CN111172070A - Preparation and application method of seed biostimulant SB-MGW9 - Google Patents
Preparation and application method of seed biostimulant SB-MGW9 Download PDFInfo
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- 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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- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
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Abstract
The invention relates to the field of seed science, in particular to a preparation and application method of seed biostimulant SB-MGW 9. The important strain Bacillus sp.MGW9 is obtained by separating, screening, purifying and identifying extremely arid soil near Ming Great Wall (Ming Great Wall) in Shandan county in Gansu province, and researches show that the strain has the capacities of salt resistance, nitrogen fixation, phosphorus dissolution, indoleacetic acid production and the like, the seed biostimulant SB-MGW9 can be directly prepared by using the bacterial liquid, and the germination and emergence capacities of the corn seeds in the saline-alkali soil can be effectively improved by the seed soaking and drying treatment. The invention can be widely applied to the production of the corn in the saline-alkali soil.
Description
Technical Field
The invention relates to the field of Seed science, in particular to a preparation and application method of Seed biostimulantoff Ming Great Wall 9 (SB-MGW 9).
Background
In recent years, biostimulants have become an extremely fashionable term in the global agricultural market. By international definition, biostimulant is a substance that contains certain components and microorganisms whose effects, when applied to a plant or rhizosphere, are stimulating for the plant's natural processes, including enhancing/benefiting nutrient uptake, nutritional efficacy, abiotic stress tolerance, and crop quality, regardless of nutrient content. In europe, the use of biostimulants has been directed to fruit trees (citrus, olive, grape, etc.), vegetables and fruits (broccoli, capsicum, cucumber, strawberry, tomato, melon, etc.), food crops (potato, wheat, corn, rape, etc.), flowers, nurseries, etc., and has achieved good results.
Seed vigor is a generic term that determines the potential ability of seeds to emerge quickly and neatly and grow into normal seedlings under a wide range of field conditions (McDonald, 1980). The seed treatment and coating means that after the seeds are harvested and before the seeds are sown, various effective treatments are adopted, including sterilization and disinfection, warm soup seed soaking, fertilizer seed soaking and dressing, trace element treatment, low-temperature stratification, growth regulator treatment, coating and other strengthening methods, so that the method has the advantages of preventing the seeds from carrying pathogenic bacteria and plant diseases and insect pests in soil, and protecting the seeds from normal germination and emergence growth; the stress resistance of the seeds to unfavorable soil and climate conditions is improved, and the seedling rate is increased; the storage resistance of the seeds is improved, and the deterioration of the seeds is prevented; the size and the shape of the seeds are changed, so that the mechanical sowing is facilitated; increasing seed vigor, promoting whole and strong seedlings, increasing crop yield, improving product quality and the like. Earlier researches find that the biological stimulin can effectively improve the seed activity to ensure that uniform, uniform and strong seedlings are realized after field sowing, and the later yield and quality of plants can be continuously influenced, particularly, the biological stimulin for seeds is researched and developed as a new industry, and has wide development and application prospects.
Population growth, soil degradation and pesticide abuse are major challenges that must be faced by agriculture for decades to come. Meanwhile, in recent years, development, improvement and utilization of medium and low yield fields such as dry thin lands, saline-alkali lands and the like in China play an important role in enlarging crop planting area and improving yield, wherein high-quality seeds are also an important foundation and guarantee for adapting to soil degradation environment, meeting green development and utilization of saline-alkali lands and realizing sustainable development of seed industry, so that improvement of seed quality, especially continuous breakthrough of key technology of seed quality guarantee, becomes one of the most important subjects of development of new-era seed industry. Corn is used as the first large grain crop in China, and the key that the corn production is well caught and the grain is continuously and stably developed is particularly emphasized in the text of corn treatise by the Hanchang of the country. Therefore, techniques for enhancing seed quality are continuously developed, such as: the novel seed biostimulant product is developed and applied to the production of crops such as corn and the like, so that the seed space is improved, the capability of crop seed adversity seedling emergence and the like is improved, the crop yield and quality are guaranteed, and the method has great significance for accelerating the conversion of new and old kinetic energies in China, guaranteeing the effective supply of future grains in China and assisting the world grain safety.
At present, few reports of improving the germination capacity of corn seeds in adverse situations (soil salinization, soil impoverishment, drought and the like) by using a seed biostimulant product in China are reported. Among them, soil salinization is the most common problem in agricultural production worldwide, and the generated ion toxicity and osmotic stress have great influence on the growth and development of plants, directly influencing the yield and quality. Breeding new stress-resistant crop varieties is a feasible solution, but the period is long and the difficulty is high; in contrast, the development of a biostimulant product for seeds of a target variety has become a more efficient strategy. The scientific application of the product can effectively improve the seed boundary, improve the (anti) adverse growth resistance of crop seeds, realize the quality improvement and the efficiency improvement and realize the green production.
Based on the separation and identification of beneficial microorganisms, reports and products for developing seed biostimulant products to improve the saline-alkali tolerance (resistance) capability of germination of crop seeds such as corn and the like are few, and the product has huge market space and development potential and is very worthy of research and development.
Disclosure of Invention
The invention aims to meet the market demand of seed biostimulant and overcome the defects of the prior art, and provides a preparation and application method of the seed biostimulant SB-MGW 9.
In order to achieve the purpose, the invention is based on that the biostimulant stimulates the natural process of the plant, and particularly has the efficacy of improving the abiotic stress resistance of the plant growth and the like. The method aims at improving the salt stress resistance of the germination of the corn and other crop seeds, and the like, and widely collects possible source samples of target beneficial microorganisms, obtains target strains through a series of operations such as separation and identification, and completes research, development and application work of the salt stress resistance biostimulant for improving the germination of the corn seeds. The invention adopts the following technical scheme to obtain a preparation and application method of seed biostimulant SB-MGW9, which mainly comprises the following operations: (1) ecological investigation and collection of bacteria source samples; (2) separating and screening strains; (3) purifying the strain; (4) researching the characteristics of the target strain; (5) measuring the growth promoting capability of the strain; (6) identifying the strain; (7) preparing seed biostimulant; (8) and (3) detecting the actual effect of the application of the seed biostimulant.
In the operation (1), according to a research and development target, ecological research is carried out, a sampling scheme is formulated, bacteria source sample collection is carried out, and identification, information remark and low-temperature storage are carried out on the collected sample. The bacterial source of the invention is near Ming Great Wall (100.88E, 38.84N) of Shandan county of Gansu province, and extremely arid soil (about 1 kg) (Zhao Yangxiao agricultural university of Thailand province delays teaching help and takes samples).
In the operation (2), endogenous bacteria in the soil sample are separated and screened step by step on solid beef extract peptone containing NaCl with different concentrations by a homogenization method.
In the above-mentioned operation (3), the candidate strain is obtained by purifying the strain by the plate streaking method, and the salt tolerance of the candidate strain and the like are further confirmed.
In the above-mentioned operation (4), the target strain is measured for its own nitrogen fixation ability, phosphorus dissolution ability, production hormone indoleacetic acid, and the like.
In the operation (5), a target bacterial solution is prepared, and standard germination test, seedling growth determination, measurement of simulated field emergence rate and the like are carried out after seed soaking treatment of the corn variety to be tested so as to identify the growth promotion effect.
In the above-mentioned operation (6), morphological feature identification and 16S rDNA species molecular identification are performed on the objective colonies.
In the operation (7), the target seed biostimulant is prepared by taking the target bacterial liquid as a main effective component.
In the operation (8), the practical effect verification and the use method research of the saline-alkali soil corn production are carried out on the biological stimulin of the target seeds.
The invention has the beneficial effects that: the biological stimulin can stimulate the natural process of plant, and has especially the functions of raising the resistance to abiotic stress. In order to improve the salt stress resistance and other abilities of corn seed germination, the target seed bio-stimulant is obtained through flows of ecological investigation, strain source sample collection, strain separation and screening, strain purification, target strain characteristic research, strain growth promoting ability determination, strain identification, seed bio-stimulant preparation, seed bio-estrogen application actual effect detection and the like, and the use method is established by combining the saline-alkali soil application actual effect. The method can be widely applied to the production of the corn in the saline-alkali soil, so as to improve the activity of the corn seeds, ensure the rapid and orderly emergence of the seeds under the conditions of salt stress and the like, and assist the yield and the efficiency increase of the corn in the saline-alkali soil.
Drawings
FIG. 1 is a flow chart of the operation of the present invention.
FIG. 2 shows the salt tolerance measurement of the strain MGW9 of the present invention.
FIG. 3 shows the nitrogen-fixing and phosphorus-dissolving capacity of the strain MGW9 of the present invention.
FIG. 4 is a standard curve of the variation of IAA concentration with absorbance in accordance with the present invention.
FIG. 5 shows the effect of MGW9 bacterial liquid treatment on maize seed germination vigor and germination rate under salt stress according to the present invention.
FIG. 6 shows the effect of MGW9 bacterial liquid treatment on the growth of maize seedlings under salt stress of the present invention.
FIG. 7 shows the effect of MGW9 bacterial liquid treatment on the simulated field emergence rate of corn seeds.
FIG. 8 is a gram stain map of strain MGW9 of the present invention.
FIG. 9 shows the 16S rDNA sequence of the strain MGW9 of the present invention.
FIG. 10 is phylogenetic tree evolutionary tree of strain MGW9 of the present invention.
Detailed Description
The invention is described in detail below with reference to the figures and examples.
As shown in figure 1, the biological stimulator can stimulate the natural process of plants, and substances such as beneficial bacteria of microorganisms and the like are important components of the biological stimulator for seeds, so that the development and the utilization of the biological stimulator are beneficial to improving the vitality of the seeds, enhancing the stress resistance of the germinated seeds, and continuously influencing the yield and the quality of the plants in the later period. The preparation and application method of the salt-tolerant biostimulant for improving the germination of the corn seeds comprises the operations of ecological investigation, strain source sample collection, strain separation and screening, strain purification, target strain characteristic research, strain growth promoting capability determination, strain identification, preparation of the seed biostimulant, actual effect detection of the seed biostimulant application and the like.
The invention relates to a preparation and application method of seed biostimulant, and the specific implementation mode is as follows.
(1) Ecological investigation and collection of bacteria source samples: according to the research and development target of the seed biostimulant, based on ecological research, a microbial beneficial bacterium source is selected, and information such as the name, specific time, location and the like of a collected sample is registered; the bacterial source of the invention is an extremely arid soil sample (about 1 kg) obtained near Ming Great Wall (100.88E, 38.84N) of Shandan county of Gansu province in 8 and 17 days of 2017 for later use.
(2) Separating and screening strains: beef extract peptone medium: 3.0g of beef extract, 10.0g of peptone, 15-25g of agar, 1000mL of distilled water and pH 7.4-7.6; weighing 20g of soil sample by adopting a dilution method, putting the soil sample into a triangular flask, adding 80mL of sterile water, shaking up to prepare strain suspension, and diluting the strain suspension into strain solutions with different concentrations for later use. 1-2 drops of 10-fold serial diluted strain suspension are dipped by an inoculating loop, and are inoculated on a solid beef extract peptone medium plate containing 10 NaCl with different concentrations (salt concentration is increased continuously until no bacterial colony appears) of 5%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% and the like by a coating method, the concentration is repeated for 6 times, the solid beef extract peptone medium plate is placed in a constant-temperature incubator at 28 ℃ for 6-7 days, and the growth condition of the strain is observed (figure 2).
(3) Strain purification: based on the observation of the growth condition of plate bacteria, if bacterial colonies appear on the solid beef extract peptone medium plates with 5%, 7%, 8%, 9%, 10%, 11% and 12% of NaCl and no bacterial colonies appear on the solid beef extract peptone medium plates with 13%, 14% and 15% of NaCl, selecting the solid beef extract peptone medium plates with 12% of NaCl, selecting the colonies with vigorous growth, and inoculating the colonies onto the solid beef extract peptone medium by using a plate streaking method; and putting the culture medium into an incubator for continuous culture, and when a large number of bacterial colonies grow out, continuously streaking and purifying until a single bacterial colony is purified (figure 2). Note: after the strains are separated, different strains are numbered, and the strain with the number MGW9 (Ming Great Wall 9) is selected in the invention.
(4) Characteristic study of target strains: culturing at 28 deg.C for 48h to obtain strain MGW9, and performing salt tolerance, nitrogen fixation, phosphorus solubility, and hormone-producing indoleacetic acid supplementing tests.
Determination of salt resistance: adding 0.1mL of MGW9 bacterial solution with growth logarithmic phase into beef extract peptone liquid culture medium with 40mL of NaCl content of 5%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% and 15%, culturing at 28 deg.C with constant temperature shaking table 200 r/min for 24h, measuring absorbance of bacterial solution OD600nm, and repeating for 3 times with beef extract peptone liquid culture medium without inoculation as control.
The results further show that the strain MGW9 has certain salt tolerance, and the salt tolerance range is 0% -12.0% (figure 2).
The MGW9 strain uses 25% glycerin as freezing protective agent, the MGW9 strain is preserved in an ultra-low temperature refrigerator at-80 ℃, the preservation places are: the agricultural college of Qingdao agricultural university seed science and engineering laboratory. Meanwhile, the culture medium is also preserved in the common microorganism center of China Committee for culture Collection of microorganisms; address: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the registration number of the preservation center is CGMCC No.18690, and the preservation date is: year 2019, month 10 and day 16.
And (3) nitrogen fixation determination: on an aseptic operation platform, the MGW9 strain is streaked and inoculated into a nitrogen-free culture medium, the nitrogen-free culture medium is placed in a dark incubator for culture at 30 ℃, the growth condition of the nitrogen-free culture medium is observed for 4-6 days, and whether the strain can fix nitrogen per se is checked according to the existence of bacterial colonies on a flat plate, so that the normal growth of the nitrogen-free culture medium is ensured.
The results showed that MGW9 strain was inoculated on nitrogen-free medium, colonies were observed at 4d and at 6d, colonies were more distinct, indicating that MGW9 strain has nitrogen fixation ability (FIG. 3).
And (3) phosphorus solubility determination: on an aseptic operation platform, the MGW9 strain is point-connected in inorganic phosphorus and organic phosphorus culture media, and is placed in a dark incubator for culture at 30 ℃, whether the strain has a transparent ring (phosphorus dissolving ring) is observed for about 7 days, whether the strain has the capability of decomposing inorganic phosphorus and organic phosphorus is judged according to the size of the transparent ring (if the ratio of the diameter of the phosphorus dissolving ring to the diameter of a bacterial colony is 1, the capability of decomposing phosphorus is not existed), and the steps are repeated for 3 times.
The results show that the MGW9 strain can observe transparent circles at the 3 rd stage after being inoculated with organic phosphorus and inorganic phosphorus culture media until the transparent circles at the 7 th stage increase; in which transparent circles were produced on the organophosphorus medium plates, the ratio of the diameter of the phosphorus-solubilizing circle to the diameter of the colony was 1.7, indicating that the MGW9 strain had the ability to solubilize inorganic phosphorus and organophosphorus (FIG. 3).
Hormone production indoleacetic acid determination: firstly, shaking and culturing the bacterial liquid on a King's B liquid culture medium at the constant temperature of 28 ℃ for 48h at 200 r/min according to the specification of a Kovacs indigo substrate kit (Qingdao Haibo biotechnology Co., Ltd.), wherein the color turns red to indicate that MGW9 can generate indoleacetic acid, and the darker the color is, the more indoleacetic acid is generated, and the 3 times of repetition are carried out; secondly, preparing a standard curve by using an indoleacetic acid standard substance, centrifuging the bacterial suspension cultured for 48 hours and a control 10000 r/min for 10min, taking a supernatant, adding an equal volume of Salkowsk color developing agent, performing light-shielding color development for 20min at room temperature, and measuring the light absorption value of OD530 nm; and finally, calculating the content of indole acetic acid secreted by the endophytic bacteria according to a standard curve.
The result of hormone-producing indoleacetic acid determination shows that Kovacs indigo substrate reagent turns red after the bacterium is added into a King's B liquid culture medium containing 100 mg/L tryptophan, which indicates that MGW9 has the capability of producing indoleacetic acid, and that OD530nm is 0.482 after Salkowsk color development, and the standard curve equation y =0.025x +0.001 (theR 2 =0.985, y represents absorbance, and x represents concentration) was calculated to have an indole acetic acid content of 19.24 mg/L (fig. 4).
(5) And (3) measuring the growth promoting capability of the strain: treating corn seeds by using MGW9 bacterial liquid, and performing standard germination test, seedling growth determination, field emergence rate simulation determination and the like on the treated corn seeds; statistical analysis of the results statistical analysis of the data single-factor analysis of variance was performed using SPSS17.0 statistical software and the Duncan method was used to test the significance of differences between different treatments for each parameter (P < 0.05).
Standard germination test and seedling growth determination: taking samples of the variety of the corn of Zhongdi 175, Zhengdan 958 and Ludan 981 as materials (collected in 2018 without coating), and performing paper rolling germination according to the crop seed inspection regulation and the international seed inspection regulation; selecting healthy and plump seeds with uniform size, sterilizing the seeds with 1% NaClO solution for 10min, cleaning the seeds with sterile distilled water for 3 times, and absorbing floating water on the surfaces of the seeds with absorbent paper for later use; the concentration of MGW9 bacterial liquid which is kept at the constant temperature of 28 ℃ for 200 r/min and is shake-cultured for 48 hours is adjusted to be 1.0 multiplied by 108—1.5×108cfu/mL; soaking the sterilized corn seeds in the bacterial liquid for 6h (Treatment, T), preserving moisture for 12h, taking out, and drying for later use (the preliminary test is completed at the early stage). Based on earlier stage study, 200mmol/L NaCl solution is selected to wet germination paper (American Anchor Anke germination paper) and is placed in a staggered bed, then the germination paper is rolled up and placed into a self-sealing bag, the germination paper is vertically placed in an artificial climate box at 25 ℃ in a dark place, 3 times of repetition are carried out, each repetition is carried out for 100 grains (3 rolls), and under the salt stress condition, the non-soaked part is taken as the non-soaked partThe result is Control1 (Control 1, C1), the result is Control2 (Control 2, C2), the 4 th statistical Germination vigor (GE), and the 7 th statistical Germination Percentage (GP); the number of germinated seeds/tested seeds in GE ═ 4d is multiplied by 100 percent; the number of sprouted seeds/tested seeds in GP 7d is multiplied by 100 percent; while the germination rate is counted, 10 seedlings are randomly selected from each repetition, and the measurement of indexes such as bud (seedling) length, main root length, bud (seedling) fresh weight/10 strains, root fresh weight/10 strains, bud (seedling) dry weight/10 strains (105 ℃, 17h +/-1 h, low-temperature drying, International Rules for selected Testing Editon 2012 and ISTA) and root dry weight/10 strains (105 ℃, 17h +/-1 h, low-temperature drying, International Rules for selected Testing Editon 2012 and ISTA) is referred.
And (3) measuring the emergence rate of the simulated field: enough saline-alkali soil is transported from Shandong Yingcity, and a simulated field test is carried out at room temperature. Firstly, saline-alkali soil is put into a germination box, a soil bed is made flat by a flat base plate, and the soil depth is about 10 cm; secondly, orderly placing the treated or untreated seeds in a bed through seed placing holes in a seed placing plate, placing the seed embryos downwards, removing the seed placing plate, and covering soil for 3 cm; finally, the earth is covered flatly by a flat base plate (note: the flat base plate, the seed placing plate and the seed placing hole used in the step are introduced in the utility model patent of Jiangqun and Li Heqin, a corn seed germination box, the patent number is 201320432096. X), and the germination box is sealed by a preservative film. Each treatment was repeated 3 times, each time 100 seeds were repeated, and the rate of emergence was counted on day 15.
The test is completed in 2018 in the seed science and engineering laboratory of Qingdao agricultural university from 9 months to 10 months.
From the standard germination test results: compared with C1 and C2, T can obviously improve the germination potential and the germination rate of 3 corn variety samples under the condition of salt stress, and after the 3 corn variety samples are treated (T), the germination potential and the germination rate are obviously different from those of C1 (figure 5).
From the results of the seedling growth test: compared with C1 and C2, T can obviously improve 6 indexes of bud (seedling) length, main root length, bud (seedling) fresh weight/10 plants, root fresh weight/10 plants, bud (seedling) dry weight/10 plants, root dry weight/10 plants and the like under the condition of salt stress of 3 corn variety sample varieties. Compared with C1, the 6 indicators of T all reached significant levels (fig. 6).
According to the result of the simulated field emergence rate measurement: the T can obviously improve the emergence rate of the simulated field of the seeds of the 3 corn variety samples, and the difference is obvious (figure 7).
(6) And (3) strain identification: the strain MGW9 was identified both morphologically and molecularly.
And (3) morphological identification: the strain MGW9 is cultured on a beef extract peptone solid medium at 28 ℃ for 48h, and the characteristics of the colony, such as morphology, gloss, texture, edge characteristics, surface characteristics, bump shape, colony color and the like, are observed and recorded.
The morphological identification result shows that the colony is circular, has the diameter of about 3-5 mm, and is milky white; the surfaces of the bacterial colonies are smooth and wet, and the edges are neat, opaque and convex; gram staining was positive and the cells were rod-shaped (fig. 8).
And (3) molecular identification: inoculating into beef extract peptone liquid culture medium, keeping the temperature at 28 deg.C for 200 r.min-1Carrying out 16SrDNA molecular identification on the bacterial liquid cultured for 48 hours by shaking, and repeating for 3 times; the MGW9 genome is extracted by adopting a bacterial genome DNA rapid extraction kit (purchased from Biotechnology engineering (Shanghai) Co., Ltd.), PCR amplification is carried out by taking the genome as a template, the general primers are 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGT-3'), the reaction system is 20uL, and the PCR amplification conditions are as follows: pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1.5min, and 30 cycles; after 10min extension at 72 ℃ the samples were stored at 4 ℃; sequencing the PCR reaction product is completed by Beijing Sanbo Polygala tenuifolia Biotech Limited liability company; performing Blast comparison on the sequencing result of the PCR reaction product on GeneBank to obtain a sequence with higher similarity with a target strain, then performing Clustalx comparison, and constructing an NJ phylogenetic tree by adopting an MEGA5.1 tool.
The molecular identification result shows that the MGW9 strain 16SrDNA gene sequence fragment has the size of 1424 bp; NCBI is logged in for Blast comparison, 11 strains with higher similarity are selected to construct an NJ phylogenetic tree by 16SrDNA gene sequences, MGW9 and Bacterium wsb-1(KJ950500.1) are gathered in one branch, the similarity rate reaches 99.0%, and the fact that the MGW9 is closest to the genetic relationship of the strains is shown (FIGS. 9 and 10).
The MGW9 was judged to be Bacillus, named as Bacillus, based on the morphological characteristics and the results of the 16S rDNA gene sequence analysis (FIGS. 8 and 9) Bacillus sp. MGW 9. Now preserved in the China general microbiological culture Collection center; address: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the registration number of the preservation center is CGMCC No.18690, and the preservation date is: year 2019, month 10, day 16; the survival is detected by the collection center in 2019 at 11 and 6 days; the proposed classification is named:Bacillus sp.。
(7) preparing seed biostimulant: tests show that the MGW9 bacterial liquid treatment has good effect on improving the salt stress resistance of the corn seed germination. The seed biological stimulator consists of bacillusBacillus sp.MGW9（1.0×108—1.5×108cfu/mL), adjusting the pH value of the bacterial liquid to 7.0-8.0, and further researching other formulas; the using method comprises the following steps: soaking seeds for 6h, and keeping moisture for 12 h.
(8) Detecting the practical effect of the seed biological stimulin application: in 2019, saline-alkali soil field emergence rate tests are carried out in Dongying and Nethereto in Shandong, the seed spacing is 0.06m, the row length is 0.6m, and the tests are repeated for 3 times. Diluting the biological stimulin of the seeds by 2-3 times before sowing, taking 175 corn varieties of seeds in the middle ground as test materials, soaking the seeds for 6 hours, preserving moisture for 12 hours, sowing, taking the seeds which are not soaked as a control, and counting the field emergence rate 15 days after sowing. The implementation effect is as follows: compared with the contrast, the treated seeds have faster emergence of seedlings and more regular emergence of seedlings, and the emergence rates of the seedlings in the field of two fields are respectively improved by 7.8 percent and 9.4 percent.
In conclusion, the invention can stimulate the natural process of plants based on the biostimulant, and particularly has the effects of improving the tolerance to abiotic stress and the like. In order to improve the salt resistance of corn seed germination, a biological stimulator for improving the salt resistance of seed germination is developed, wherein an important strain bacillus isBacillus sp.MGW9 has the functions of salt resistance, nitrogen fixation, indoleacetic acid production and phosphorus dissolving capacity, can effectively improve the germination activity of corn seeds under the condition of salt stress, and successfully prepares a strain for improving the seed growth based on MGW9The stimulating hormone SB-MGW9 lays an important foundation for the later formal commercial use of the product. The invention can be widely applied to the production of the corn in the saline-alkali soil.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting, and although the present invention is described in detail with reference to the embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions implemented by the present invention without departing from the design spirit and scope of the present invention, which should be covered by the claims of the present invention.
Sequence listing
<110> Qingdao agricultural university
Dry farming agricultural research institute of academy of agriculture and forestry of Hebei province
<120> preparation and application method of seed biostimulant SB-MGW9
<141>2020-01-03
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>1424
<212>DNA
<213> Bacillus MGW9(Bacillus sp. MGW9)
<400>1
atacatgcag tcgagcggac agatgggagc ttgctccctg atgttagcgg cggacgggtg 60
agtaacacgt gggtaacctg cctgtaagac tgggataact ccgggaaacc ggggctaata 120
ccggatggtt gtttgaaccg catggttcaa acataaaagg tggcttcggc taccacttac 180
agatggaccc gcggcgcatt agctagttgg tgaggtaatg gctcaccaag gcaacgatgc 240
gtagccgacc tgagagggtg atcggccaca ctgggactga gacacggccc agactcctac 300
gggaggcagc agtagggaat cttccgcaat ggacgaaagt ctgacggagc aacgccgcgt 360
gagtgatgaa ggttttcgga tcgtaaagct ctgttgttag ggaagaacaa gtaccgttcg 420
aatagggcgg caccttgacg gtacctaacc agaaagccac ggctaactac gtgccagcag 480
ccgcggtaat acgtaggtgg caagcgttgt ccggaattat tgggcgtaaa gggctcgcag 540
gcggtttctt aagtctgatg tgaaagcccc cggctcaacc ggggagggtc attggaaact 600
ggggaacttg agtgcagaag aggagagtgg aattccacgt gtagcggtga aatgcgtaga 660
gatgtggagg aacaccagtg gcgaaggcga ctctctggtc tgtaactgac gctgaggagc 720
gaaagcgtgg ggagcgaaca ggattagata ccctggtagt ccacgccgta aacgatgagt 780
gctaagtgtt agggggtttc cgccccttag tgctgcagct aacgcattaa gcactccgcc 840
tggggagtac ggtcgcaaga ctgaaactca aaggaattga cgggggcccg cacaagcggt 900
ggagcatgtg gtttaattcg aagcaacgcg aagaacctta ccaggtcttg acatcctctg 960
acaatcctag agataggacg tccccttcgg gggcagagtg acaggtggtg catggttgtc 1020
gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttgatctta 1080
gttgccagca ttcagttggg cactctaagg tgactgccgg tgacaaaccg gaggaaggtg 1140
gggatgacgt caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggac 1200
agaacaaagg gcagcgaaac cgcgaggtta agccaatccc acaaatctgt tctcagttcg 1260
gatcgcagtc tgcaactcga ctgcgtgaag ctggaatcgc tagtaatcgc ggatcagcat 1320
gccgcggtga atacgttccc gggccttgta cacaccgccc gtcacaccac gagagtttgt 1380
aacacccgaa gtcggtgagg taacctttag gagccagccg ccga 1424
Claims (4)
1. A method for preparing and using seed biostimulant SB-MGW9 is characterized in that the seed biostimulant SB-MGW9 is coatedComprises (1) ecological investigation and collection of bacteria source samples; (2) separating and screening strains; (3) purifying the strain; (4) researching the characteristics of the target strain; (5) measuring the growth promoting capability of the strain; (6) identifying the strain; (7) preparing seed biostimulant; (8) detecting the actual effect of the application of the seed biostimulant; in the operation (1), according to the development target of the biological stimulin, the bacterial source is extremely arid soil (100.88E, 38.84N) near Ming Great Wall (Ming Great Wall) in Shandan county in Gansu province; in the operation (2), 1-2 drops of strain suspension liquid with 10 times of serial dilution concentration are dipped in an inoculating loop by adopting a dilution method, the inoculating loop is inoculated on a solid beef extract peptone culture medium plate containing NaCl with different concentrations by adopting a coating method, and after the culture is carried out for 6-7 days at constant temperature of 28 ℃, the growth condition of the strain is observed; in the operation (3), based on the growth condition of flat plate bacteria, colonies growing on solid beef extract peptone containing 12% NaCl and originating from extreme drought soil homogenate of great wall of Ming are taken, purified by adopting a flat plate marking method, and cultured for 48 hours at 28 ℃ to obtain a strain MGW 9; in the operation (4), the strain MGW9 has the capability of fixing nitrogen, decomposing inorganic phosphorus and organic phosphorus and producing indoleacetic acid; in the above operation (5), MGW9 bacterial solution (1.0X 10)8—1.5×108cfu/mL) for soaking the corn seeds for 6h before sowing, keeping the moisture for 12h, and effectively improving the germination activity of the corn seeds under the condition of salt stress after the corn seeds are dried; in the above-mentioned operation (6), MGW9 was determined to be Bacillus by morphological identification and molecular identification, and was namedBacillus sp.MGW9, CGMCC No. 18690; in the operation (7), the biostimulant of the target seed is BacillusBacillus sp.MGW9（1.0×108—1.5×108cfu/mL), and adjusting the pH value of the bacterial liquid to 7.0-8.0; in the above operation (8), the diluted seed biostimulant (SB-MGW 9, 1.0X 10)8—1.5×108cfu/mL), soaking the corn seeds for 6h, preserving moisture for 12h, and sowing after drying, thereby effectively improving the field emergence rate of the corn seeds in the saline-alkali soil.
2. The method of claim 1 for preparing and using a seed biostimulant SB-MGW9, wherein: in the operation (4), the strain MGW9 has salt tolerance, and the salt tolerance range of the strain MGW9 is 0% -12.0%.
3. The method of claim 1 for preparing and using a seed biostimulant SB-MGW9, wherein: in the operation (4), the bacterial strain MGW9 is added into a King's B liquid culture medium containing 100 mg/L tryptophan, and the Kovacs indigo substrate reagent turns red after the bacterial strain is cultured; after Salkowsk color development, OD530nm is 0.482, and the content of indoleacetic acid is 19.24 mg/L; inoculating a strain MGW9 on a solid organophosphorus culture medium, culturing at 28 ℃ for 7d to generate a transparent ring, wherein the ratio of the diameter of the phosphorus-dissolving ring to the diameter of a bacterial colony is 1.7; the indexes of germination vigor, germination rate, bud (seedling) length, main root length, bud (seedling) fresh weight/10 plants, root fresh weight/10 plants, bud (seedling) dry weight/10 plants (105 ℃, 17h +/-1 h), root dry weight/10 plants (105 ℃, 17h +/-1 h), simulated field emergence rate and the like of the MGW9 bacterial solution after the treatment of the corn seeds are obviously higher than those of a control (without seed soaking treatment).
4. The method of claim 1 for preparing and using a seed biostimulant SB-MGW9, wherein: in the operation (6), the morphological identification result of the strain MGW9 is that the bacterial colony is circular, the diameter is about 3-5 mm, and the bacterial colony is milky white; the surfaces of the bacterial colonies are smooth and wet, and the edges are neat, opaque and convex; gram staining is positive, and cells are rod-shaped; the molecular identification result of MGW9 strain is that the 16SrDNA gene sequence fragment size is 1424bp, and the classified named Bacillus is suggestedBacillus sp.。
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