Enterobacter saccharum and application thereof

Abstract

The invention discloses enterobacter sacchari and application thereof. Relates to the technical field of plant functional microorganisms. The invention provides a strain as well as a fermentation product and application thereof. The enterobacter saccharum pnrt01 provided by the invention has the functions of fixing nitrogen, dissolving phosphorus and dissolving potassium, and can effectively promote the growth of peanuts and increase the yield. Under a potted plant, the pnrt01 promotes the fresh weight and the dry weight of a peanut plant to be increased by 11.86% and 9.09% respectively; the fresh weight and the dry weight of the stems are respectively increased by 23.22% and 13.64%; the fresh weight and the dry weight of the leaves are respectively increased by 10.48% and 9.45%; the fresh weight and the dry weight of pods are respectively increased by 14.36% and 30.02%. Under the field condition, the pnrt01 promotes the peanut legume number to be increased by 43.64%, the legume fresh weight to be increased by 29.57%, and the legume dry weight to be increased by 20.37%. The pnrt01 strain has the characteristics of low cost, no residue and safety to human and livestock when being used for dressing peanuts, and the yield of the peanuts can be obviously increased.

Description

Technical Field

[0001] The invention relates to the technical field of plant functional microorganisms, and more particularly to a strain of Enterobacter saccharolyticus and application thereof. Background Art

[0002] Peanut (Arachis hypogaea L.) is an important leguminous crop used for both food and oil. Its planting area is second only to soybean and rapeseed. Its yield, total output and export volume are the highest among oil crops in China, and it occupies an important position in the food structure of my country and even the world. Nitrogen is an indispensable element for promoting plant growth and development. Excessive nitrogen fertilizer application seriously limits the nodulation of peanuts and their nitrogen fixation function. How to achieve sustainable management of soil nitrogen cycle and the utilization of biological nitrogen fixation (BNF) has become a top priority.

[0003] Plant growth-promoting bacteria (PGPB) are a type of beneficial bacteria that colonize the rhizosphere and roots of plants, effectively promote plant growth and development, and help plants cope with external biological and abiotic stresses. They are of great significance to the green development of crops. For leguminous plants, the discovery of efficient nitrogen-fixing probiotic strains for inoculation can restore their own nodulation and nitrogen-fixing ability, thereby effectively promoting plant growth and development and ensuring stable and high yields of peanuts while reducing the amount of fertilizer applied.

[0004] Screening highly efficient multifunctional microbial strains (such as bacteria that can fix nitrogen, dissolve phosphorus, and dissolve potassium) from the natural ecological environment can effectively regulate the content ratio of key nutrients in the rhizosphere soil of plants, meet the sustainable demand for nutrients in different developmental stages of plants, reduce the application of fertilizers, especially nitrogen fertilizers, and thus achieve green agricultural development.

[0005] Therefore, whether it is possible to provide a strain of bacteria that can be used to promote plant growth and overcome the above-mentioned technical deficiencies is an urgent problem that technical personnel in this field need to solve. Summary of the invention

[0006] In view of this, the present invention provides a strain of Enterobacter sacchariflorus and its application, which has the effect of promoting peanut growth and increasing peanut yield. The use of the strain to promote peanut growth has a good prospect. Using the microorganism to dress peanut seeds can effectively reduce the application of nitrogen fertilizer and increase peanut yield.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] Deposit information: pnrt01 (Enterobacter sacchari), deposited in the General Microbiology Center of China Culture Collection Administration, the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCC: NO.32445, and the deposit date is October 31, 2024.

[0009] A strain of Enterobacter sacchari pnrt01, classified and named Enterobacter sacchari, is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC: NO.32445, and the deposit date is October 31, 2024.

[0010] The present invention also provides a fermentation product of the Enterobacter saccharolyticus pnrt01.

[0011] The present invention also provides a bacterial agent containing the above-mentioned Enterobacter saccharolyticus pnrt01 or the above-mentioned fermentation product.

[0012] The invention also provides a seed dressing agent containing the above-mentioned Enterobacter saccharigenes pnrt01 or the above-mentioned fermentation product.

[0013] The present invention also provides the use of the Enterobacter saccharolyticus pnrt01, the fermentation product, the bacterial agent or the seed dressing agent in agricultural production.

[0014] Preferred: Seed dressing is used to promote peanut growth, reduce the application of chemical fertilizers, and increase peanut yield.

[0015] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a strain of Enterobacter saccharolyticus and its application, and the technical effects achieved are:

[0016] Enterobacter saccharigenicus pnrt01 has the functions of nitrogen fixation, phosphorus solubilization and potassium solubilization.

[0017] Enterobacter saccharigenicus pnrt01 can effectively promote the growth and yield of peanuts. Under potted conditions, pnrt01 promoted the increase of fresh weight and dry weight of peanut plants by 11.86% and 9.09% respectively; stem fresh weight and dry weight increased by 23.22% and 13.64% respectively; leaf fresh weight and dry weight increased by 10.48% and 9.45% respectively; pod fresh weight and dry weight increased by 14.36% and 30.02% respectively. Under field conditions, pnrt01 promoted the increase of peanut pod number, pod fresh weight, and pod dry weight by 43.64%, 29.57%, and 20.37% respectively. Peanut seed dressing with pnrt01 strain has the characteristics of low cost, no residue, and safety for humans and animals, which can significantly increase peanut yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0019] Figure 1 The accompanying drawing is a diagram for functional identification of the strain provided by the present invention.

[0020] Figure 2 The accompanying drawing is an analysis diagram of the effects of different nitrogen levels and soil sterilization conditions on the growth-promoting properties of peanuts provided by the present invention.

[0021] Figure 3 The attached figure is a diagram showing the effect of the strain provided by the present invention on the biomass of peanut.

[0022] Figure 4 The attached figure shows the effect of the strain provided by the present invention on the lateral roots, needles, pods and nodules of peanut.

[0023] Figure 5 The accompanying drawing is a diagram showing the effect of the strain provided by the present invention on peanut yield. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] The embodiment of the invention discloses a strain of Enterobacter saccharolyticus and application thereof.

[0026] The invention uses a selective culture medium to separate the Enterobacter sacchari (strain) pnrt01 strain from the root tip of peanut. The colony on the LB culture medium (10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride (NaCl), 18 g of agar, supplemented with water to 1000 mL, pH 7) is milky white, convex, and has irregular edges. The colony diameter is 0.2-1.3 mm after being cultured at 37° C. for 96 hours, the optimum growth temperature is 35° C., and the optimum growth pH value is 7.2. The strain is preserved by patent procedure.

[0027] The raw materials not mentioned in the examples are all commercially available, and the methods not mentioned are conventional experimental methods. The data are summarized as follows:

[0028] During the fruiting period of peanuts, complete peanut plants were selected, and the substrate on the surface of the roots and the dust on the surface of the leaves were washed with deionized water. After wiping off the surface moisture, the peanut plants were divided into four parts: roots, stems, leaves, and pods. The fresh weight of each part was weighed using a balance. Subsequently, the samples were placed at 105°C for 30 minutes, and then dried at 80°C to constant weight, and the dry weight of the roots, stems, leaves, and pods was weighed respectively. The software Graphpad Prism 9.0 was used for data analysis.

[0029] There are no requirements for the peanut varieties used in the verification test, as long as they meet the experimental needs.

[0030] Example 1

[0031] Functional identification of strains

[0032] Nitrogen-fixing function: The isolated and purified pnrt01 strain was inoculated into Ashby medium (mannitol or glycerol) supplemented with Congo red solution 10.0 g, KH2PO4 0.2 g, MgSO4·7H2O 0.2 g, NaCl 0.2 g, CaSO4·7H2O 0.1 g, CaCO3 5.0 g, and the pH was adjusted to 7.0 with 1 mol / L NaOH. After sterilization, 10 mL of 0.5% Congo red solution (sterilized with a 0.22 μm filter membrane) was added to the culture medium at 50-60°C and mixed. After culturing in a microbial constant temperature incubator for 3 days, it was taken out and observed. Those with transparent circles have nitrogen-fixing function.

[0033] Potassium-solubilizing function: The isolated and purified pnrt01 strain was dipped into the prepared Alexander Rof medium (sucrose 5.0 g, Na2HPO4 2.0 g, FeCl3 0.005 g, MgSO4·7H2O 0.5 g, CaCO3 0.1 g, potassium feldspar powder (washed 5 times with deionized water) 1.0 g, bromothymol blue (BTB) 0.1 g, agar 18 g, pH adjusted to 7.0 with 1 mol / L NaOH) five times with an inoculation loop. After inoculation, the plate was placed in a microbial constant temperature incubator for 3 days, and then the plate was taken out for observation. If the area around the strain turned yellow, it had potassium-solubilizing function.

[0034] Phosphorus-dissolving function: The isolated and purified pnrt01 strain was inoculated with an inoculation loop into the pre-prepared NBRIP inorganic phosphorus-dissolving medium: glucose 10.0 g, Ca3(PO4)25.0 g, MgCL25.0 g, MgSO40.25 g, KCl 0.2 g, (NH4)2SO40.1 g, agar 18 g, and the pH was adjusted to above 7.0 with 1 mol / L NaOH. After inoculation, the strain was placed in a microbial constant temperature incubator for 3 days. A transparent circle appeared in the culture medium around the strain, indicating that it had the ability to dissolve phosphorus.

[0035] Figure 1 The results showed that the pnrt01 strain has the functions of nitrogen fixation, potassium solubilization and phosphorus solubilization.

[0036] Example 2

[0037] Cultivation of strains and peanut seed dressing

[0038] Test tube slant seed culture of pnrt01 strain: The strain was cultured on a slant of modified beef extract peptone solid medium (10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride (NaCl), 18 g of agar, made up to 1000 mL with water, pH 7.0-7.3), and cultured at 25-37°C for 2-4 days to obtain slant seeds. The appropriate culture condition is culture at 35°C for 3 days.

[0039] Liquid culture of pnrt01 strain liquid seeds: inoculate the slant seeds into LB liquid medium (without agar) in a triangular flask, and culture on a shaker at 28-37°C and 100-250rpm for 3-6 days. The optimal culture condition is 35°C and 250rpm for 3 days.

[0040] Large-scale culture of pnrt01 strain in fermenter: Inoculate the liquid seed into the LB liquid medium in the fermenter at a ratio of 1-10‰ (V / V), and control the culture conditions in the 10-10000L fermenter at: temperature 25-37°C, stirring speed 100-200rpm, fermentation time 2-4d. The optimal culture conditions are 35°C, 250rpm, and 3d.

[0041] Peanut seed dressing: The pnrt01 strain and its metabolites obtained by fermentation tank culture were concentrated (the number of viable bacteria was 2-5×10 9 / mL or more), and store at low temperature (4°C) to obtain bacterial liquid.

[0042] Then add the bacterial solution and peanut seeds into the seed mixer and stir to ensure that the bacterial strain is evenly attached to the peanut seeds.

[0043] Technical effect verification

[0044] Pot experiment 1: Application of pnrt01 in nitrogen reduction in peanut.

[0045] Experimental setup:

[0046] Treatment 1 (soil sterilization + nitrogen N4.5 + pnrt01 seed dressing): The peanut seeds were first sterilized with 3% H2O2, and then treated with >10 8The seeds were treated with the fermentation liquid of the strain with CFU / mL, and then the peanut seeds were planted in high-temperature sterilized soil. Nitrogen fertilizer (N4.5: 1.1622g (4.5kg / mu) per pot) was added in the early stage of flowering.

[0047] Treatment 2 (soil sterilization + nitrogen N0 + pnrt01 seed dressing): The peanut seeds were first sterilized with 3% H2O2, and then treated with >10 8 The seeds were treated with the fermentation liquid of the strain with a concentration of 100 CFU / mL and then planted in sterilized soil. No nitrogen fertilizer was added in the early stage of flowering.

[0048] Treatment 3 (normal soil + nitrogen N4.5 + pnrt01 seed dressing): The peanut seeds were first sterilized with 3% H2O2, and then treated with >10 8 The seeds were treated with the fermentation liquid of the strain with a concentration of CFU / mL, and then planted in non-sterile soil and nitrogen fertilizer was added in the early stage of flowering (N4.5: 1.1622g of fertilizer per pot).

[0049] Plant height was measured 14 days after peanut seedlings emerged, and the plant height (height from cotyledon node to plant top / cm) was measured every three days with a ruler, and the total measurement time was 60 days.

[0050] Results Figure 2 .

[0051] Figure 2 (a) Results: Under sterile conditions and without nitrogen fertilizer application, the plant height of the pnrt01 seed treatment was higher than that of the pnrt01 seed treatment with nitrogen (N4.5) at 14-44 days of peanut growth. This indicates that nitrogen affects the growth-promoting function of the pnrt01 strain on peanuts.

[0052] Figure 2 (b) Results: Under the same nitrogen condition (N4.5), peanut plants were taller in the non-sterilized soil than in the sterilized soil when the peanuts were growing for 14-44 days. The results showed that the pnrt01 strain can adapt to complex soil environments, promote peanut growth, and has development and application value.

[0053] Potted plant experiment 2:

[0054] Treatment group: Peanut seeds (Xinbaihua 15) were soaked in the bacterial solution prepared in Example 2 for 5 min to ensure that the surface of the seeds was fully adhered to the bacterial solution (B: Enterobacter sacchari strain pnrt01).

[0055] CK group: using sterile fermentation liquid (LB liquid medium) to mix seeds (A: CK);

[0056] The seeds that have completed the seed dressing treatment are planted in pots of the same size (unsterilized soil), and one month after planting, a fertilizer with a pure nitrogen content of 4.5 kg / mu is applied to the peanuts (Note: the pure nitrogen content in production is usually about 10 kg / mu).

[0057] Results Figure 3 :

[0058] Seed dressing with pnrt01 bacterial solution has a significant promoting effect on the growth and development of peanuts. The results of peanuts treated with pnrt01 compared with the control (CK) are shown in Figure 3 :

[0059] Figure 3 Middle (a) Results: pnrt01 promotes peanut growth and development phenotype;

[0060] Figure 3 Middle (b, f) Results: The average root fresh weight and dry weight of the aboveground part of peanut increased by 11.86% and 9.09%, respectively;

[0061] Figure 3 Middle (c, g) Results: Stem fresh weight and dry weight increased by 23.22% and 13.64%, respectively;

[0062] Figure 3 Middle (d, h) Results: leaf fresh weight and dry weight increased by 10.48% and 9.45%, respectively;

[0063] Figure 3 Results in (e, i): The fresh weight and dry weight of pods increased by 14.36% and 30.02%, respectively.

[0064] pnrt01 promoted root development and nodule formation, and affected the number of lateral roots, needles, pods, and nodules in peanuts. The results are shown in Figure 4 :

[0065] Figure 4 (a) Results: The number of lateral roots of peanuts treated with pnrt01 increased by 12.04% compared with the control;

[0066] Figure 4 (b) Results: The average number of needles inserted in peanuts increased by 38.65%;

[0067] Figure 4 (c) Results: The average number of peanut pods increased by 42.35%;

[0068] Figure 4 (d) Results: The number of root nodules per plant increased by 12.55% on average.

[0069] Field trials:

[0070] Treatment group: The bacterial solution prepared in Example 2 was used for seed dressing at a dosage of 500 mL / 30 kg and 1000 mL / 30 kg:

[0071] Control group: seeds were mixed with sterile fermentation liquid (LB liquid culture medium sterile).

[0072] Experimental setup: The experiment was set up with three biological replicates, and the area of ​​each replicate unit plot was 3 meters wide and 15 meters long, totaling 45 square meters. During the peanut harvest season, three points were selected in each unit plot, and 20 plants were randomly selected at each point. The number of pods was counted and the average fresh weight was weighed. After drying, the average dry weight was weighed. The results are shown in Figure 5 :

[0073] Figure 5 (a) Results: 1000mL (pnrt01) / 30kg seeds significantly promoted the number of flowers and pods (P=0.0291), increasing by 43.64% relative to the control;

[0074] Figure 5 (b) Results: pnrt01 can promote the growth and increase the yield of peanuts. On average, the fresh weight of pods of 20 peanut plants increased by 29.57%, and the dry weight of pods increased by 20.37%.

[0075] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0076] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of Enterobacter sacchari pnrt01, characterized in that: It is classified and named Enterobactersacchari and deposited in the General Microbiology Center of China Culture Collection Administration. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC: NO.32445 and the deposit date is October 31, 2024.

2. The fermentation product of Enterobacter saccharolyticus pnrt01 according to claim 1.

3. A bacterial agent containing the Enterobacter saccharolyticus pnrt01 according to claim 1 or the fermentation product according to claim 2.

4. A seed dressing containing the Enterobacter saccharolyticus pnrt01 according to claim 1 or the fermentation product according to claim 2.

5. Use of the Enterobacter saccharolyticus pnrt01 according to claim 1, the fermentation product according to claim 2, the bacterial agent according to claim 3 or the seed dressing agent according to claim 4 in agricultural production.

6. The use according to claim 5, characterized in that The seed dressing agent is used for promoting the growth of peanuts, reducing the application of chemical fertilizers and increasing the yield of peanuts.

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