CN121652995A - Pantoea H1 and demonstration method and application thereof in agricultural aid - Google Patents

Abstract

This application relates to the field of microbial technology, disclosing a strain of *Pantotheca acuminata* H1 and its demonstration methods and applications in agricultural assistance. This *Pantotheca acuminata* H1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36875. The invention also provides a microbial agent containing this strain, its preparation method, and its application in crop cultivation. This strain possesses highly efficient dual phosphorus-solubilizing ability, capable of dissolving both insoluble inorganic phosphorus and mineralizing organic phosphorus, thereby improving soil phosphorus availability. Simultaneously, it exhibits excellent salt and alkali tolerance, maintaining high activity even under adverse conditions. Applying this agent during the seedling stage can significantly improve the rhizosphere microenvironment, promote root development and plant growth in crops such as corn and mung beans, and increase seedling emergence and yield in saline-alkali soils. It provides excellent microbial germplasm resources for activating soil nutrients and reducing fertilizer use while increasing yield.

Description

Pantoea H1 and demonstration method and application thereof in agricultural aid

Technical Field

The invention relates to the technical field of microorganisms, in particular to pantoea H1, an demonstration method of the pantoea H1 in agricultural assistance and application of the pantoea H1.

Background

Phosphorus is the second most essential nutrient element for limiting plant growth, and plays a vital role in the energy metabolism, signal transduction and growth and development processes of crops. In agricultural production, most of the phosphorus in the soil exists in a poorly soluble form that is difficult for crops to absorb and utilize. In order to maintain crop yield, a large amount of chemical phosphate fertilizer is typically applied during agricultural planting. However, phosphorus fertilizer applied to soil is extremely easily fixed by metal ions such as calcium, iron, aluminum and the like in the soil, and about 75% -90% of phosphorus is rapidly converted into an ineffective form and accumulated in the soil. The phenomenon not only causes extremely low utilization rate of phosphate fertilizer in seasons and serious waste of nonrenewable phosphate rock resources, but also causes loss of excessive accumulated phosphorus along with surface runoff, and is extremely easy to cause environmental pollution risks such as water eutrophication and the like.

The use of phosphorus-dissolving microorganisms to convert poorly soluble inorganic and organic phosphorus in soil to soluble phosphorus that is absorbable by crops is considered a safe, economical and effective biological route to solve the above-mentioned problems. Activated sludge is used as a resource library rich in microorganisms and is reserved with rich functional strain resources, but the development of phosphorus-dissolving strains with agricultural application potential is not enough at present. Although a variety of phosphorus-solubilizing bacteria have been isolated in the prior art, many challenges remain in practical agricultural applications. On one hand, the existing phosphorus-dissolving microorganism strain is relatively limited in variety, and the problem of poor environmental adaptability generally exists, particularly under the condition of stress soil such as saline-alkali soil, the exogenous strain is difficult to effectively colonize or maintain high biological activity, so that the field application effect is unstable. On the other hand, the phosphorus activation mechanism of part of strains is single, and the efficient dissolution of inorganic phosphorus and the full mineralization of organic phosphorus in soil are difficult to realize simultaneously, so that the accumulated phosphorus in the soil cannot be excavated and utilized to the greatest extent. Therefore, the method screens and obtains an excellent strain with high-efficiency double phosphorus dissolving capability and good environmental tolerance, and has important application value for developing novel high-efficiency biofertilizer, activating soil nutrients and guaranteeing grain safety.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a pantoea H1 and an demonstration method and application thereof in agricultural assistance, and the problems that the existing phosphorus-dissolving microorganism has poor environmental adaptability (especially saline-alkali tolerance) and single phosphorus-dissolving mechanism, so that indissolvable phosphorus accumulated in soil cannot be effectively activated and utilized and the yield-increasing effect of crops is unstable are solved.

In order to achieve the aim, the invention is realized by the following technical scheme that a pantoea H1 and an exemplary method and application thereof in agricultural assistance are provided, and the invention provides an obvious pantoea (Pantoea conspicua) H1 by adopting the following technical scheme:

The strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.36875 and the preservation address of the strain is No.3 of Xila No. 1, north Star in the Korean region of Beijing, and the preservation date of the institute of microorganisms of the national academy of sciences of China is 2025, 12 months and 02 days.

By adopting the technical scheme, the pantoea H1 provided by the invention has the following remarkable technical effects:

The double phosphorus dissolving property is that the strain has double capacities of effectively dissolving insoluble inorganic phosphorus (such as tricalcium phosphate) and mineralizing organic phosphorus (such as calcium phytate). In the metabolic process, the strain can change the pH value of the environment and secrete related enzymes, and the phosphorus immobilized in the soil is converted into water-soluble phosphorus which can be absorbed and utilized by crops, so that the effectiveness of the phosphorus in the soil is improved.

The strain sieve is selected from specific environments and has good tolerance to environments with high pH value (8.5) and high salt concentration (60 mmol/L mixed saline-alkali). Under the stress of saline and alkaline, the strain can still keep higher biological activity and reproductive capacity, and is suitable for colonization in the soil environment of saline and alkaline land.

Preferably, the 16SrRNA gene sequence of the pantoea H1 is shown as SEQ ID No. 1. By adopting the technical scheme, the molecular biological classification characteristics of the strain are clarified, the singleness of strain sources and the stability of genetic background are ensured, and the quick identification and tracking of the strain are facilitated by a genetic means.

In a second aspect, the invention provides a microbial agent containing the pantoea H1, which adopts the following technical scheme:

The microbial agent comprises the pantoea H1 thallus and an agriculturally acceptable carrier, wherein the carrier is a liquid culture medium carrier or a solid matrix carrier.

By adopting the technical scheme, the carrier is utilized to provide necessary nutrition support and physical living space for the strain, the shelf life of the strain is prolonged, and the carrier is convenient for transportation and application in field production.

Preferably, the microbial agent is prepared by inoculating a strain of pantoea H1 into a liquid culture medium, culturing for 8-24 hours at the temperature of 25-30 ℃ and the rotating speed of 160-200 rpm, or mixing the liquid microbial agent with a solid substrate carrier according to the mass ratio of 1:2-1:4, piling and fermenting at the temperature of 30-35 ℃ and drying to obtain the solid microbial agent. By adopting the technical scheme, the specific temperature control and fermentation parameters (25 ℃ to 30 ℃ and 8 to 24 hours) enable the thalli to be in the middle and later stages of the logarithmic growth phase, so that the thalli are vigorous in metabolism and biomass accumulation reaches the peak value, the effective viable count is high, and the thalli decay caused by overcultivation is avoided. The stacking technology in the solid fermentation process promotes the adsorption and combination of thalli and carriers, and improves the survival rate of the microbial inoculum in soil.

Preferably, the liquid culture medium carrier comprises tryptone, yeast extract powder and sodium chloride, the solid matrix carrier comprises turfy soil and wheat bran, and the mass ratio of the turfy soil to the wheat bran is (2-4): 1. By adopting the technical scheme, the liquid carrier component provides rich carbon and nitrogen sources, high activity of thalli is maintained, the turfy soil in the solid carrier has good adsorptivity and water retention, the wheat bran provides slow-release organic nutrition, and the microenvironment formed by the combination of the turfy soil and the wheat bran is favorable for quick colonization of the strain at the early stage of soil application.

In a third aspect, the present invention provides an exemplary method for agricultural cultivation of the pantoea strain H1, which adopts the following technical scheme:

An exemplary method for using the pantoea H1 in agricultural aid comprises the following steps of S1 preparing a microbial agent, namely providing the pantoea H1 or the microbial agent, S2 applying the pantoea H1 or the microbial agent to crops or planting soil in a seed soaking, root irrigation or soil accompanying mode.

By adopting the technical scheme, the invention utilizes the rhizosphere growth promotion action mechanism of the pantoea H1 to realize crop yield increase and soil improvement, and the specific action mechanism is as follows:

Rhizosphere colonization and acidolysis phosphorus, namely colonization of an applied pantoea H1 in crop rhizosphere soil. During the metabolic process, the strain secretes small molecular organic acids such as gluconic acid, citric acid and the like, which results in the reduction of the pH value of the rhizosphere microenvironment. The high concentration of hydrogen ions (H ⁺) exchanges with calcium ions in the poorly soluble phosphates (such as Ca ₃(PO₄)₂) to disrupt the phosphate lattice structure, thereby releasing hydrogen phosphate ions (HPO ₄ ^2- or H ₂PO₄ ^-).

The bacterial strain secretes acid phosphatase and phytase and other extracellular enzymes, acts on organic phosphorus compounds (such as phytate) in soil, catalyzes the hydrolytic cleavage of phosphate bonds, converts the organic phosphorus into inorganic phosphorus, and remarkably improves the quick-acting phosphorus content of the soil.

The method improves saline-alkali microecology, namely, in saline-alkali soil, the organic acid secreted by the strain can neutralize the too high alkalinity around the root system, reduce the pH value of the rhizosphere, relieve the corrosion and poison of carbonate to the root system of crops, and simultaneously, the improved rhizosphere physicochemical environment promotes the absorption of nutrient elements such as nitrogen, phosphorus and the like by the crops, and improves the tolerance of the crops to saline-alkali stress.

Preferably, in step S2, the specific manner of application treatment is selected from any one of the following:

(1) Root irrigation treatment, namely diluting a liquid microbial agent by 100-200 times with water, and irrigating along the root soil of crops, wherein the application amount is reduced to 1-2L of stock solution used per mu;

(2) Immersing crop seeds in the liquid microbial agent, immersing for 2-4 hours in the dark, taking out, drying in the shade and sowing;

(3) And (3) carrying out soil tracing treatment, namely mixing the solid microbial agent with the organic fertilizer and applying the mixture in the planting ditch.

By adopting the technical scheme, different application modes are suitable for different agricultural production scenes. The seed soaking treatment ensures that the strain is colonized at the seed coat and radicle at the initial stage of seed germination to provide early protection, the root irrigation treatment ensures that bacterial liquid directly acts on a root system dense area, the utilization efficiency of the strain is improved, and the soil accompanying treatment is combined with the application of organic fertilizer to provide continuous nutrition sources for the strain and prolong the action time.

Preferably, the application treatment is performed during the seedling stage of the crop, and the application treatment comprises at least two consecutive applications performed at intervals of 5-7 days. By adopting the technical scheme, the application is selected in seedling stage (such as corn three-leaf one-heart stage and mung bean first-pair true leaf stage), and the key window period of crop root system development and nutrient requirement is grasped. The continuous application at intervals can supplement the quantity of flora lost due to environmental competition, maintain the population advantage of a strain of pantoea H1 in rhizosphere soil, and ensure the continuous phosphorus dissolving and growth promoting effect.

Preferably, the crops are corn or mung beans, the planting soil is saline-alkali soil, and the saline-alkali soil is saline-alkali soil with pH of 8.0-9.0. By adopting the technical scheme, the applicability of the method under specific crop and adverse soil conditions is verified, and particularly in moderate saline-alkali soil, the problem of low utilization rate of chemical fertilizer is solved by a biological improvement means.

In a fourth aspect, the invention provides an application of the pantoea strain H1 in preparing biofertilizer or soil conditioner, which adopts the following technical scheme:

application of Pantoea H1 in preparing biofertilizer or soil conditioner is provided.

By adopting the technical scheme, the wild strain pantoea H1 with specific phosphorus dissolving and stress resisting functions is converted into standardized agricultural input products. The application not only develops new microorganism germplasm resources, but also provides an environment-friendly and efficient biological solution for solving the problems of soil phosphorus solidification, difficult saline-alkali soil utilization, excessive chemical fertilizer application and the like in agricultural production.

Preferably, the effective viable count of the pantoea H1 in the biofertilizer or the soil conditioner is more than or equal to 1.0X10 ⁸ CFU/mL or more than or equal to 2.0X10 ⁸ CFU/g.

By adopting the technical scheme, the quality index of the product is limited. The high concentration of the effective viable bacteria ensures that the flora after being applied into the soil occupies the advantages of ecological niches in competition with indigenous microorganisms, and ensures the stability and the effectiveness of the biofertilizer or the soil conditioner in practical field application.

The invention provides a pantoea H1, an exemplary method for assisting agriculture and application thereof. The beneficial effects are as follows:

The pantoea H1 provided by the invention has excellent phosphorus activation capability, and shows remarkable dissolution and mineralization effects on common insoluble inorganic phosphorus (tricalcium phosphate) and organic phosphorus (calcium phytate) in soil. The strain reduces the pH value of the environment by secreting organic acid in the metabolic process, destroys inorganic phosphate lattices by acidolysis, and simultaneously secretes phosphatase substances to catalyze the hydrolysis of organic phosphorus. The dual mechanism can effectively release the fixed potential phosphorus resources in the soil, and convert ineffective phosphorus into water-soluble phosphorus which can be directly absorbed and utilized by crops, so that the dependence on chemical phosphorus fertilizer in agricultural production is reduced, and soil hardening and degradation caused by excessive fertilization are relieved.

The strain sieve is selected from specific environments, has natural tolerance to high pH value and salt stress, and can still keep higher reproductive activity and colonisation ability under a moderate saline-alkali environment (pH is 8.5). The strain is applied to the saline-alkali soil, and the metabolic acid-producing activity of the strain in the rhizosphere micro-domain can effectively neutralize alkaline substances, regulate the acid-base balance of the rhizosphere and improve the physicochemical environment of root system growth. The improvement effect of the rhizosphere microecology relieves the poison of saline-alkali stress to crop root systems, improves the germination rate and biomass accumulation of crops such as mung beans and the like under adverse conditions, and provides an effective technical means for the bioremediation and utilization of saline-alkali soil.

The invention is based on a microbial agent prepared by pantoea H1, and can improve the agronomic characters and yield constitution of crops by matching with the application processes of root irrigation or seed soaking in the seedling stage, and the field test shows that the technical scheme can effectively promote the root elongation and plant height of corn and mung bean, optimize the root-cap ratio, reduce the length of the corn bald tip and improve the weight of single spike and hundred grains. Through the synergistic effect of improving rhizosphere nutrition supply and enhancing plant stress resistance, the strain can remarkably improve the acre yield of crops and realize the quality improvement and the efficiency improvement of agricultural production.

Drawings

FIG. 1 is a schematic diagram of a phylogenetic tree of Pantoea H1 of the present invention based on the 16SrRNA gene sequence;

Wherein, the evolution relation analysis of the strain H1 of the figure 1 and other bacterial groups is based on the gene sequence of the 16SrRNA, a phylogenetic tree is constructed by adopting an adjacent method (Neighbor-training), and all phylogenetic analysis is completed by MEGA11 software;

Through BLAST comparison and phylogenetic analysis, the homology between the 16SrRNA sequence of the strain H1 and Pantoea conspicua (obvious pantoea) reaches 100%, the two sequences are gathered into one branch in a phylogenetic tree, and the H1 is initially identified as the species by combining a classical classification threshold;

FIG. 2 is a schematic diagram showing the determination of the phosphorus-dissolving effect of Pantoea H1 of the present invention;

FIG. 3 is a schematic diagram showing the result of gram staining of Pantoea H1 of the present invention;

FIG. 4 is a schematic diagram showing the growth promoting effect of Pantoea sativa H1 on potted mung beans;

FIG. 5 is a schematic diagram showing the growth of corn after pantoea H1 is implemented;

FIG. 6 is a schematic representation of the effect of pantoea H1 of the invention on maize fruits after implementation.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Preparation examples 1-5:

Preparation example 1 screening, separation and identification of Pantoea H1 Strain

This preparation describes the acquisition of an apparent Pantoea (Pantoea conspicua) H1 strain. Activated sludge in an aeration tank of a sewage treatment plant was collected, 1.0g of activated sludge was weighed and put into a conical flask containing 20mL of sterile distilled water, and shake-cultured at room temperature for 30 minutes to prepare a suspension. The supernatant was aspirated and serially diluted to 10-fold, 100-fold and 1000-fold concentrations using sterile water. 100 mu L of each gradient dilution is sucked and coated on LB solid medium plates respectively, and the plates are placed in a 28 ℃ constant temperature incubator reversely for standing culture for 24 hours. After the colony of the plate grows out, single colony is selected according to colony morphology, three rounds of streak purification are carried out on the LB plate, and purified strains are obtained. The purified strain was inoculated onto a plate of an organic phosphorus screening medium (containing calcium phytate) and cultured at 28℃for 72 hours, and the diameter (D) of the phosphate solubilizing circle and the diameter (D) of the colony were measured, and an excellent strain having a D/D value of 4.25 was selected and designated as H1.

The strain H1 was identified by culturing on LB plates for 3 days, then subjecting to gram staining and microscopic examination, and the result showed that the cells were red, and the cells were judged to be gram-negative. The genomic DNA of the strain was extracted, and PCR amplification and Sanger sequencing were performed using the 16SrRNA universal primers 27F and 1942R to obtain the sequence shown in SEQ ID No. 1. BLAST alignment of the sequences in NCBI database and construction of phylogenetic tree confirmed that strain H1 was significantly Pantoea (Pantoea conspicua). The strain is mixed with 80% glycerol according to the volume ratio of 1:4, and the mixture is placed in a refrigerator at-80 ℃ for preservation.

Preparation example 2 preparation of a Pantoea H1 liquid microbial agent (reference Process)

The preparation example corresponds to the preparation method of bacterial liquid mainly used in the subsequent examples and test examples. Taking out a stored pantoea H1 glycerol tube from a refrigerator at-80 ℃, streaking and inoculating the pantoea H1 glycerol tube on an LB solid plate through an inoculating loop, and performing inverted culture at 37 ℃ for 24 hours for activation. Single colonies were picked and inoculated into 250mL triangular flasks containing 50mLLB liquid medium and cultured with shaking at 37℃and 180rpm for 12 hours to obtain seed solutions. The seed solution was transferred to a 500mL Erlenmeyer flask containing 200mL of fresh LB liquid medium at an inoculum size of 1% (v/v), and cultured under shaking at 37℃and 180rpm until the optical density value OD ₆₀₀ of the bacterial solution reached 1.0, and the culture was stopped. The obtained fermentation broth is a pantoea H1 liquid microbial agent, and the detection result of the effective viable count is 1.5X10 ⁸ CFU/mL.

Preparation example 3 preparation of a Pantoea H1 liquid microbial agent (Low temperature Long time Process)

This preparation was intended to verify the viability of the strain in culture at lower temperatures. Inoculating the activated pantoea H1 strain in preparation example 2 into an LB liquid medium to prepare seed liquid. The seed solution was inoculated into fresh LB liquid medium at an inoculum size of 2% (v/v), the culture temperature was adjusted to 28℃and the shaking table rotation speed was set to 160rpm, and the culture time was prolonged to 24 hours. After the completion of the cultivation, the OD ₆₀₀ of the bacterial liquid was measured and found to be 1.1, and the number of viable bacteria of the obtained liquid was measured and found to be 1.2X10. 10 ⁸ CFU/mL. The microbial inoculum prepared by the process is used for supporting the generalization of the lower fermentation temperature range in the claims.

Preparation example 4 preparation of a Pantoea H1 liquid microbial agent (high temperature short time Process)

This preparation was designed to verify the rapid fermentation capacity of the strain at higher inoculum sizes. Inoculating the activated pantoea H1 strain in preparation example 2 into an LB liquid medium to prepare seed liquid. The seed solution was inoculated into fresh LB liquid medium at an inoculum size of 5% (v/v), the culture temperature was set at 30℃and the shaking table rotation speed was set at 200rpm, and the culture time was shortened to 10 hours. After the completion of the culture, the bacterial liquid OD ₆₀₀ was measured to be 0.95, and the number of viable bacteria of the obtained liquid was measured to be 1.4X10. 10 ⁸ CFU/mL. The microbial inoculum prepared by the process is used for summarizing different inoculum sizes and culture times in the claims.

Preparation example 5 preparation of Pantoea H1 solid microbial fertilizer

The preparation example shows the process of preparing solid preparation with the strain. Commercial turfy soil and wheat bran are mixed according to the mass ratio of 3:1 to be used as a carrier, crushed and sieved by a 60-mesh sieve, and then sterilized by high-pressure steam for 30 minutes at 121 ℃, and dried for standby. Mixing a pantoea H1 liquid microbial inoculum with OD ₆₀₀ =1.0 obtained in preparation example 2 with sterilized and cooled carrier according to the mass ratio of liquid to solid being 1:3. Fully and uniformly stirring under the aseptic condition, and stacking and fermenting for 48 hours in a 30 ℃ aseptic room, wherein the stacking is carried out for 2 times. After fermentation, drying at 35 ℃ until the water content is 10%, crushing and packaging to obtain the pantoea H1 solid microbial fertilizer, wherein the effective viable count is 2.0X10 ⁸ CFU/g. This preparation is used to support the generalization of the claims with respect to microbial agent dosage forms and carriers.

Examples 1 to 5:

As shown in FIG. 1, example 1 this example provides a method for dissolving inorganic phosphorus by using a pantoea H1 strain, comprising the steps of sucking 1mL of a bacterial liquid from a pantoea H1 liquid microbial agent obtained in preparation example 2, placing in a sterile centrifuge tube, centrifuging at 8000rpm for 5min, discarding the supernatant, resuspending the washed cells with sterile distilled water, repeating washing for 3 times, and finally resuspending to the original volume with sterile distilled water to obtain an inoculated bacterial suspension. A250 mL triangular flask containing 50mL of inorganic phosphorus screening culture medium (3.0 g/L-5.0g/L tricalcium phosphate is used as the only phosphorus source) is taken, and the inoculum suspension is inoculated according to an inoculum size of 1% (v/v). The inoculated triangular flask is placed in a constant temperature shaking table at 28 ℃ plus or minus 1 ℃ and is subjected to shaking culture for 5-7 days at a rotating speed of 180rpm in a dark place. Treatment with an equal volume of sterile distilled water was set as a blank. Samples were taken periodically during the incubation period for subsequent determination of the water-soluble phosphorus content in the fermentation broth.

As shown in figure 2, the embodiment 2 provides a method for dissolving organic phosphorus by using a pantoea H1 strain, which comprises the following steps of washing thalli and preparing an inoculated fungus suspension, wherein the method is the same as that of the embodiment 1. A250 mL triangular flask containing 50mL of organic phosphorus screening culture medium (3.0 g/L-5.0g/L calcium phytate is used as the only phosphorus source) is taken, and inoculating bacteria suspension is inoculated according to an inoculum size of 1% (v/v). The triangular flask is placed in a constant temperature shaking table at 28 ℃ plus or minus 1 ℃ and is cultivated for 5 to 7 days at 180rpm in a dark place. Treatment with an equal volume of sterile distilled water was set as a blank. After the cultivation is finished, the supernatant is collected by centrifugation and used for measuring the water-soluble phosphorus content in the fermentation broth.

As shown in figure 3, the embodiment 3 provides a potting application method for promoting mung bean growth under saline-alkali stress by using a pantoea H1 strain, which comprises the following steps of selecting commercial mung bean seeds, selecting seeds which are full in particle, uniform in size and free from diseases and insect pests, sterilizing the surfaces, and flushing with sterile water. Preparing planting soil, and filling the sieved soil into planting pots, wherein the filling amount of each pot is consistent. Preparing a saline-alkali stress simulation solution, wherein the solute molar ratio is NaCl, na ₂SO₄:NaHCO₃:Na₂CO₃ =1:9:9:1, the total concentration is 60mmol/L, and the pH is regulated to 8.5+/-0.1. Before sowing, the saline-alkali solution is used for soaking basin soil, sowing is carried out when the soil humidity is proper, and 5 grains are sowed in each basin. And after the seedlings grow out of the first pair of true leaves, fixing seedlings, and reserving 3 seedlings with consistent growth vigor in each pot. And (3) taking the pantoea H1 liquid microbial inoculum prepared in preparation example 2 as a treating agent, and carrying out root irrigation inoculation along soil around the root of the seedling, wherein the application amount of each pot is 10mL. A blank group to which an equal amount of sterile water was applied was set. After inoculation, the pot was placed in an illuminated incubator and incubated at 28 ℃. The root irrigation is carried out for the second time on the 7 th day after the first inoculation, and the application amount is the same as the previous application amount. The culture period was 30 days in total, and after expiration, the plant height, root length, biomass and other indicators were measured.

As shown in FIG. 4, example 4 this example provides a field application method for improving corn yield and soil by using a pantoea strain H1, comprising the step of testing a test corn variety Zhengdan 958. The test uses a random block design, and sets a treatment group and a control group. In the maize seedling stage, a pantoea H1 liquid microbial inoculum (the effective viable count is more than or equal to 1.0X10 ⁸ CFU/mL) provided in preparation example 2 is used, and diluted 100-200 times by clean water to prepare diluted microbial inoculum. The method adopts a root irrigation mode, the diluted bacterial liquid is applied to the corn root soil of the treatment group, and the application amount is reduced to 1L-2L of stock solution used per mu. The control group was given an equal amount of clear water. The field management is carried out according to the conventional agronomic operation, and no chemical phosphate fertilizer is additionally applied in the whole growth period. In the mature harvesting period of corns, the corns in each district are measured for yield, including plant height, spike position height, spike length, spike thickness, bald tip length, spike weight and acre yield, and rhizosphere soil samples are collected to measure the content of available phosphorus and alkaline hydrolysis nitrogen.

As shown in FIG. 5 and FIG. 6, example 5 this example provides a method for seed soaking treatment using a pantoea H1 strain, comprising the step of using a pantoea H1 liquid microbial inoculum prepared in preparation example 3 or preparation example 4. Selecting corn or mung bean seeds, sterilizing and cleaning the surfaces of the corn or mung bean seeds, immersing the seeds in a container containing H1 liquid microbial inoculum, wherein the liquid level of the seeds is 1cm-2cm higher than that of the seeds. Soaking in dark at room temperature for 2-4 hr while stirring gently to ensure uniform contact. And after the soaking is finished, taking out the seeds, airing the seeds at a shade place until no water stain exists on the surface, and sowing the seeds immediately. Seeds soaked with sterile water were set as blank. In this example, the strain was colonized on the seed surface by seed soaking in order to provide growth promotion and stress resistance protection at the early stage of seed germination, and the method of using the seed soaking in the claims was supported.

Comparative examples 1 to 5:

Comparative example 1:

compared with example 1, the method is characterized in that an equal volume of sterile distilled water is used to replace a pantoea H1 liquid microbial agent, and the rest are the same.

Comparative example 2:

compared with example 1, the same procedure was followed except that an equal volume of sterile LB liquid medium (without thallus) was used instead of one pantoea H1 liquid microbial inoculum, the remainder being the same.

Comparative example 3:

Compared with example 3, the method is characterized in that an equal volume of sterile distilled water is used to replace a pantoea H1 liquid microbial agent, and the rest are the same.

Comparative example 4:

Compared with the example 3, the method is characterized in that one pantoea H1 liquid microbial agent is subjected to inactivation treatment (namely, inactivated bacterial liquid) by high-pressure steam sterilization at 121 ℃ for 20 minutes before use, and the rest are the same.

Comparative example 5:

Compared with example 4, the method is characterized in that an equal volume of clear water is used to replace a strain of pantoea H1 diluted bacterial liquid, and the rest are the same.

Test examples 1-3:

Test example 1 determination of phosphorus-dissolving Capacity of Pantoea H1

Experiments prove that the phosphorus dissolution characteristic of a strain of pantoea H1 is verified by adopting a flat plate phosphorus dissolution ring method and a liquid fermentation method.

Plate phosphorus dissolution test the activated H1 strain was inoculated onto solid screening media containing tricalcium phosphate (inorganic phosphorus) and calcium phytate (organic phosphorus) according to the method of preparation example 1, and cultured at 28℃for 72 hours. The diameter (D) of the phosphate solubilizing ring and the colony diameter (D) were measured, and the D/D value was calculated. Treatment with equal amounts of sterile water was used as a blank.

Liquid fermentation experiments were performed according to the procedure of example 1 (inorganic phosphorus) and example 2 (organic phosphorus). The experimental setup was grouped as follows:

and inoculating a pantoea H1 live bacterial liquid into the H1 treatment group.

CK control group inoculated with an equal volume of sterile distilled water (corresponding to comparative example 1).

Matrix control group an equal volume of sterile LB liquid medium (corresponding to comparative example 2) was inoculated. 3 replicates were set for each treatment. After 5d of culture, taking fermentation liquor for centrifugation, taking supernatant to measure pH value, and adopting a molybdenum-antimony colorimetric method to measure the content of soluble phosphorus.

The test results are shown in Table 1.

The results analysis and conclusion show that under the condition that indissolvable inorganic phosphorus (tricalcium phosphate) or organic phosphorus (calcium phytate) is taken as the sole phosphorus source, the pH value of fermentation liquor of a CK control group and a matrix control group is not changed obviously, the content of soluble phosphorus is maintained at an extremely low level (< 10 mg/L), and the method shows that the content of background phosphorus introduced by natural environment or culture medium is extremely low and spontaneous phosphorus dissolution phenomenon is avoided.

After 5 days of culture, the pH value of the fermentation broth is significantly reduced in the treatment group inoculated with a strain of Pantoea H1. Wherein the pH of the inorganic phosphorus system is reduced from 7.20 to 4.42, and the pH of the organic phosphorus system is reduced to 5.12. With the decrease of the pH value, the content of soluble phosphorus in the fermentation broth is greatly improved, the inorganic phosphorus system reaches 421.3mg/L, and the organic phosphorus system reaches 315.7mg/L. The clear phosphorus-dissolving circles (D/D values of 4.25 and 3.68, respectively) observed in the plate experiments were consistent with the results of the liquid fermentation.

The data show that the pantoea H1 can secrete organic acid substances in the metabolic process, so that the culture environment is acidified, insoluble inorganic phosphate is converted into soluble phosphorus by acidolysis, and meanwhile, the pantoea H1 has mineralization capacity on organic phosphorus sources such as calcium phytate. The pantoea H1 has high-efficiency double phosphorus dissolving activity and can activate potential phosphorus resources in soil.

Test example 2 mung bean efficacy-promoting effect determination under saline-alkali stress

The experiment shows that the test aims to verify the actual promotion effect of the pantoea H1 on the growth of crops in the saline-alkali stress environment and eliminate the nutritional interference of the organic matter components of the bacteria.

Experimental grouping and treatment potting experiments were performed exactly as in example 3, comparative example 3 and comparative example 4.

Example 3 group (live bacteria treatment) A live bacterial solution of Pantoea H1 was applied by root irrigation.

Comparative example 3 group (blank) root drenching with sterile water.

Comparative example 4 group (inactivated control) H1 bacterial liquid after root irrigation and high temperature inactivation is applied.

Stress conditions, namely, soaking all the treated soil by 60mmol/L saline-alkali simulation solution (pH 8.5), and simulating the environment of the moderate saline-alkali soil.

Measuring the index:

germination rate, counting the number of seedlings at 7 days after sowing, and calculating the germination rate.

Biomass index, namely carefully taking out the plants after culturing for 30 days, cleaning root soil, and sucking up surface moisture. Plant height (stem base to growth point) and root length (main root length) were measured using a ruler, and plant fresh weight was measured using an electronic balance. 3 strains were averaged for each pot and repeated 3 times.

The experimental results are shown in Table 2.

The result analysis and conclusion are that according to the data in the table 2, the mung bean plant growth of the comparative example 3 (blank control) is obviously inhibited, the germination rate is low, the root system development is poor and the biomass accumulation is less under the environment of pH8.5 and 60mmol/L saline-alkali mixed stress. The index data of comparative example 4 (inactivated bacteria) is similar to that of a blank control group, and no difference exists between the index data and the blank control group, which shows that the simple addition of the cell lysate or the culture medium component is insufficient for relieving the damage of saline-alkali stress to crops.

Example 3 (H1 viable) showed significantly better agronomic performance than the two control groups. Compared with a blank control, after the pantoea H1 viable bacteria is applied, the germination rate of mung beans is improved by about 40%, the plant height is increased by about 71.8%, the root length is increased by about 90.4%, and the fresh weight is increased by about 144.7%.

The results prove that the pantoea H1 can still maintain higher biological activity in a saline-alkali environment. The growth promoting mechanism is characterized in that living microorganisms colonize and metabolize at the rhizosphere, on one hand, pH value of the rhizosphere microenvironment is regulated through secretion of organic acid to relieve alkali stress, and on the other hand, living bacteria continuously convert fixed phosphorus in soil into plant absorbable forms, so that root elongation and nutrient absorption are promoted, and the survivability and biomass accumulation of plants under adverse conditions are enhanced. The fact that the inactivated bacterial liquid is ineffective further determines that the technical effect is derived from the vital activity of the strain rather than the substance component itself.

Test example 3 corn field yield and rhizosphere soil nutrient determination

The experiment shows that the test aims to verify the influence of the pantoea H1 on the agronomic characters, the yield constitution and the soil fertility of corn under the actual field planting condition.

Experimental arrangement field trials were conducted according to the methods of example 4 (H1 treatment group) and comparative example 5 (blank control group). The ground force is uniform in test and the conventional management is consistent.

Sampling and assay sampling assays were performed during the mature harvest period of corn.

Agronomic character investigation, namely randomly selecting 10 corn plants in each cell, and measuring plant height (from the ground to the top of the tassel), spike length and bald tip length.

And (5) measuring the yield, namely carrying out actual yield measurement on each cell. Counting the effective spike number of the community, weighing fresh spike weight, threshing, drying, calculating hundred grain weight and converting acre yield (calculated according to 14% standard water content).

And (3) analyzing soil nutrients, namely collecting 0-20cm of plough layer soil of corn rhizosphere in each cell by adopting a multipoint mixing method. And (3) after air drying and sieving, measuring the quick-acting phosphorus content of the soil by adopting a sodium bicarbonate leaching-molybdenum-antimony colorimetric method, and measuring the alkaline hydrolysis nitrogen content of the soil by adopting an alkaline hydrolysis diffusion method.

The results of the experiment are shown in Table 3.

The results analysis and conclusion show that the treatment group applying the pantoea H1 liquid microbial inoculum is superior to the clear water control group in all indexes according to the field measured data in the table 3.

In the aspect of soil nutrient, the quick-acting phosphorus content of the rhizosphere soil of the treatment group in the example 4 reaches 26.95mg/kg, and is improved by 46.31 percent compared with the control group. The data directly prove that after the pantoea H1 is colonized in a complex field soil environment, organic acid and phosphatase substances can be effectively secreted, and insoluble phosphorus fixed by calcium, iron and aluminum in the soil is converted into quick-acting phosphorus which can be directly absorbed and utilized by crops. Meanwhile, the alkaline hydrolysis nitrogen content of the soil is also improved to a certain extent, which indicates that the strain activity improves the rhizosphere microecological environment and promotes the nitrogen circulation.

The improvement of the soil nutrient availability is directly reflected on the growth and development of corn. Compared with the control group, the corn plant height of the H1 microbial inoculum is increased, the nutrition growth is vigorous, the spike length is obviously increased in the reproduction growth stage, the bald tip length is greatly shortened (about 64.84 percent is reduced), and the seed filling is full, and the pollination fertilization condition and the nutrition supply are improved. In the final yield constitution factors, the dual lifting of the weight of a single spike and the weight of hundred grains enables the yield per mu to reach 734.8kg, and the yield is increased by 19.44% compared with the conventional planting.

In conclusion, the pantoea H1 has excellent phosphate-dissolving and growth-promoting characteristics, and the nutrition condition of the root system is improved by activating the soil stock nutrient, so that the yield increase of crops is realized.

The additional strain 16SrRNA sequence (SEQ ID No. 1):

AGAGCGAGTGGTAAGCGCCCTCCCGAAGGTTAAGCTACCTACTTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATTCTGATCCACGATTACTAGCGATTCCGACTTCACGGAGTCGAGTTGCAGACTCCGATCCGGACTACGACGCACTTTGTGAGGTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATGCGCCATTGTAGCACGTGTGTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCCTTTGAGTTCCCGACCGAATCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAGCGTTCCCGAAGGCACCAAGGCATCTCTGCCAAGTTCGCTGGATGTCAAGAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTAACGCGTTAGCTCCGGAAGCCACTCCTCAAGGGAACAACCTCCAAGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTCGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACAAGACTCAAGCCTGCCAGTTTCAAATGCAGTTCCCAGGTTAAGCCCGGGGATTTCACATCTGACTTAACAGACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGGGTAACGTCAATCGACGCGGTTATTAACCGCATCGCCTTCCTCCCCGCTGAAAGTACTTTACAACCCGAAGGCCTTCTTCATACACGCGGCATGGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCTGGTCATCCTCTCAGACCAGCTAGGGATCGTCGCCTAGGTGGGCCATTACCCCGCCTACTAGCTAATCCCATCTGGGTTCATCCGATAGTGAGAGGCCCGAAGGTCCCCCTCTTTGGTCTTGCGACGTTATGCGGTATTAGCCACCGTTTCCAGTGGTTATCCCCCTCTATCGGGCAGATCCCCAGACATTACTCACCCGTCCGCCACTCGTCACCCAAGAGCAAGCTCTCTGTGCTACCGTCCGACTTGCATGTGTTAGGCCTGCCGCCAGCGTTCAATCTGAGCCAGGAGCAAACTCTCAA.

Claims (10)

1. The Pantoea H1 is characterized in that the Pantoea H1 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.36875.

2. The pantoea strain H1 as claimed in claim 1, wherein the 16SrRNA gene sequence of the pantoea strain H1 is shown in SEQ ID No. 1.

3. A microbial agent comprising a pantoea strain H1 as claimed in claim 1 or 2, characterized in that the microbial agent comprises a pantoea strain H1 and an agriculturally acceptable carrier;

the carrier is a liquid culture medium carrier or a solid matrix carrier.

4. The microbial agent according to claim 3, wherein the microbial agent is prepared by a method comprising the steps of:

Inoculating a strain of pantoea H1 into a liquid culture medium, and culturing for 8-24 hours at the temperature of 25-30 ℃ and the rotating speed of 160-200 rpm to obtain a liquid microbial agent;

Mixing the liquid microbial agent with a solid matrix carrier according to the mass ratio of 1:2-1:4, stacking and fermenting at 30-35 ℃ and drying to obtain the solid microbial agent.

5. A microbial agent according to claim 3, wherein the liquid medium carrier comprises tryptone, yeast extract and sodium chloride;

The solid matrix carrier comprises turfy soil and wheat bran, and the mass ratio of the turfy soil to the wheat bran is (2-4): 1.

6. An exemplary method of using a pantoea strain H1 as claimed in any one of claims 1 or 2 in agriculture, comprising the steps of:

s1, preparing a microbial agent, namely providing the pantoea H1 or the microbial agent;

S2, applying treatment, namely applying the pantoea H1 or microbial inoculum to crops or planting soil in a seed soaking, root irrigation or soil accompanying mode.

7. The method according to claim 6, characterized in that in step S2, the specific manner of application treatment is selected from any one of the following:

(1) Root irrigation treatment, namely diluting a liquid microbial agent by 100-200 times with water, and irrigating along the root soil of crops, wherein the application amount is reduced to 1-2L of stock solution used per mu;

(2) Immersing crop seeds in the liquid microbial agent, immersing for 2-4 hours in the dark, taking out, drying in the shade and sowing;

(3) And (3) carrying out soil tracing treatment, namely mixing the solid microbial agent with the organic fertilizer and applying the mixture in the planting ditch.

8. An exemplary method of using Pantoea H1 in agriculture according to claim 6, wherein said application treatment is carried out in the seedling stage of the crop, and said application treatment comprises at least two consecutive applications carried out at intervals of 5-7 days.

9. An exemplary method of using Pantoea H1 in agriculture according to claim 6, wherein said crop is corn or mung bean, said planting soil is saline-alkali soil, and said saline-alkali soil is saline-alkali soil with pH of 8.0-9.0.

10. Use of a strain of pantoea H1 according to any one of claims 1-2 for the preparation of biofertilizer or soil amendment.