provides application of the adhesive sword bacteria in any one or more of improving crop yield, improving crop quality and enhancing crop salt resistance. The adhesive sword bacteria CN-02 can produce extracellular polysaccharide, and the extracellular polysaccharide has water absorption, can maintain the osmotic pressure inside plants under the condition of high permeability through symbiosis with the plants, so that the extracellular polysaccharide has the capability of enhancing the salt resistance of the plants. The extracellular polysaccharide producing strain can be used together with organic fertilizer or independently, improves the microbial environment of rhizosphere, promotes plant growth, enhances the salt resistance of plants and reduces the using amount of fertilizer.
Preferably, the crop is a grain crop, a vegetable crop or a melon and fruit crop.
The invention further provides application of the adhesive sword bacteria in preparing extracellular polysaccharide by fermentation, and the preparation method comprises the following steps: inoculating the adhesive sword bacteria into LB culture medium, activating for 12-24h at 25-35 ℃, transferring the activated adhesive sword bacteria into fermentation culture medium, and culturing for 36-50h at 30-35 ℃ to obtain fermentation liquor, wherein the extracellular polysaccharide yield in the fermentation liquor is up to 7.4g/L.
The formula of the fermentation medium is as follows: 10-100g/L of carbon source, 1-30g/L of nitrogen source, 0.01-50g/L of inorganic salt and pH value of 5.0-9.0, wherein the solvent is water.
The carbon source is any one or the combination of a plurality of glucose, lactose, xylose, citric acid and starch; the nitrogen source is peptone, yeast extract, corn steep liquor, (NH) 4 ) 2 SO 4 、NH 4 Cl、(NH 4 ) 2 HPO 4 And NH 4 NO 3 Any one or a combination of a plurality of the above; the inorganic salt is any one or the combination of a plurality of sulfate, phosphate, dihydrogen phosphate, hydrogen phosphate and hydrochloride.
The invention further provides an active microbial agent comprising said adhesive sword bacteria.
The invention further provides a preparation method of the microbial inoculum, which comprises the following steps: inoculating the adhesive sword bacteria into an LB culture medium, activating for 12-24 hours at 25-35 ℃, transferring the activated adhesive sword bacteria into a fermentation culture medium, and culturing for 36-50 hours at 30-35 ℃ to obtain a fermentation liquor, wherein the fermentation liquor or a system obtained by post-treatment of the fermentation liquor is a microbial inoculum, namely the fermentation liquor, a concentrated solution obtained by concentrating the fermentation liquor or powder obtained by drying the fermentation liquor is the microbial inoculum.
The microbial inoculum is in the form of solution, suspension, granule, powder or stick, and the concentration of thallus in the microbial inoculum is 1×10 8 -9×10 9 CFU/g。
The formula of the LB culture medium is as follows: 10g/L of peptone, 5g/L of yeast powder, 10g/L of sodium chloride and pH of 6.0-8.0, wherein the solvent is water.
The formula of the fermentation medium is as follows: 10-100g/L of carbon source, 1-30g/L of nitrogen source, 0.01-50g/L of inorganic salt and pH value of 5.0-9.0, wherein the solvent is water.
The carbon source is any one or the combination of a plurality of glucose, lactose, xylose, citric acid and starch; the nitrogen source is peptone, yeast extract, corn steep liquor, (NH) 4 ) 2 SO 4 、NH 4 Cl、(NH 4 ) 2 HPO 4 And NH 4 NO 3 Any one or a combination of a plurality of the above; the inorganic salt is any one or the combination of a plurality of sulfate, phosphate, dihydrogen phosphate, hydrogen phosphate and hydrochloride.
The invention further provides application of the microbial inoculum in any one or more of improving crop yield, improving crop quality and enhancing crop salt resistance; the microbial inoculum is applied to crops and garden crops through root irrigation or spraying treatment. The microbial inoculum can be used by mixing with organic fertilizer, or can be used independently, so as to improve the rhizosphere microbial environment, promote plant growth, enhance the salt resistance of plants and reduce the use amount of fertilizer.
Preferably, the crop is a grain crop, a vegetable crop or a melon and fruit crop.
The beneficial effects are that: (1) The invention screens out the adhesive sword bacteria CN-02 which can strengthen the salt resistance of plants and produce extracellular polysaccharide from soil, and prepares the adhesive sword bacteria CN-02 microbial inoculum by taking the adhesive sword bacteria CN-02 as a strain for fermentation culture. The adhesive sword bacteria CN-02 can adhere to plant root systems, can enter plants to form symbiosis with the plants, can improve the absorption of the plants to external nutrient elements, can improve the self metabolic system, can maintain the internal water potential of the plants, and the like, so that the growth and development of the plants are promoted, the yield is improved, and meanwhile, the drought resistance of the plants is enhanced. Has extremely important significance and application value for fully exerting the ecological fertility of soil, keeping the balance of agricultural ecological environment and the like. (2) The adhesive sword bacteria CN-02 provided by the invention has no pollutant discharged to the environment in the production process, and the adhesive sword bacteria CN-02 is an environment-friendly fertilizer in the true sense; the preparation method of the adhesive sword bacteria CN-02 microbial inoculum provided by the invention has the advantages of simple production process and high production efficiency, can realize industrial mass production, and has good economic and social effects.
Detailed Description
Example 1 selection and identification of adhesive Sword
The adhesive sword bacteria CN-02 is a strain selected by the inventor from the soil of the paddy field in the Nanjing Shipu kou area Shang Quan (taking polysaccharide production as a standard), and the screening process is as follows:
and dissolving the collected soil sample in sterile water, vibrating and uniformly mixing. Subjecting the sample solution to gradient dilution to obtain 10 -4 、10 -5 、10 -6 Three concentration gradient dilutions were spread evenly on solid medium (medium composition: sucrose 20g/L, peptone 5g/L, beef extract 1g/L, naCl 5g/L, agar 20 g/L). Culturing in a constant temperature incubator at 30 ℃ for 48 hours, checking the plate every day during the culture period, observing colony characteristics, and taking the viscous colony as a screening target. From which single colonies capable of producing high-viscosity extracellular polysaccharide are picked and purified by streaking on a solid culture plate.
Inoculating the single colonies obtained in the previous step into liquid culture medium (medium components: 20g/L of sucrose, 5g/L of peptone, 1g/L of beef extract and 5g/L of NaCl) respectively, culturing, performing shake fermentation culture at 30 ℃ on a shaking table, and comparing the viscosities of fermentation broths of different strains after 48 hours, wherein the strain with the highest viscosity of the fermentation broth is the adhesive sword bacteria CN-02.
The adhesive sword bacteria CN-02 has the following properties:
after culturing for 16-24h on LB solid medium, observing that the cells are single cells by microscope, the length of the thallus is 1.0-1.5 μm, and the width is 0.4-0.6 μm. Belongs to gram-negative bacteria, has no flagellum and no spore. The bacterial colony is milky white, opaque, smooth, sticky, convex and regular in edge after being cultured for 12 hours at 30 ℃ in the culture medium. The 16S rDNA of the adhaerensCN-02 is shown in a sequence table SEQ ID No.1, and the total length of the sequence is 1388bp. The strain of the present invention was identified as Sword-adhesion and designated CN-02 by comparison with the blastN sequence alignment tool in the NCBI database (https:// www.ncbi.nlm.nih.gov /).
Example 2 adhesive Sword CN-02 bacteria agent
E.adhaerensCN-02 preserved at 4 ℃ in inclined plane is activated and inoculated on eggplant bottle culture medium (namely activation culture medium), wherein the activation temperature is 30 ℃, and the activation time is 16h. Selecting a large amount of strains on eggplant bottles, inoculating into a fermentation medium, wherein the aeration rate of sterile air in a fermentation tank is 0.2vvm, the stirring speed is 200rpm, and culturing at 30deg.C for 48h to obtain fermentation liquor, wherein the number of thallus in the microbial inoculum can reach 5×10 9 CFU/g or more.
The activating culture medium is LB culture medium, and the fermentation culture medium comprises the following components: glucose 40g/L, beef extract 8g/L, naCl 6g/L, peptone 10g/L, and pH 7.0.
Example 3 determination of the ability of Swiss adhesion CN-02 to produce exopolysaccharides
The extracellular polysaccharide content in the fermentation broth of example 2 was quantitatively measured using a phenol sulfuric acid method, and the extracellular polysaccharide production amount of the adhesive Sword CN-02 was calculated from a standard curve made of anhydrous glucose. The result shows that the yield of the adhesive sword bacteria CN-02 extracellular polysaccharide reaches 7.4g/L.
Example 4 adhesion of Sword CN-02 can significantly increase cabbage yield
The preparation method of the microbial inoculum used in the invention is the same as that of the example 2.
And (5) taking Chinese cabbage as an experimental object, and developing a potting experiment.
The test is carried out by 4 treatment modes:
treatment 1 (T) 1 ) Normally watering; treatment 2 (T) 2 ) Adding 0.6% salt when watering for the first time; treatment 3 (T) 3 ) Normally watering and applying; the microbial inoculum prepared in example 2 (5 mL microbial inoculum is added into each basin, and the water content of the microbial inoculum is calculated into the water casting amount); treating 4%T 4 ) 0.6% salt was added at the first watering and the microbial inoculum prepared in example 2 was applied (5 mL of microbial inoculum was added per pot, the microbial inoculum water content was calculated as the amount of water to be poured).
The microbial agents of treatment 3 and treatment 4 are applied by a spraying method.
The application amount and daily management of the four treated other conventional fertilizers are kept consistent, and various indexes such as yield and the like are detected in the harvest period of the cabbages, and the experimental results are shown in table 1.
TABLE 1 growth conditions under different treatment conditions in pot experiments of Chinese cabbage
Treatment of | Root length (cm) | Leaf length (cm) | Fresh weight (g) | Yield increase% |
T 1 | 6.9±0.4 | 29.3±0.3 | 142.3±1.6 | — |
T 2 | 6.2±0.6 | 18.9±0.5 | 96.7±4.3 | -32.04 |
T 3 | 13.2±0.5 | 32.3±0.6 | 170.5±1.4 | 19.8 |
T 4 | 12.6±0.7 | 28.6±0.4 | 149.7±2.5 | 5.2 |
In the experiment of Chinese cabbage potting, compared with the treatment 1, the treatment 3 under normal watering quantity has obvious improvement of root length, leaf length and fresh weight, and the yield is increased by 19.8%; process 2 yield was reduced by 32.04%; when the microbial inoculum is applied, the yield of the treatment 4 is increased by 5.2%, and compared with the treatment 2, the root length, the leaf length and the fresh weight are greatly improved. The adhesive sword bacteria CN-02 can promote the growth of crops and has very obvious excellent performance in enhancing the salt resistance of crops.
Example 5 adhesion of Sword CN-02 can significantly increase wheat yield
The preparation method of the microbial inoculum used in the invention is the same as that of the example 2.
Taking wheat as a research object, and carrying out a cell experiment.
The experiment sets 3 treatment modes, each treatment mode is repeated for 3 times, 9 cells are total, and the cell area is 5m multiplied by 4 m=20m 2 Each cell is randomly distributed.
Treatment 1 (T1) is a blank group without adding a microbial inoculum; treatment 2 (T2) application of the microbial inoculum prepared in example 2 (16 kg/hm 2 ) The method comprises the steps of carrying out a first treatment on the surface of the Treatment 3 (T3) application of an inactivated microbial agent (16 kg/hm 2 The microbial inoculum prepared in example 2 was inactivated at high pressure and high temperature).
Treatments 2 and 3 were applied by root irrigation.
The application amount and daily management of other conventional fertilizers for the three treatments are consistent. Various indexes such as wheat yield and the like are detected in the mature period of wheat, and the experimental results are shown in table 2.
TABLE 2 wheat yield status under different treatment conditions
The result shows that in the wheat experiment, the adhesion sword bacteria CN-02 obviously promotes the growth of wheat; the yield is improved by 37.1%, the thousand kernel weight is also obviously improved, and the yield is almost the same as that of a control group after the adhesive sword bacteria CN-02 is inactivated. This demonstrates that the adhesion of Sword CN-02 can significantly improve crop yield and quality.
The adhesive sword bacteria of the embodiment 6 can obviously improve the yield and quality of watermelons
Taking watermelons as research objects, carrying out cell experiments.
The experiment sets 3 treatment modes, each treatment mode is repeated for 3 times, 9 cells are total, and the cell area is 5m multiplied by 4 m=20m 2 Each cell is randomly distributed.
T 1 No microbial inoculum is added for treatment, namely a blank group; t (T) 2 The seeds are placed in a container, and then the microbial fertilizer (200 ml/mu) is diluted by 50 times for seed dressing.
The application amount and daily management of other conventional fertilizers for both treatments are consistent. Various indexes such as watermelon yield and the like are detected in the mature period of the watermelon, and the experimental results are shown in table 3.
TABLE 3 yield and quality status of watermelons under different treatment conditions
The result shows that in the watermelon experiment, compared with a control group, the adhesive sword bacteria CN-02 obviously improves the content of vitamin C in the watermelon, and simultaneously improves the sugar degree, fruit setting rate and yield of the watermelon. This demonstrates that the adhesive Sword CN-02 significantly improved the yield and quality of watermelon.
Sequence listing
<110> Suzhou Konning polyol Co., ltd
<120> A strain of adhesive sword bacteria and application thereof in preparation of microbial fertilizer
<141> 2020-12-28
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1388
<212> DNA
<213> 16SrDNA(2 Ambystoma laterale x Ambystoma jeffersonianum)
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aaccaactcc catggtgtga cgggcggtgt gtacaaggcc cgggaacgta ttcaccgcag 120
catgctgatc tgcgattact agcgattcca acttcatgca ctcgagttgc agagtgcaat 180
ccgaactgag atggcttttg gagattagct cgacctcgcg gtctcgctgc ccactgtcac 240
caccattgta gcacgtgtgt agcccagccc gtaagggcca tgaggacttg acgtcatccc 300
caccttcctc tcggcttatc accggcagtc cccttagagt gcccaaccaa atgctggcaa 360
ctaagggcga gggttgcgct cgttgcggga cttaacccaa catctcacga cacgagctga 420
cgacagccat gcagcacctg tctccgatcc agccgaactg aaggaaaacg tctccgtaat 480
ccgcgatcgg gatgtcaagg gctggtaagg ttctgcgcgt tgcttcgaat taaaccacat 540
gctccaccgc ttgtgcgggc ccccgtcaat tcctttgagt tttaatcttg cgaccgtact 600
ccccaggcgg aatgtttaat gcgttagctg cgccaccgaa cagtaaactg cccgacggct 660
aacattcatc gtttacggcg tggactacca gggtatctaa tcctgtttgc tccccacgct 720
ttcgcacctc agcgtcagta atggaccagt gagccgcctt cgccactggt gttcctccga 780
atatctacga atttcacctc tacactcgga attccactca cctcttccat actctagaca 840
cccagtatca aaggcagttc cggggttgag ccccgggatt tcacccctga cttaaatgtc 900
cgcctacgtg cgctttacgc ccagtaattc cgaacaacgc tagccccctt cgtattaccg 960
cggctgctgg cacgaagtta gccggggctt cttctccggt taccgtcatt atcttcaccg 1020
gtgaaagagc tttacaaccc tagggccttc atcactcacg cggcatggct ggatcaggct 1080
tgcgcccatt gtccaatatt ccccactgct gcctcccgta ggagtttggg ccgtgtctca 1140
gtcccaatgt ggctgatcat cctctcagac cagctatgga tcgtcgcctt ggtaggcctt 1200
taccccacca actagctaat ccaacgcggg ctcatccttt cccgataaat ctttccccca 1260
aagggcttat acggtattag cacaagtttc cctgcgttat tccgtagaaa agggtagatt 1320
cccacgcgtt actcacccgt ctgccgctcc ccttgcgggg cgctcgactg catgggaagc 1380
tgccgcaa 1388
Claims (9)
1. A strain of adhesive sword bacteria CN-02, which is classified and named as adhesive sword bacteria @, is providedEnsifer adhaerens) The strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation time of 2020, 12 months and 11 days and a preservation number of CGMCC NO. 21357.
2. The use of the adhesive sword CN-02 as defined in claim 1 for preparing microbial fertilizer.
3. Use of the adhesive sword bacteria CN-02 as claimed in claim 1 for any one or more of improving crop yield, improving crop quality and enhancing salt resistance of crops.
4. Use of the adhesive sword CN-02 according to claim 1 for producing extracellular polysaccharide.
5. An active microbial agent comprising the adhesive sword CN-02 of claim 1.
6. The method for preparing the active microbial agent according to claim 5, wherein the adhesive sword bacteria CN-02 is inoculated in LB culture medium and activated at 25-35 ℃ for 12-24h, the activated adhesive sword bacteria is transferred in fermentation culture medium, and the fermentation broth is obtained by culturing at 30-35 ℃ for 36-50h, and the fermentation broth or a system obtained by post-treatment of the fermentation broth is the microbial agent.
7. The method according to claim 6, wherein the bacterial agent is in the form of solution, suspension, granule or powder, and the bacterial agent has a bacterial cell concentration of 1X 10 8 -9×10 9 CFU/g。
8. The use according to claim 3, wherein the application to crops is by root irrigation, spraying or seed soaking.
9. The use according to claim 8, wherein the crop comprises a food crop, a vegetable crop.
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EP3058825A1 (en) * | 2015-02-19 | 2016-08-24 | Futureco Bioscience, S.A. | Bacteria with nematicidal activity |
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Title |
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Chemical characterization and potential application of exopolysaccharides produced by Ensifer adhaerens JHT2 as a bioemulsifier of edible oils;Vanessa Marques Alvarez et al.;《International Journal of Biological Macromolecules》;20180314;第114卷;摘要,第18页左栏第1段至第24页右栏第2段 * |
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