阅读说明：本技术 一种提高玉米种子萌发耐盐性生物刺激素制备及应用方法 (Preparation and application method of biological stimulin for improving salt tolerance of corn seed germination ) 是由 江绪文 李贺勤 于 2019-05-29 设计创作，主要内容包括：本发明涉及种子科学领域,特别是关于一种提高玉米种子萌发耐盐性生物刺激素制备及应用方法。从青岛崂山藿香植株根系中分离筛选、纯化鉴定获得重要菌株<I>Pantoea agglomerans</I> strain HX-1,研究发现其具有产吲哚乙酸、溶磷、耐盐等能力,其菌液浸种能有效提高玉米种子活力,与菌株<I>Bacillus megaterium</I> HX-2制备的生物刺激素能有效提高盐碱地玉米种子萌发出苗能力。本发明基于生物刺激素可对植物的自然进程起到刺激作用,特别是具有能够提高非生物胁迫抗力等功效,从藿香植株根系成功获得菌株HX-1,并用于目标生物刺激素的制备及应用方法研究。本发明可广泛应用于盐碱地玉米生产中。(The invention relates to the field of seed science, in particular to a preparation method and an application method of biological stimulin for improving the salt tolerance of corn seed germination. Important bacterial strain is obtained by separating, screening, purifying and identifying from roots of Laoshan agastache rugosus Pantoea agglomerans The strain HX-1 is researched and found to have the capabilities of producing indoleacetic acid, dissolving phosphorus, resisting salt and the like,the bacterial liquid can effectively improve the activity of corn seeds by soaking seeds, and the bacterial strain Bacillus megaterium The biological stimulator prepared by HX-2 can effectively improve the germination and seedling emergence capability of the corn seeds in the saline-alkali soil. The invention can stimulate the natural process of plants based on the biological stimulin, particularly has the efficacy of improving abiotic stress resistance and the like, successfully obtains the strain HX-1 from the root system of the agastache rugosus plants, and is used for the research of the preparation and application methods of the target biological stimulin. The invention can be widely applied to the production of the corn in the saline-alkali soil.)

1. A preparation and application method of biological stimulin for improving salt tolerance of corn seed germination is characterized by comprising (1) collecting a bacterium source sample; (2) bacterial strainsSeparating and screening; (3) purifying the strain; (4) measuring the growth promoting capability of the strain; (5) identifying the strain; (6) preparing biological stimulin; (7) detecting the actual effect of the application of the biostimulant; in the operation (1), according to the research and development target of the biological stimulin, the bacterial source is the fresh agastache plant tissue grown in the Laoshan of Qingdao; in the operation (2), after the surfaces of fresh agastache rugosus plant tissue samples are successfully sterilized, 100 mu L of homogenate of different tissue samples diluted by 10 times in series is taken by a homogenate method and coated on a solid beef extract peptone culture medium flat plate containing NaCl with different concentrations, and the growth condition of bacteria is observed after culturing at constant temperature of 28 ℃ for 48 hours; in the operation (3), based on the growth condition of flat plate bacteria, colonies growing on solid beef extract peptone containing 7% NaCl and derived from root system tissue homogenate of agastache rugosus plants are taken, purified by adopting a flat plate scribing method, and cultured for 48 hours at 30 ℃ to obtain a strain HX-1; in the operation (4), the strain HX-1 has the capacity of producing indoleacetic acid and dissolving organic phosphorus; HX-1 bacterial liquid (1.0X 10)⁸—1.5×10⁸cfu/mL) for 12 hours before sowing, the germination activity of the corn seeds under the condition of salt stress can be effectively improved; in the above-mentioned operation (5), the determination that HX-1 is Pantoea agglomerans, named "Pantoea agglomerans", by combining morphological identification and molecular identification is madePantoea agglomeransstrain HX-1; in the operation (6), the target biostimulant is Pantoea agglomeransPantoea agglomeransstrainHX-1（2.0×10⁸—2.5×10⁸cfu/mL), Bacillus megateriumBacillus megateriumHX-2（2.0×10⁸—2.5×10⁸cfu/mL) in a volume ratio of 1: 1, fully mixing, and adjusting the pH of the mixed bacterial liquid to 5.0-8.0; in the operation (7), the target biological stimulin is diluted by 2-3 times before use, and is taken out after the corn is soaked for 12 hours, drained and sown, so that the emergence rate of the corn in the saline-alkali soil can be effectively improved.

2. The method for preparing and using the biostimulant for improving the salt tolerance of corn seed germination as claimed in claim 1, wherein: in the operation (3), the strain HX-1 has certain salt tolerance, and the salt tolerance range is 0.5% -7.0%; the pH range of suitable growth of strain HX-1 is 4.0-8.0.

3. The method for preparing and using the biostimulant for improving the salt tolerance of corn seed germination as claimed in claim 1, wherein: in the operation (4), the strain HX-1 is added into a King-chi B liquid culture medium containing 100 mg/L tryptophan, and the Kovacs indigo substrate reagent turns red after the strain is cultured; after Salkowsk color development, OD530nm is 0.151, and the content of indoleacetic acid is 29 mg/L; inoculating the strain HX-1 on a solid organophosphorus culture medium, and culturing at 30 ℃ for 7D to generate a transparent ring, wherein the ratio of the diameter (D) of the phosphorus-dissolving ring to the diameter (D) of a bacterial colony is 1.5; after the corn is treated by the HX-1 bacterial liquid, indexes such as germination potential, germination rate, seedling (bud) length, main root length, seedling (bud) fresh weight/10 plants, root fresh weight/10 plants, seedling (bud) dry weight/10 plants (105 ℃, 8 h), root dry weight/10 plants, simulated field emergence rate and the like are all obviously higher than those of a control (non-seed soaking treatment).

4. The method for preparing and using the biostimulant for improving the salt tolerance of corn seed germination as claimed in claim 1, wherein: in the operation (5), the morphological identification result of the strain HX-1 is that the bacterial colony is circular, yellow, neat in edge, wet and smooth in surface, negative in gram stain and short rod-shaped; the molecular identification result of HX-1 strain is that the size of 16SrDNA gene sequence fragment is 1444bp, and the fragment is matched with Pantoea agglomeransPantoea agglomerans(FJ 592996.1) the similarity rate reaches 99.0%.

Technical Field

The invention relates to the field of seed science, in particular to a preparation method and an application method of biological stimulin for improving the salt tolerance of corn seed germination.

Background

Biostimulant is an extremely fashionable term that has emerged in the global agricultural market in recent years. By international definition, a biostimulant is a substance that contains certain components and microorganisms whose effects, when applied to a plant or rhizosphere, are stimulatory for the plant's natural processes, including enhancing/benefiting nutrient uptake, nutritional efficacy, abiotic stress resistance, and crop quality, regardless of nutritional composition. 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, seed soaking in warm soup, seed soaking and dressing in fertilizers, trace elements, 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 so as to ensure that uniform, uniform and strong seedlings are realized after field sowing, and the yield and the quality of plants in the later period can be continuously influenced.

In recent years, development, improvement and utilization of low-yield fields such as dry thin lands, saline-alkali lands and the like in China are one of effective ways for increasing crop planting area and increasing yield, particularly, high-quality seeds are also an important basis and guarantee for improving yield per unit through green development and utilization of saline-alkali lands and the like, so that deep improvement of seed quality becomes one of the most important subjects for development of new-era breeding. Corn is used as the first large grain crop in China, and the key of grabbing corn production and grabbing continuous and stable development of grains is particularly emphasized in the text of corn treatise by the Hanchang of the country. Therefore, new products of the seed biostimulant are continuously researched and developed and are applied to the production of crops such as corn and the like, so that the capability of crop seeds in adversity of emergence of seedlings and the like is improved to guarantee the yield and the quality of the crops, and the method has great significance for accelerating the conversion of new and old kinetic energies in China and guaranteeing the effective supply of future grains in China.

At present, few reports are reported for improving the adversity germination and seedling emergence capability of corn seeds by using a seed biostimulant product in China. Soil salinization is the most common problem in agricultural production worldwide, and ion toxicity and osmotic stress generated by the soil salinization have great influence on the growth and development of plants, and directly influence the yield and the quality. The breeding of salt-tolerant crops is a feasible solution, but the successful examples at present are few, and the improvement of the salt-tolerant growth capability of the existing cultivated crops becomes an effective strategy. Few reports are reported for improving the saline-alkali resistance of crops such as corn and the like by separating substance components such as microorganisms and the like to research and develop biological stimulin products, and huge market space and development potential are provided.

Disclosure of Invention

The invention aims to meet the market demand of the seed biostimulant and overcome the defects of the prior art, and provides a preparation and application method of the biostimulant for improving the salt tolerance of corn seed germination.

In order to achieve the purpose, the invention is based on that biological hypohormones have stimulation effect on the natural process of plants, and particularly have the effect of improving the resistance of the plant growth to abiotic stress and the like. Around the aim of improving the salt resistance of corn seed germination, possible source samples of target beneficial microorganisms are widely collected, and research, development and application work of improving the salt resistance of corn seed germination of biostimulant is completed through a series of operations such as separation and identification. The invention adopts the following technical scheme to obtain a preparation and application method of biological stimulin for improving the salt tolerance of corn seed germination, which mainly comprises the following operations: (1) collecting a bacteria source sample; (2) separating and screening strains; (3) purifying the strain; (4) measuring the growth promoting capability of the strain; (5) identifying the strain; (6) preparing biological stimulin; (7) and (5) detecting the actual effect of the application of the biostimulant.

In the operation (1), according to a research and development target, collecting a bacteria source sample, and identifying and storing the sample; the bacterial source of the invention is a root system of a Laoshan mountain Agastache rugosa plant in Qingdao.

In the operation (2), salt-tolerant strains are separated and screened step by step on the agastache rugosus root endophytic bacteria on the solid beef extract peptone containing NaCl with different concentrations by adopting a homogenization method.

In the operation (3), a plate-streaking method is adopted to purify the strain to obtain a candidate strain, and the salt resistance, the acid resistance, the alkali resistance and the like of the candidate strain are measured.

In the operation (4), hormone-producing indoleacetic acid, phosphorus dissolving capacity and the like are carried out on the candidate strain; preparing a bacterial solution, and performing standard germination test, seedling growth determination, field emergence rate simulation determination and the like after seed soaking treatment of test corn seeds so as to identify the growth promotion effect.

In the above-mentioned operation (5), morphological feature identification and 16S rDNA species molecular identification are performed on the objective colonies.

In the operation (6), the target biological stimulin is prepared by taking the target bacterial liquid as a main effective component.

In the operation (7), the practical effect verification and the use method research of the production and application of the target biological stimulin saline-alkali soil corn are carried out.

The invention has the beneficial effects that: the biological stimulin can stimulate the natural process of plant, and has especially the effect of raising abiotic stress resistance. In order to improve the salt resistance of corn seed germination, a target biostimulant is prepared by collecting a bacteria source sample, separating and screening strains, purifying the strains, measuring the growth promoting capacity of the strains, identifying the strains and the like, and a use method is established by combining with the application effect verification of the saline-alkali soil; 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 condition of salt stress 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 effect of NaCl and pH on the growth of strain HX-1 according to the invention.

FIG. 3 shows the effect of HX-1 bacterial liquid treatment on germination potential and germination rate of corn seeds under salt stress.

FIG. 4 shows the effect of HX-1 bacterial liquid treatment on the growth of maize seedlings under salt stress.

FIG. 5 shows the effect of HX-1 bacterial liquid treatment on the simulated field emergence rate of corn seeds under salt stress.

FIG. 6 shows the 16S rDNA gene sequence of the strain HX-1 of the present invention.

FIG. 7 is a phylogenetic tree of the strain HX-1 according to the invention.

Detailed Description

The invention is described in detail below with reference to the figures and examples.

As shown in figure 1, the invention is based on that the biostimulant can stimulate the natural process of plants, and substances such as beneficial bacteria of microorganisms are used as an important composition form of the biostimulant, so that the development and the utilization of the biostimulant are beneficial to improving the seed vigor and continuously influencing the yield and the quality of the plants in the later period. The preparation and application method of the biological stimulin for improving the salt tolerance of the corn seed germination comprises the operations of strain source sample collection, strain separation and screening, strain purification, strain growth promoting capability determination, strain identification, biological stimulin preparation, biological stimulin application actual effect detection and the like.

The invention relates to a preparation method and an application method of biological stimulin for improving salt tolerance of corn seed germination.

(1) Collecting a bacteria source sample: according to a biological stimulator research and development target, selecting a plant endophyte source, and registering information such as a collected sample name, specific time, a collected sample location and the like; the bacterial source is fresh agastache rugosus plant tissue collected from 9-month Qingdao Laoshan (longitude 120.47; dimensionality 36.10) in 2015 (the sample is identified as an agastache rugosus plant by Tanyong professor of Yangxi traditional Chinese medicine university in Guangxi province of original Qingdao agricultural university).

(2) Separating and screening strains: bacteria were separated and screened on solid beef extract peptone with different concentrations of NaCl by homogenization. Firstly, cleaning the surface of a fresh agastache rugosus plant tissue sample (comprising roots, stems, leaves, flowers and the like); secondly, soaking the sample in 70% ethanol for 1min under a sterile environment, taking out the sample, and washing the sample for 3 times by using sterile water; the sample was then placed in 2% NaClO containing 0.5 mL/L Tween-20₃After the solution is shaken and soaked for 20min, the solution is taken out and washed for 3 times by sterile water (the washing liquid for the 3 rd time is collected), and the surface floating water of the sample is sucked by sterile absorbent paper for standby; and finally, taking 100 mu L of the 3 rd washing liquid to coat on a solid beef extract peptone culture medium flat plate, culturing for 48h to observe whether the culture medium has bacterial growth, if not, indicating that the surface of the sample is successfully sterilized, otherwise, re-sterilizing. Different tissue samples (including roots, stems, leaves, flowers and the like) of the agastache rugosus plants with sterilized surfaces are respectively placed in a sterile mortar for fully grinding, then the grinding fluid is gradually diluted by 10 times in series, 100 mu L of homogenate of the tissues of the roots, the stems, the leaves, the flowers and the like is respectively taken and coated on a solid beef extract peptone culture medium plate containing 6 NaCl with different concentrations, such as 0.5%, 1%, 3%, 5%, 7%, 9% and the like, and the solid beef extract peptone culture medium plate is placed in a constant-temperature culture box for culture at 28 ℃ for 48 hours for observing the growth condition of bacteria.

(3) Strain purification: based on the observation of the growth of flat plate bacteria, the root tissue samples are of great interest, and include that a small amount of bacterial colonies grow on the solid beef extract peptone containing 7% NaCl, and no bacterial colonies grow on the solid beef extract peptone containing 9% NaCl. Selecting a colony growing on solid beef extract peptone containing 7% NaCl, purifying the colony by adopting a plate marking method, culturing for 48 hours at 30 ℃ to obtain a strain HX-1 (HuoXiang-1), and carrying out supplementary tests such as salt resistance, acid and alkali resistance and the like aiming at the strain.

Salt resistance: adding 0.1mL of HX-1 bacterial liquid in the logarithmic phase of growth into 40 mL of beef extract peptone liquid culture medium with the NaCl content of 0.5%, 1.0%, 3.0%, 5.0%, 7.0% and 9.0%, culturing for 24h at the constant temperature of 30 ℃ by using a shaking table at 200 r/min, measuring the light absorption value of the bacterial liquid OD600nm, and repeating for 3 times by using the beef extract peptone liquid culture medium without inoculation as a control.

The result shows that the strain HX-1 has certain salt tolerance, and the salt tolerance range is 0.5-7.0% (see figure 2).

Acid and alkali resistance: adding 0.1mL of HX-1 bacterial liquid in the logarithmic phase of growth into 40 mL of beef extract peptone liquid culture medium with pH of 4, 5, 6, 7, 8, 9, 10, culturing for 24h at a constant temperature of 30 ℃ by a shaking table at 200 r/min, measuring the light absorption value of the bacterial liquid OD600nm, and repeating for 3 times by using the beef extract peptone liquid culture medium without inoculation as a control.

The results showed that strain HX-1 grew properly at a pH in the range of 4.0 to 8.0 (see FIG. 2).

The strain HX-1 takes 25% glycerol as a freezing protective agent, and the strain HX-1 is stored in an ultra-low temperature refrigerator at-80 ℃, wherein the storage places are as follows: the agricultural college of Qingdao agricultural university seed science and engineering laboratory.

(4) And (3) measuring the growth promoting capability of the strain: firstly, measuring the capability of producing hormone indoleacetic acid and dissolving phosphorus of a strain HX-1; secondly, treating corn seeds by using HX-1 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).

Hormone production indoleacetic acid determination: firstly, carrying out shake culture on a 30 ℃ constant temperature 200 r/min bacterial liquid for 48h on a King's B liquid culture medium according to the specification of a Kovacs indigo substrate kit (Qingdao Haibo biotechnology Co., Ltd.), wherein the color turns red to indicate that the endogenous bacterium HX-1 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 measuring the production of hormone indoleacetic acid 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 endophyte HX-1 has the capability of producing indoleacetic acid. After Salkowsk color development, OD530nm was 0.151, and the indoleacetic acid content was 29mg/L as calculated by the standard curve equation y =0.005x +0.006 (R2 =0.9994, y represents absorbance and x represents concentration).

And (3) measuring the phosphorus dissolving capacity: the HX-1 strain is spotted on a solid organophosphorus culture medium, the culture is carried out for 7D at the temperature of 30 ℃, the existence of a phosphorus-dissolving ring is observed, the ratio of the diameter (D) of the phosphorus-dissolving ring to the diameter (D) of a bacterial colony is determined, the ratio is 1, the phosphorus-dissolving capacity is not existed, and 3 times of repetition are carried out.

The determination result of the phosphorus dissolving capacity is as follows: a transparent ring is generated on an organophosphorus culture medium plate, and the ratio of the diameter (D) of a phosphorus-dissolving ring to the diameter (D) of a bacterial colony is 1.5, which indicates that the endophyte HX-1 has the capacity of dissolving organophosphorus.

Standard germination test and seedling growth determination: using Zhengdan 958, Denghai 618 and Ludan 981 corn variety samples as materials (collected in 2017 without coating), and performing paper rolling germination according to crop seed inspection regulation and 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; adjusting the concentration of HX-1 bacterial liquid which is cultured by shaking at the constant temperature of 30 ℃ and 200 r/min for 48h to be 1.0 multiplied by 108-1.5 multiplied by 108 cfu/mL; soaking sterilized corn seeds in a bacterial solution for 12h (Treatment, T), taking out, selecting 200mmol/L NaCl solution to wet germinating paper (American Anchor Anke germinating paper) on the basis of earlier research, alternately placing the germinating paper on a bed, rolling the germinating paper, vertically placing the germinating paper in an artificial climate box in a dark place at 25 ℃, germinating for 3 times, taking the non-soaked germinating paper as a Control1 (Control 1, C1) and the liquid culture medium as a Control2 (Control 2, C2) for 100 repeated times (3 rolls), counting the Germination vigor (Germination Energy, GE) at 4d and counting the Germination rate (Germination Percentage, GP) at 7 d; 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 percentage is counted, 10 seedlings are randomly selected from each repetition, and the indexes of seedling (bud) length, main root length, seedling (bud) fresh weight/10 seedlings, root fresh weight/10 seedlings, seedling (bud) dry weight/10 seedlings (105 ℃, 8 h), root dry weight/10 seedlings (105 ℃, 8 h) and the like are measured.

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 by using a plastic germination box. 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, placing the seeds in a bed in order 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 was carried out in 2017 in the seed science and engineering laboratory of Qingdao agricultural university from 8 months to 9 months.

From the standard germination test results (see fig. 3): compared with C1 and C2, T can obviously improve the germination vigor and the germination rate of 3 corn variety-like varieties under the condition of salt stress, and the difference is obvious.

From the results of the seedling growth test (see fig. 4): 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 the salt stress of seeds of 3 corn variety samples, and the difference of other indexes is obvious except that the difference of T and C1 and C2 is not obvious in the indexes of 3 corn varieties including Lu single 981 root dry weight/10 plants.

According to standard germination tests and seedling growth determination results, after the seeds are soaked in HX-1 bacterial liquid for 12 hours, the germination of corn seeds and the seedling growth capacity under the salt stress condition can be effectively promoted.

In view of simulating field rate of emergence measurements (see fig. 5): compared with C1 and C2, T can obviously improve the emergence rate of the seeds of 3 corn variety samples in the simulated field, and the difference is obvious.

(5) And (3) strain identification: the strain HX-1 is identified from both morphological and molecular aspects.

And (3) morphological identification: the strain HX-1 is cultured for 48h at 30 ℃ on a beef extract peptone solid medium, 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 bacterial colony is round and yellow, the edge is neat, the surface is wet and smooth, gram staining is negative, and the bacterial colony is short rod-shaped.

And (3) molecular identification: carrying out 16SrDNA molecular identification on the bacterial liquid inoculated into the beef extract peptone liquid culture medium and subjected to 30-1 constant temperature 200 r.min-shaking culture for 48h, and repeating for 3 times; extracting HX-1 genome by using a bacterial genome DNA rapid extraction kit (purchased from Biotechnology engineering (Shanghai) Co., Ltd.), performing PCR amplification by using the genome as a template, wherein the universal 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 size of the 16SrDNA gene sequence fragment of the HX-1 strain is 1444bp (shown in figure 6); logging in NCBI to carry out Blast comparison, selecting 10 bacterial strains with higher similarity 16SrDNA gene sequences to construct NJ phylogenetic tree, HX-1 and Pantoea agglomeransPantoea agglomerans(FJ 592996.1) was clustered in one branch with a similarity of 99.0%, indicating that HX-1 was closest to the strain (see FIG. 7).

Combining morphological characteristics and 16S rDNA gene sequence analysis results, judging HX-1 to be Pantoea agglomerans namedPantoea agglomeransstring HX-1. 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 collection center registration number (CGMCC number 17646); survival is detected by the collection center in 2019, 4 and 29 days; the proposed classification is named:Pantoeaagglomerans。

(6) preparing the biological stimulator: based on the previous stage, the endophytic bacterium HX-2 Bacillus megaterium separated from the healthy agastache leaf (HX-2)Bacillus megaterium) The bacterial strain HX-2 can secrete indoleacetic acid and dissolve inorganic phosphorus, the pH range of the bacterial strain HX-2 suitable for growth is 5.0-9.0, the NaCl content can grow under the condition of 0.5% -8.0%, and other important conclusions (Jiangyou, Li Shijiu, Tantang Yong. the identification and tolerance of the wrinkled giant hyssop endophytic bacteria HX-2 and the growth promoting effect on host plants. the herbage academic newspaper, 2018, 27: 161-168). Tests show that the effect of improving the salt stress resistance of corn seed germination is better when HX-1 and HX-2 are mixed, so HX-2 is also used for preparing the target biological stimulin.

The biostimulant is Pantoea agglomeransPantoea agglomeransStrain HX-1 (2.0X 108-2.5X 108 cfu/mL), Bacillus megateriumBacillus megateriumHX-2 (2.0X 108-2.5X 108 cfu/mL) in a volume ratio of 1: 1, fully mixing, and adjusting the pH of the mixed bacterial liquid to 5.0-8.0.

And (3) detecting the actual effect of the application of the biostimulant: in 2018, a field emergence rate test of saline-alkali soil was carried out in Dongying and Nethereto in Shandong, wherein the seed spacing was 0.03m, the row spacing was 0.05m, the row length was 0.3m, and the test was repeated for 3 times. Diluting the biostimulant by 2-3 times before sowing, taking seeds of the Zhengdan 958 corn variety as a test material, soaking the seeds for 12h, draining, sowing, taking the seeds which are not soaked as a control, and counting the rate of emergence in the field 15 days after sowing. The implementation effect is as follows: compared with the contrast, the treated seeds have faster emergence and more regular emergence, and the emergence rates in the fields of two fields are respectively improved by 8.3 percent and 10.2 percent.

In conclusion, the biological hypohormone can stimulate the natural process of plants, and particularly has the effects of improving abiotic stress resistance and the like. In order to improve the salt resistance of corn seed germination, a biological stimulator for improving the salt resistance of corn seed germination is developed, wherein an important strain pantoea agglomeransPantoea agglomeransStrain HX-1 has the capability of producing indoleacetic acid and dissolving phosphorus, can effectively improve the germination activity of corn seeds under the condition of salt stress, and is combined with the bacillus megaterium obtained in the earlier stageBacillus megateriumHX-2 successfully prepares the improved jadeThe rice seed germination salt-tolerant biostimulant lays an important foundation for later-stage 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

<120> preparation and application methods of biological stimulin for improving salt tolerance of corn seed germination

<130>2019104555632

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>1444

<212>DNA

<213> Pantoea agglomerans HX-1(Pantoea agglomerans strain HX-1)

<400>1

tgaatgcgac ccgcagctac catgcagtcg gacggtagca cagagagctt gctctcgggt 60

gacgagtggc ggacgggtga gtaatgtctg gggatctgcc cgatagaggg ggataaccac 120

tggaaacggt ggctaatacc gcataacgtc gcaagaccaa agagggggac cttcgggcct 180

ctcactatcg gatgaaccca gatgggatta gctagtaggc ggggtaatgg cccacctagg 240

cgacgatccc tagctggtct gagaggatga ccagccacac tggaactgag acacggtcca 300

gactcctacg ggaggcagca gtggggaata ttgcacaatg ggcgcaagcc tgatgcagcc 360

atgccgcgtg tatgaagaag gccttcgggt tgtaaagtac tttcagcggg gaggaaggcg 420

acggggttaa taaccctgtc gattgacgtt acccgcagaa gaagcaccgg ctaactccgt 480

gccagcagcc gcggtaatac ggagggtgca agcgttaatc ggaattactg ggcgtaaagc 540

gcacgcaggc ggtctgttaa gtcagatgtg aaatccccgg gcttaacctg ggaactgcat 600

ttgaaactgg caggcttgag tcttgtagag gggggtagaa ttccaggtgt agcggtgaaa 660

tgcgtagaga tctggaggaa taccggtggc gaaggcggcc ccctggacaa agactgacgc 720

tcaggtgcga aagcgtgggg agcaaacagg attagatacc ctggtagtcc acgccgtaaa 780

cgatgtcgac ttggaggttg ttcccttgag gagtggcttc cggagctaac gcgttaagtc 840

gaccgcctgg ggagtacggc cgcaaggtta aaactcaaat gaattgacgg gggcccgcac 900

aagcggtgga gcatgtggtt taattcgatg caacgcgaag aaccttacct actcttgaca 960

tccacggaat ttggcagaga tgccttagtg ccttcgggaa ccgtgagaca ggtgctgcat 1020

ggctgtcgtc agctcgtgtt gtgaaatgtt gggttaagtc ccgcaacgag cgcaaccctt 1080

atcctttgtt gccagcgatt cggtcgggaa ctcaaaggag actgccggtg ataaaccgga 1140

ggaaggtggg gatgacgtca agtcatcatg gcccttacga gtagggctac acacgtgcta 1200

caatggcgca tacaaagaga agcgacctcg cgagagcaag cggacctcac aaagtgcgtc 1260

gtagtccgga tcggagtctg caactcgact ccgtgaagtc ggaatcgcta gtaatcgtgg 1320

atcagaatgc cacggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccatgg 1380

gagtgggttg caaaagaagt aggtagctta accttcggga gggcgctacc acatgataca 1440

gtac 1444