A heterotrophic nitrifying strain Brevundimonas DB-4 and its application

Abstract

The present invention discloses a heterotrophic nitrifying Brevundimonas DB‑4 and its application, belonging to the field of microbial technology. The heterotrophic nitrifying Brevundimonas (Brevundimonas) DB‑4 was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee on December 11, 2023, with a deposit number of CGMCC No.29279. The Brevundimonas DB‑4 provided by the present invention is derived from the decomposed matter of the mixed compost of bean cake and vegetable cypress, has a strong heterotrophic nitrification ability, can promote the conversion of ammonium nitrogen in the rhizosphere soil to nitrate nitrogen, and can effectively colonize in the soil, increase the nitrate nitrogen content in the soil and plants; at the same time, it has the ability to depotassium, thereby increasing the content of available potassium in the rhizosphere soil, can promote the improvement of crop quality, and can be widely used in the preparation of microbial fertilizers to improve the nitrogen conversion efficiency of fertilizers and reduce the application of chemical fertilizers.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a heterotrophic nitrifying Brevundimonas DB-4 and an application thereof. Background Art

[0002] Fertilizer is an important component of agricultural production materials and plays a vital role in the development of my country's agriculture. However, the efficiency-enhancing ability of fertilizers is constrained by the law of diminishing returns and has certain limitations in agricultural production. At present, the phenomenon of blind fertilization in the agricultural production stage is quite serious, resulting in reduced crop yields, soil compaction, and agricultural resource pollution. Therefore, how to research and produce new fertilizers while stabilizing crop yields, so as to achieve fertilizer reduction and efficiency increase and improve the fertilization output ratio, is a problem that needs to be solved urgently.

[0003] Nitrogen fertilizer is the fertilizer with the largest production and usage in the world. According to the nitrogen group, nitrogen fertilizer can be divided into ammonium nitrogen fertilizer, nitrate nitrogen fertilizer, ammonium nitrate nitrogen fertilizer and amide nitrogen fertilizer. After nitrogen fertilizer is applied to the soil, only 30%-40% of the applied amount is absorbed and utilized by crops, and most of the nitrogen fertilizer is lost to the environment through various channels. Nitrification is NH4 + or NH3 via NO2 - Oxidized to NO3 - The process of soil nitrogen conversion can be generally divided into autotrophic nitrification (ammonium oxidizing bacteria oxidize ammonium nitrogen to nitrate nitrogen) and heterotrophic nitrification (heterotrophic microorganisms oxidize organic nitrogen or ammonium nitrogen to nitrate nitrogen). Among them, heterotrophic nitrification can directly convert soil organic nitrogen into nitrite nitrogen and nitrate nitrogen that can be used by plants, which is beneficial to improving the soil's nitrogen supply capacity. Due to the particularity of heterotrophic nitrification, its regulatory role in soil nitrogen conversion has attracted much attention. Using heterotrophic nitrifying microorganisms to improve soil nutrient supply capacity has become an important research topic for reducing fertilizer application and increasing efficiency.

[0004] Nitrogen is one of the essential elements for plant growth and development. 3- -N) and ammonium nitrogen (NH 4+ -N) is the main form of inorganic nitrogen absorbed and utilized by plants. Nitrogen form can affect crop growth, mainly in terms of affecting crop yield and quality, crop biological traits and soil physical and chemical properties. Reasonable application of nitrogen in different forms can effectively increase crop yield and improve crop quality. Among them, nitrate nitrogen can directly participate in the synthesis of important substances such as protein, enzymes and chlorophyll in plants, thereby improving the nutritional level and photosynthetic efficiency of plants, and then promoting plant growth and increasing production. Secondly, nitrate nitrogen can also affect the quality indicators of plants such as aroma, taste, color, etc., thereby having the function of improving the quality of agricultural products, especially in fruit and vegetable production, nitrate nitrogen can make fruits sweeter and more delicious, and improve the nutritional value and taste of vegetables.

[0005] Generally speaking, dryland plants prefer nitrate, such as corn and wheat, while aquatic plants or plants growing on strongly acidic soils prefer ammonium. For open-air crops, nitrate nitrogen fertilizers are easily leached and lost after being applied to the soil, increasing the nitrogen loss rate. Compared with open-air cultivation, the supply of water, nutrients, etc. for crops grown in facility cultivation can also be effectively regulated and managed. Its yield is higher than that of traditional open-field cultivation, and it has a more stable quality guarantee, which can provide the market with a steady stream of best-selling crops. According to statistics, the current planting area of ​​modern facilities in the country has reached 40 million mu, which shows obvious characteristics of high input and high output compared with open-field cultivation. For crops, in the sealed environment of the greenhouse, compound fertilizers with excessive ammonium nitrogen and amide nitrogen content are easily decomposed and release a large amount of ammonia in the mulch film, especially in the greenhouse with double-film structure, causing ammonia damage; and after the application of nitrate nitrogen fertilizers such as ammonium nitrate and potassium nitrate, it is easy for crops to accumulate nitrates. Therefore, isolating microorganisms with strong heterotrophic nitrification ability and promoting the conversion of ammonium nitrogen is not only beneficial to improving the quality of crops that prefer nitrate nitrogen in facility cultivation, but also can reduce ammonia harm and fertilizer application, which is of great significance for the sustainable development of agriculture.

[0006] Microbial fertilizers are based on growth-promoting agents. As a safe, environmentally friendly and efficient disease prevention and growth promotion product, they can prevent and control crop diseases and reduce fertilizer input. They have become a research hotspot for crop disease prevention and growth promotion. As a new type of fertilizer, microbial fertilizers are special in that they do not directly provide nutrients to plants. Instead, they inoculate the soil with beneficial bacteria, which affect the changes in nutrients in the soil through their life activities - fixation, decomposition and secretion of bacteria, so as to provide the required nutrients for crops, thereby promoting the healthy growth of crops, increasing yields, improving the quality of agricultural products, and ultimately improving the soil environment. In the past, research on microbial fertilizers has focused on the development of growth-promoting agents with functions such as growth promotion, disease prevention and soil fertility improvement. Growth-promoting bacteria discovered at home and abroad include more than 20 species such as Pseudomonas, Bacillus, Agrobacterium, Flavobacterium, Serratia and Brevundimonas. At present, there are many types of Brevundimonas that can dissolve algae and promote plant growth, but there are few studies on the heterotrophic nitrification ability of Brevundimonas, especially on the application of Brevundimonas in promoting the release of soil nitrate nitrogen and improving crop quality. Summary of the invention

[0007] The purpose of the present invention is to provide a heterotrophic nitrifying Brevundimonas DB-4 and its application to solve the problems existing in the above-mentioned prior art. The Brevundimonas DB-4 provided by the present invention has strong heterotrophic nitrification ability and phosphorus and potassium solubilization effect, can promote the conversion of ammonium nitrogen in rhizosphere soil into nitrate nitrogen, and thus promote the improvement of crop quality.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides a heterotrophic nitrifying Brevundimonas DB-4, which was deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration on December 11, 2023, with a deposit number of CGMCC No.29279.

[0010] The present invention also provides a microbial agent, the active ingredient of which is the bacterial liquid of the heterotrophic nitrifying Brevundimonas DB-4.

[0011] Furthermore, in the microbial agent, the concentration of the heterotrophic nitrifying Brevundimonas DB-4 is 10 8 -10 9 cfu / mL.

[0012] The present invention also provides the use of the heterotrophic nitrifying Brevundimonas DB-4 or the microbial agent in improving the nitrate nitrogen content in soil.

[0013] The present invention also provides the use of the heterotrophic nitrifying Brevundimonas DB-4 or the microbial agent in improving the yield and quality of dryland plants.

[0014] Furthermore, the dryland plants include corn and wheat.

[0015] The present invention also provides the use of the heterotrophic nitrifying Brevundimonas DB-4 or the microbial agent in improving soil fertility.

[0016] Furthermore, the improving soil fertility includes promoting the conversion of ammonium nitrogen in the soil into nitrate nitrogen and promoting the content of effective phosphorus and available potassium in the soil.

[0017] The present invention discloses the following technical effects:

[0018] The Brevundimonas DB-4 provided by the present invention is derived from the mature material of the mixed compost of bean cake and Chinese cypress, has strong heterotrophic nitrification ability, can promote the conversion of ammonium nitrogen in the rhizosphere soil into nitrate nitrogen, and can effectively colonize in the soil, thereby increasing the nitrate nitrogen content in the soil and plants; at the same time, it has the ability to release potassium, thereby increasing the content of available potassium in the rhizosphere soil, and can promote the improvement of crop quality.

[0019] The Brevundimonas DB-4 provided by the present invention can be widely used in the preparation of microbial fertilizers to improve the nitrogen conversion efficiency of fertilizers and reduce the application of chemical fertilizers. The cultivation operation of the strain is simple, the cost is low, and it is safe to the environment, and it is suitable for industrial production and market promotion applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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 will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 This is the colony morphology of Brevundimonas DB-4;

[0022] Figure 2 NH4 + -N standard working curve diagram;

[0023] Figure 3 Degradation of NH4 by Brevundimonas DB-4 + -N measurement result diagram;

[0024] Figure 4 This is a graph showing the potassium solubilization ability of Brevundimonas DB-4;

[0025] Figure 5 This is the effect of Brevundimonas DB-4 on the vitamin C content of fruit corn;

[0026] Figure 6 This is a graph showing the effect of Brevundimonas DB-4 on the soluble solids content of fruit corn. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] Example 1 Isolation, screening and identification of heterotrophic nitrifying strains

[0033] The mixed compost samples of bean cake, rice bran and wheat straw were inoculated into enrichment and acclimation medium (2.0g ammonium sulfate, 14.31g sodium succinate, 50mL Weiss salt solution, 1000mL deionized water, pH 7.0) for enrichment and acclimation. The enrichment solution was transferred to a new enrichment and acclimation medium at a 10% inoculation rate every 4 days. After repeating this 4 times, the enrichment solution was diluted 10 times in a gradient, and 0.2mL of the dilution was applied to the separation medium plate. A single colony was picked and streaked for separation and purification, inoculated on a solid beef extract peptone medium (18g / L agar powder was added to the beef extract peptone liquid medium) slant, and stored at 4°C for subsequent screening. The purified strain was inoculated into beef extract peptone liquid culture medium (peptone 10g, beef extract 3g, NaCl 5g, fixed to 1000mL, pH7.0-7.2), and cultured at 160r / min and 30°C for 20h. The inoculum size of 1-3% was inoculated into a conical flask containing screening culture medium (heterotrophic nitrification culture medium, composed of ammonium sulfate 1g / L, sodium succinate 7.16g / L, Wei's salt solution 50mL, deionized water 1000mL, pH 7.0), and cultured at 160r / min and 30°C for 24h. After centrifugation, the supernatant was taken for determination of ammonia nitrogen concentration, and the strain slant with low ammonia nitrogen concentration was selected and preserved for later use.

[0034] A single colony with low ammonia nitrogen concentration was selected and streaked on a solid beef extract peptone medium for separation and purification to obtain bacterial strain DB-4. The strain was yellow in the middle with white edges on the beef extract peptone medium, opaque, moist, slightly convex, and with neat edges (see Figure 1 ). The results of 16SrDNA sequencing and BLAST comparison showed that the strain had 99% similarity with Brevundimonas, and it was identified as Brevundimonas. Its 16SrDNA base sequence is shown in SEQ ID NO.1, which was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on December 11, 2023, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 29279.

[0035] SEQ ID NO.1:

[0036]

[0037] Pick a single colony of Brevundimonas DB-4 and inoculate it into 10mL of liquid beef extract peptone medium, and culture it at 30℃ and 1500rpm for 24h to obtain seed solution. Inoculate the seed solution into a triangular flask containing 300mL of liquid LB medium at a 5% inoculation rate, and culture it at 30℃ and 150rpm for 24h to obtain Brevundimonas DB-4 bacterial solution with a concentration of 10 7 -10 9 cfu / mL. Depending on the amount of bacterial solution used, the culture can be scaled up step by step in this way.

[0038] Example 2 Determination of heterotrophic nitrification capacity of Brevundimonas

[0039] The strains obtained by preliminary separation and screening were inoculated into beef extract peptone liquid culture medium, and cultured at 30℃ and 150r / min for 24h. After the culture, the bacterial solution was centrifuged at 5000r / min for 10min, the supernatant was discarded, and the solution was washed and resuspended with sterile water. This was repeated 3 times to adjust the OD 600nm value of the bacterial solution to 0.8. The solution was inoculated into heterotrophic nitrification medium, with sterile water as the control. The culture was carried out at 30℃ and 150r / min for 48h, and the NH4 in the fermentation broth was determined. + -N content, the specific steps are as follows:

[0040] ① Take 5 mL of fermentation liquid, add 95 mL of sterile water, and dilute 20 times. Take 1 mL of the diluted liquid and place it in a 20 mL colorimetric tube, add 2 mL each of phenol solution and sodium hypochlorite alkaline solution, and shake well.

[0041] ② After placing at room temperature of 25℃ for 30 minutes, add 1mL of masking agent to dissolve possible precipitates, and then dilute to scale with distilled water. Use a 1cm colorimetric cell at a wavelength of 625nm to read the absorbance. Measure the blank control group at the same time.

[0042] ③ Experimental drawing of NH4 + -N standard curve, respectively absorb 2.5μg / mL NH4 + -N standard solution 0, 0.8, 1.6, 2.4, 3.2 and 4 mL were placed in a 20 mL colorimetric tube, and 2 mL of phenol solution and sodium hypochlorite alkaline solution were added and shaken to obtain 0, 0.1, 0.2, 0.3, 0.4 and 0.5 μg / mL of NH4 + -N standard series solution, after being placed at room temperature of 25℃ for 30min, add 1mL of masking agent to dissolve possible precipitates, and then dilute to scale with distilled water. Use a 1cm colorimetric cell at a wavelength of 625nm to read the absorbance. Draw a standard working curve ( Figure 2), and then substitute the absorbance value measured in step ② into the standard working curve to obtain the NH4 + -N content is 5.04mg / L( Figure 3 ).

[0043] ④ Calculate the degradation rate, the calculation formula is as follows:

[0044] Degradation rate = (C0-C) / C0×100%,

[0045] Where: C0 is NH4 + -N initial content, mg / L; C final is NH4 in the fermentation liquid after fermentation + -N content, mg / L. + Substituting the -N content into the formula, we can obtain the NH4 + The degradation rate of -N reached 97.6%, indicating that Brevundimonas DB-4 had a strong nitrification ability.

[0046] Example 3 Determination of the Potassium-Solving Ability of Brevundimonas

[0047] Brevundimonas was inoculated into silicate solid culture medium (2g disodium hydrogen phosphate, 5g sucrose, 0.5g magnesium sulfate heptahydrate, 0.005g ferric chloride, 0.1g calcium carbonate, 1g feldspar powder, 14g agar, 1L distilled water, pH 7.0-7.5), with 4 inoculation points per dish, repeated 3 times, and cultured at 30℃ for 5 days; if the strain forms a potassium-solubilizing circle on it, it proves that it has the ability to dissolve potassium, otherwise, it has no potassium-solubilizing ability. The results are shown in Figure 4 , Brevundimonas DB-4 formed a yellow transparent potassium-solubilizing circle on the silicate solid culture medium, indicating that Brevundimonas can convert insoluble potassium into effective potassium, which helps plants absorb potassium and thus promotes the improvement of plant quality.

[0048] Example 4 Effect of Brevundimonas DB-4 on improving corn quality

[0049] (1) The organic fertilizer + urea + microbial agent treatment was set up and divided into three treatments: ① Control treatment (CK1): base fertilizer (organic fertilizer 1000 kg / mu, compound fertilizer 20.8 kg / mu), topdressing (urea 16.047 kg / mu, potassium sulfate 9.64 kg / mu), and water as a control; ② Beef extract peptone liquid culture medium treatment (CK2): on the basis of fertilization in the control treatment, sterilized beef extract peptone liquid culture medium and water (liquid culture medium: water = 1:4, v / v); ③ Microbial agent 1 treatment (DB-4 microbial agent): on the basis of fertilization in the control treatment, a suspension of Brevundimonas DB-4 (concentration 10 8 -10 9cfu / mL) of beef extract peptone liquid medium and water (liquid medium: water = 1:4, v / v).

[0050] On September 12 and October 8, respectively, each corn plant was directly irrigated with an equal amount of different treatment agents (water, sterilized beef extract peptone liquid culture medium and water, beef extract peptone liquid culture medium inoculated with Brevundimonas DB-4 bacterial suspension and water) in each treatment. The irrigation volume was 50-100 mL. The irrigation position was the soil close to the corn root system. Other field cultivation management and pest and disease control measures were the same.

[0051] (2) Sow seeds on August 24, 2023, and grow seedlings in trays; use a 12×6 seedling tray + peat substrate, one seed per hole, water thoroughly once, cover with a layer of soil, and place the seedlings outdoors in a ventilated place.

[0052] 2023.8.30 Application of base fertilizer + tillage and ridge making: Planting in small plots, each treatment group is divided into a plot, 20m×8m, and each plot is fertilized and tilled according to the amount of base fertilizer. Each plot has 5 beds, with a wide furrow of 1.5m, and two rows are planted in each bed, with a plant spacing of 45cm, and reasonable planting.

[0053] 2023.9.5 Corn planting: When the corn seedlings grow to three leaves and one heart, select the seedlings with the same growth trend for transplanting and planting. Water them once and allow them to grow for 3 days. If any dead seedlings are found during this period, replant them in time. Remove tillers in time, leaving only one ear per corn plant, ensure adequate nutrition, and prevent and control diseases and pests in time.

[0054] (3) On October 31, during the pollination period, the biological traits of corn (stem diameter, plant height, leaf length, leaf width, ear width, ear length, number of rows per ear, and kernels per ear) were observed and recorded in each plot. On November 14, before the corn was harvested, 30 corn samples were collected from each plot, weighed, and the weight of a single ear was calculated. At the same time, the quality (soluble solids and vitamin C) was determined.

[0055] (4) Soil samples were collected from different plots. Eight sampling points were selected in each plot along an S-shaped line with a sampling depth of 0-20 cm. The collected fresh soil samples were thoroughly mixed and passed through a 2 mm sieve, placed in a sterile plastic bag, and stored in a refrigerator at 4°C for the determination of soil inorganic nitrogen, available phosphorus, and available potassium. The strain was re-isolated from the inoculated soil, but not from the control soil, confirming the colonization of Brevundimonas DB-4 in the soil.

[0056] The results of the above experiments are shown in Tables 1-3 and Figure 5-Figure 6 .

[0057] Table 1 Effects of different treatments on the biological morphology of corn

[0058]

[0059] Note: Different letters indicate significant differences among treatments at the 5% level.

[0060] Table 2 Effects of different treatments on corn yield

[0061]

[0062] Note: a indicates no significant difference among treatments.

[0063] Table 3 Effects of different treatments on soil available nitrogen, available phosphorus and available potassium

[0064]

[0065] Note: Different letters indicate that the differences among treatments reached a significant level of 1%.

[0066] As shown in Table 1, Brevundimonas DB-4 significantly changed the plant height and stem thickness of fruit corn, and there were significant differences in the experimental group (DB-4 inoculant) and the control group (CK1 and CK2) (p<0.05, t test). Compared with the control, the stem thickness increased significantly and the plant height decreased significantly. As shown in Table 2, although there were no significant differences in the ear weight, ear length, ear width, ear row and ear grain between the different treatments of corn (p>0.05, t test), the single ear weight of Brevundimonas DB-4 was 190.92g, which showed an increasing trend compared with the control (188.21g). It shows that the application of Brevundimonas DB-4 has the potential to increase corn yield. As shown in Table 3, the increase of soil nitrate nitrogen content and available potassium content can be promoted after the soil is inoculated with Brevundimonas DB-4.

[0067] Figure 5 and Figure 6 It showed that Brevundimonas DB-4 had a significant effect on improving the quality of fruit corn, and there were significant differences between the experimental group (DB-4 agent) and the control group (CK1 and CK2) (p<0.01, t test). Compared with the control, the vitamin C content ( Figure 5 ) and soluble solids content ( Figure 6 ) increased by 24.7% and 12.0% respectively.

[0068] In summary, the results of the above examples show that the Brevundimonas DB-4 isolated and obtained in the present invention has a strong heterotrophic nitrification ability, that is, the ability to promote the conversion of ammonium nitrogen into nitrate nitrogen, and also has the ability to release potassium. Field experiments have verified that soil inoculation with Brevundimonas DB-4 can promote the increase of soil nitrate nitrogen content and available potassium content, thereby promoting the improvement of plant quality, and can be widely used in the preparation of microbial fertilizers, improve the nitrogen conversion efficiency of fertilizers, and reduce the application of chemical fertilizers.

[0069] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A heterotrophic nitrifying bacterium Brevundimonas DB-4, characterized in that: It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 11, 2023, with the deposit number CGMCC No.29279.

2. A microbial agent, characterized in that: The active ingredient is the bacterial liquid of the heterotrophic nitrifying Brevundimonas DB-4 described in claim 1.

3. The microbial agent according to claim 2, characterized in that: In the microbial agent, the concentration of the heterotrophic nitrifying Brevundimonas DB-4 is 10 8 -10 9 cfu / mL.

4. Use of the heterotrophic nitrifying Brevundimonas DB-4 according to claim 1 or the microbial agent according to claim 2 in increasing the nitrate nitrogen content in soil.

5. Use of the heterotrophic nitrifying Brevundimonas DB-4 according to claim 1 or the microbial agent according to claim 2 in improving the yield and quality of dryland plants.

6. The use according to claim 5, characterized in that: The dryland plants include corn and wheat.

7. Use of the heterotrophic nitrifying Brevundimonas DB-4 according to claim 1 or the microbial agent according to claim 2 in improving soil fertility.

8. The use according to claim 7, characterized in that: The improving soil fertility includes promoting the conversion of ammonium nitrogen in the soil into nitrate nitrogen and promoting the content of effective phosphorus and quick-acting potassium in the soil.

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