Application of heterotrophic nitrifying bacteria combined with nitrogen fertilizer in improving soil habitat and promoting the growth of young apple trees

Abstract

The present invention provides an application of a heterotrophic nitrifying bacteria agent combined with nitrogen fertilizer in improving soil habitat and promoting the growth of young apple trees. The heterotrophic nitrifying bacteria agent is composed of pure cultured bacterial strains H7-5, H9-7 and H11-4, H7-5 is Microbacterium, H9-7 is Pseudomonas, H11-4 is Arthrobacter, and the nitrifying bacteria agent can improve the microecological environment of fruit tree soil and promote the growth of fruit trees. The present invention isolates 26 strains of dominant nitrifying bacteria from apple orchard soil, and after screening, the bacterial strains with good nitrification activity are made into a mixed agent, which can change the distribution of soil microbial populations and nutrient supply conditions after combining soil nitrogen fertilizer treatment, and affect the growth of young apple trees. The soil habitat and the growth of young apple trees show that the inoculation of the agent with nitrate nitrogen fertilizer can increase the soil nitrate nitrogen, available potassium content and pH value, change the diversity characteristics of the soil bacterial population, and show a positive effect on the growth-promoting effect of young apple trees.

Description

Technical Field

[0001] The invention belongs to the technical field of fruit tree growth, and specifically relates to an application of a heterotrophic nitrifying bacteria agent combined with nitrogen fertilizer in improving soil habitat and promoting the growth of young apple trees. Background Art

[0002] The apple industry is highly intensive, and water and fertilizer regulation are usually used in the production process to improve the vitality of apple tree roots. However, excessive nitrogen fertilizer causes soil acidification and microbial population replacement. [1，2] , causing changes in the morphology and function of apple roots, such as the plant's roots becoming short and thick and their number decreasing, becoming one of the important factors restricting apple yield and quality. [3-5] .

[0003] Soil microorganisms are closely related to the transformation of nutrients such as nitrogen in the orchard ecosystem, and play a vital role in balancing soil fertility, improving the stress resistance of fruit trees, and optimizing the orchard soil ecological environment. [6，7] Nitrification is a key step in soil nitrogen transformation, including the oxidation of ammonia to nitrite and nitrite to nitric acid, which are driven by nitrite bacteria and nitrifying bacteria, respectively, collectively known as nitrifying bacteria. [8，9] Among them, the oxidation of ammonia (or ammonium) to nitrite, namely nitrification, is the rate-limiting step in the soil nitrification process.

[10] Farmland management measures such as fertilization can cause changes in soil environmental factors, thereby changing the nitrogen transformation process in which soil nitrifying bacteria participate. [11-13] Although most nitrifying bacteria are autotrophic, their ammonium oxidation efficiency is significantly higher than that of heterotrophic bacteria. Some studies have found that heterotrophic nitrifying microorganisms are present in large numbers in the soil. Their acid resistance and strong resistance to adverse environments make their contribution to nitrification not to be ignored [9].

[0004] Apple as a Nitrogen-loving Plant

[14] By screening the dominant heterotrophic nitrifying strains in the orchard ecological environment, applying different nitrogen fertilizers, and backgrafting them into the root zone of young apple trees, combined with the growth conditions of the fruit trees, the relationship between young apple trees, microorganisms, and soil fertilization can be evaluated. This has practical significance for regulating soil nitrogen behavior and soil nutrient supply and promoting the growth of apple plants.

[0005] Isolation and Activity Detection of Heterotrophic Nitrifying Bacteria

[0006] Cultivable heterotrophic nitrifying bacteria were obtained from orchard soil using an improved nitrite bacteria enrichment and separation medium. Bacterial strains with good nitrification activity were screened using Griess reagent detection.

[0007] Determination of excellent microbial agents for promoting growth of young apple trees

[0008] The potted grafting experiment was conducted, combined with the measurement of the aboveground and root biomass of young trees, the analysis of soil nutrient content such as different forms of nitrogen, and the changes in soil microbial population diversity, to determine the combination of nitrifying bacteria agents that had a good effect on the growth of young apple trees ( Figure 1 ).

[0009] Fertilization is a prerequisite for the healthy growth of apple trees. With the development of sustainable agriculture, the application of microbial agents to regulate the supply of nutrients in orchard soil and affect the growth of fruit trees has become an important part of the development of "green and ecological agriculture". In the process of fruit tree management, how to give full play to the synergistic effect of nitrogen fertilizer and functional microbial agents to promote the growth of fruit trees by improving the soil micro-environment is the main problem of existing technologies. Summary of the invention

[0010] The present invention aims at the deficiencies in the prior art and aims to provide an application of a heterotrophic nitrifying bacteria agent combined with nitrogen fertilizer in improving soil habitat and promoting the growth of young apple trees.

[0011] To achieve the above object, the solution of the present invention is:

[0012] Application of heterotrophic nitrifying bacteria combined with nitrogen fertilizer in improving soil habitat and promoting the growth of young apple trees.

[0013] Furthermore, the heterotrophic nitrifying bacteria agent is composed of pure cultured bacterial strains H7-5, H9-7 and H11-4.

[0014] Further, H7-5 is Microbacterium, H9-7 is Pseudomonas, and H11-4 is Arthrobacter.

[0015] Furthermore, the preparation method of the heterotrophic nitrifying bacteria agent is: the improved separation medium is sterilized at 121°C for 30 minutes, and then the bacterial strains H7-5, H9-7 and H11-4 are inoculated into the improved separation medium respectively, and cultured at 25°C for 15 days.

[0016] Furthermore, the components of the improved separation medium are: (NH4)2SO4, 2g, K2HPO4, 0.75g, NaH2PO4, 0.25g, MnSO4·4H2O, 0.01g, MgSO4·7H2O, 0.03g, CaCO3, 5g and 1% soil extract.

[0017] Furthermore, the soil extract was prepared by taking 400 g of air-dried soil from an apple orchard, filtering it through a 20-mesh sieve, adding 1 L of tap water, sterilizing it at 121° C. for 1 h, taking it out and precipitating it at room temperature for 6-7 h, centrifuging it, and taking the supernatant for later use.

[0018] Furthermore, the application of heterotrophic nitrifying bacteria inoculants in combination with nitrate nitrogen fertilizers increased the soil nitrate nitrogen and available potassium content, improved the soil pH value, and promoted the growth of young apple trees.

[0019] Furthermore, the application of heterotrophic nitrifying bacteria agent combined with nitrate nitrogen fertilizer increased soil bacterial richness (S) and Shannon diversity (H) index.

[0020] Due to the adoption of the above scheme, the beneficial effects of the present invention are:

[0021] The present invention isolates 26 strains of dominant heterotrophic nitrifying bacteria from the soil of an apple orchard, and after screening, three bacterial strains with good nitrification activity are determined. After being made into a mixed bacterial agent, combined with soil nitrate nitrogen fertilizer treatment, the soil microbial population distribution and nutrient supply conditions can be changed, affecting the growth of young apple trees. Soil bacterial population analysis shows that the bacterial agent treatment has a significant effect on the composition of soil bacterial populations. It is manifested that the combination of the bacterial agent and nitrate nitrogen fertilizer has a significant effect on improving the soil bacterial richness index. The changes in the soil environment and the growth of young apple trees show that the inoculation of the bacterial agent combined with the nitrate nitrogen fertilizer can significantly increase the soil nitrate nitrogen, available potassium content and pH value, and has a positive effect on the growth promotion of young apple trees. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a diagram of the research on bacterial agents in the prior art.

[0023] Figure 2 This is a diagram of microbial species that may be represented by the dominant nitrifying bacteria of the present invention.

[0024] Figure 3 The biomass of apple saplings treated with different methods in the present invention (Note: Figure Ⅰ and II represent root biomass and aboveground biomass, respectively; 1N, 2N and CK in the figure represent NH4 + —N, NO3 — —N and no fertilization treatment; A, B and C represent inoculated bacterial agent A, bacterial agent B and no inoculated bacterial treatment, respectively; different Roman characters and lowercase letters represent significant differences among different fertilization treatments and all treatments (P<0.05, the same below).

[0025] Figure 4 The DGGE maps of soil bacterial communities in the root zone of apples treated differently according to the present invention.

[0026] Figure 5 This is a cluster analysis of the bacterial community structure in the soil root zone of apples with different treatments according to the present invention. DETAILED DESCRIPTION

[0027] The invention provides an application of a heterotrophic nitrifying bacteria agent combined with a nitrogen fertilizer in improving soil habitat and promoting the growth of young apple trees.

[0028] The present invention isolates and identifies cultivable heterotrophic nitrifying bacteria in apple orchard soil, and identifies the best bacterial agent combination that helps improve soil environmental quality and promote the growth of young apple trees through screening of excellent functional strains.

[0029] The present invention isolates culturable heterotrophic nitrifying bacteria strains from the root zone soil of an apple orchard, and combines the strain nitrification activity screening test to determine the dominant strain with good nitrification ability. The pure culture technology is used to prepare a microbial agent and inoculate it into the rhizosphere soil of young apple trees, and combined with ammonium nitrogen fertilizer and nitrate nitrogen fertilizer treatment, by detecting the changes in the growth index of young trees, physical and chemical indicators such as soil nitrogen, and the distribution of microbial populations, the inoculated microbial agent and nitrogen fertilizer are evaluated in improving the ecological environment of the orchard and affecting the regulation effect of the growth of young apple trees.

[0030] Embodiment 1:

[0031] Isolation and identification of nitrifying bacteria

[0032] 1. Test materials

[0033] The experiment was conducted in a 5-year-old apple orchard at the Fruit Tree Research Base of Shenyang Agricultural University. The stock and scion combination was 'Hanfu' / GM256 / Shandingzi, the row and plant spacing was 0.5m×4m, and the soil type was brown soil.

[0034] 2. Test methods

[0035] 2.1 Soil sample collection: Soil samples were collected during the flowering period, rapid growth period of new shoots, fruit expansion period and leaf fall period. The sampling point was located under the tree canopy, 0.5m away from the tree trunk. When sampling, the surface covering (weeds that have not yet rotted, etc.) was removed, and a 0-20cm soil profile was taken vertically downward as a sample. The 5-point sampling was mixed and processed, repeated 3 times, and the roots and soil invaders were removed. The "four-point method" was used to select 1kg of soil samples and stored in a refrigerator at 4℃ for later use.

[0036] 2.2 Soil physical and chemical properties were tested according to Dousen method

[15] .

[0037] 2.3 Enrichment, separation and purification of nitrifying bacteria: Bacteria were isolated by dilution plate method and cultured in improved nitrosobacteria isolation medium at 25°C for 15 days. The 4-10 colonies that appeared on the highest dilution plate were taken as the dominant bacteria in the soil, and the strains were purified by plate streak method.

[16] .

[0038] 2.4 Screening of dominant nitrifying bacteria: Screening of dominant nitrifying bacteria strains is carried out according to relevant methods.

[17] . In the initial screening, pick a single colony and place it in 30mL of nitrosating bacteria isolation culture medium, culture it at 25℃ for 15d, take 2 drops of culture and place them on a white porcelain colorimetric plate, add 2 drops of Griess reagent I and II, and judge its nitrification activity based on whether a red reaction occurs and the depth of the red color. Select strains with stronger nitrification activity for rescreening. Adjust the pH values ​​of the isolation culture medium to 6.4, 7.0 and 8.0 respectively. Pipette 1mL of the strain culture medium obtained in the initial screening into 30mL of nitrosating bacteria isolation culture medium of different pH values, culture it at 25℃ for 15d, take 5mL of the bacterial solution and place it in a test tube, add 2mL of sulfonamide reagent, shake well and compare colors at a wavelength of 520nm. Select strains with stronger nitrosating ability (OD 520 >0.112) for molecular identification.

[0039] 2.5 DNA extraction and molecular identification of dominant nitrifying bacteria: 16S rDNA sequence analysis was used to identify dominant bacteria. Bacterial DNA was extracted using a kit (Bacteria Genomic DNA Kit, CWBIO). The specific method was referred to the instruction manual. Universal primers were used to amplify bacterial 16S rDNA. The forward primer was 8-27F (5′-AGAGTTTGATCMTGGCTCAG-3′) (SEQ ID NO.1), and the reverse primer was 1378-1401R (5′-CGGTGTGTACAAGGCCCGGGAACG-3′)

[15] (SEQ ID NO.2). The 50 μL PCR reaction system was composed of the following: 5 μL Taq DNA polymerase buffer, 2.5 mmol / L MgCl2, 0.2 mmol / L dNTPs (each), 0.4 μmol / L forward and reverse primers, 40 ng template, 5 U Taq DNA polymerase (TaKaRa), 32 μL ddH2O. The reaction procedure was: pre-denaturation at 95°C for 10 min, adding Taq DNA polymerase, denaturation at 95°C for 1 min, renaturation at 60°C for 30 s, extension at 72°C for 1.5 min, 30 cycles, and final extension at 72°C for 10 min. The amplified product was detected with 1.0% agarose gel. The PCR recovery kit (DNA Fragment Purification Kit Ver.2.0, TaKaRa) was used for purification. The purified and recovered PCR product was entrusted to Dalian Bao Biotechnology Co., Ltd. for sequencing. The sequencing results were submitted to the GenBank database (http: / / www.ncbi.nlm.nih.gov), and the target sequence was compared with the sequences contained in the gene bank using BLAST to obtain the sequence with the closest homology.

[18] .

[0040] Embodiment 2:

[0041] Determination of the combination of nitrifying bacteria

[0042] 1. Test materials

[0043] 1.1 Combination of bacterial agents: The strains are five dominant nitrifying bacterial strains with obvious nitrosation effects, numbered H7-5 (Microbacterium sp.), H9-7 (Pseudomonas sp.), H11-3 (Pseudomonas sp.), H11-4 (Arthrobacter sp.), and H11-5 (Sphingobacterium sp.). Among them, the combination of H11-3, H11-4 and H11-5 is bacterial agent No. 1; the combination of H7-5, H9-7 and H11-4 is bacterial agent No. 2.

[0044] 1.2 Nitrogen fertilizer: The types of nitrogen applied to potting soil are potassium nitrate and ammonium sulfate.

[0045] 1.3 Fruit tree varieties: One-year-old Aralia dasyphylla seedlings were used as test materials.

[0046] 2. Test methods

[0047] 2.1 Experimental design: The experiment was designed as a split plot, with the main experimental factors being the application of ammonium nitrogen and nitrate nitrogen respectively; the secondary plot was inoculated with nitrifying bacteria, with a total of treatments of inoculation with No. 1 bacterial agent, No. 2 bacterial agent, and no inoculation with bacterial agent. The experiment was repeated 4 times, with a total of 6 treatments and 24 pots.

[0048] 2.2 Nitrifying bacteria strains and nitrogen fertilizer application:

[0049] Preparation of soil extract: Take 400g of air-dried soil from the apple orchard, pass it through a 20-mesh sieve, add 1L of tap water, sterilize it at 121℃ for 1h, take it out and precipitate it at room temperature for 6-7h, centrifuge it and take the supernatant for later use.

[0050] Improved separation medium: (NH4)2SO4, 2g; K2HPO4, 0.75g; NaH2PO4, 0.25g; MnSO4·4H2O, 0.01g; MgSO4·7H2O, 0.03g; CaCO3, 5g; 1% soil extract, distilled water added to 1L; adjusted pH to 7.8-8.0, and 5 bacterial strains were cultured in the improved medium respectively.

[0051] The growth of each strain was detected by spectrophotometer at 620 nm, and the OD of each strain was adjusted. 620The values ​​were 0.4 respectively. After equal amounts were mixed, a mixed bacterial solution was prepared. 30 mL of the mixed bacterial solution was applied to the soil of each apple plant. The control treatment applied an equal amount of sterile culture solution. The nitrogen fertilizer applied to the soil was 0.5 g of pure nitrogen per young tree each time. The bacterial solution and nitrogen were applied once each every 15 days to the potted soil, for a total of 4 times.

[0052] A 1.5L plastic pot was used as a culture container. The pot experiment was conducted at the fruit tree base of Shenyang Agricultural University. The plastic pots were changed regularly to reduce the adverse effects of the placement on the test results. The plants were harvested after 90 days of growth since the application of the bacterial agent and fertilizer. Among them, the aboveground parts and roots of the plants were harvested separately, and the plant samples were dried at 70℃ for 48h and weighed. The soil samples were stored in a -80℃ low-temperature refrigerator for subsequent bacterial diversity analysis.

[0053] 2.3 PCR-DGGE detection of soil bacteria: DGGE denaturation gradient was 45-65% (100% denaturation is equivalent to 7 mol L -1 Urea and 40% deionized formamide), polyacrylamide concentration was 8%, electrophoresis was performed at 60°C, 1×TAE 200V for 5h, and the electrophoresis film was stained with SYBR GREEN 1 dye (1:10 000 dilution) for 30min. The staining results were analyzed under a UV gel imaging system.

[0054] DGGE gel band recovery sequencing: The DGGE band to be recovered was cut with a sterilized scalpel, and the target band was recovered using the OMEGA Poly-Gel DNA Extraction Kit.

[0055] PCR amplification was performed using 2 μL of the recovered product as a template and 341F / 518R as primers

[19] .

[0056] 2.4 Data analysis: Quantity-One software (BIO RAD) was used to analyze the DGGE map, where the Shannon Diversity index was used to measure the diversity of the community; the Pielou index was used to measure the uniformity of the community; the Margalef index was used to indicate the richness; and the Berger-Parker index was used to indicate the dominance. The diversity is shown as follows: Shannon-Wiener diversity index H' = -ΣP i ln i ; Pielou community evenness index Jsw = (-∑P i ln i ) / lnS; Margalef index D mg =(S-1) / lnN; Berger-Parker index D = Nmax / N, where P iis the number of individuals of the i-th species, S is the total number of species in the plot, Nmax is the number of individuals of the dominant species, and N is the number of individuals of all species in the sampling. SPSS23 statistical software was used to process the experimental data, and Duncan's test was used to test the significance of the difference between the mean values ​​of each treatment. EXCEL was used for plotting, and the Mega Neighbor-Joining method was used to establish the phylogenetic tree of nitrifying bacteria.

[0057] Results and Analysis

[0058] (I) Screening and molecular identification of dominant nitrifying bacteria strains in apple orchard soil

[0059] 1. Screening of dominant nitrifying bacteria strains in apple orchard soil at different growth stages

[0060] The 26 dominant nitrifying bacteria strains isolated from different growth stages were cultured in liquid shaking for 15 days, and the nitrite-forming ability of the strains was detected by Griess reagent. The preliminary screening results are shown in Table 1. As can be seen from the table, most of the 26 nitrifying bacteria strains showed nitrite-forming ability. Among them, 12 nitrifying bacteria strains had good nitrogen conversion ability and could be used for subsequent re-screening research.

[0061] Table 1 Determination of nitrite intensity of dominant strains in soil of 'Hanfu' apple orchard at different periods (preliminary screening)

[0062]

[0063]

[0064] -, (+)+, ++, and +++ represent no nitrosating ability, insignificant nitrosating ability, weak nitrosating ability, average nitrosating ability, and strong nitrosating ability, respectively.

[0065] The 12 nitrifying bacteria obtained in the initial screening were inoculated into culture media with different pH values, and the nitrosation ability of different strains was detected at a wavelength of 520nm. The results are shown in Table 2. As can be seen from the table, at pH 6.4, strain H11-5 had the highest nitrosation ability; at pH 7.0, strain H11-4 had the highest nitrosation ability; at pH 8.0, strain H9-7 had the highest nitrosation ability. Considering that the isolated strains came from different growth periods, combined with OD 520 >0.112, and finally five bacterial strains, namely H7-5, H9-7, H11-3, H11-4 and H11-5, were identified as the dominant strains with good nitrosation ability.

[0066] Table 2 Determination of nitrite intensity of dominant strains in soil of 'Hanfu' apple orchard (rescreening)

[0067]

[0068] 2. Analysis of 16S rDNA sequences of dominant nitrifying bacteria

[0069] The 16S rDNA sequences of the five dominant strains with good nitrification ability were analyzed, and the sequencing results were compared with the gene sequences registered in GenBank using the Blast program. The homology analysis results showed that ( Figure 2 ), strain H7-5 and Microbacterium sp. constitute a separate branch, and the strain similarity reaches 100%; strains H9-7, H11-3 and Pseudomonas sp. constitute a separate branch, and the similarity reaches 100%; strain H11-4 and Arthrobacter sp. constitute a separate branch, and the similarity reaches 99%; strain H11-5 and Sphingobacterium sp. constitute a separate branch, and the similarity reaches 99%.

[0070] (II) Effects of nitrifying bacteria on the growth of young apple trees

[0071] 1. Effects of inoculation with fungi and different fertilization treatments on the biomass of young apple trees

[0072] The bacterial strains with good nitrification ability were prepared into mixed strains according to the sampling period, among which strains H11-3, H11-4 and H11-5 constituted bacterial agent 1, and strains H7-5, H9-7 and H11-4 constituted bacterial agent 2. The growth and development of the saplings were tested after 90 days of growth. Figure 3 It can be seen that compared with the unfertilized control, the application of nitrogen fertilizer can significantly increase the aboveground biomass of apple saplings (P>0.05), but there is no significant difference in the effect of ammonium nitrogen fertilizer and nitrate nitrogen fertilizer on the aboveground biomass. The application of ammonium nitrogen fertilizer showed a significant inhibitory effect on root biomass, while nitrate nitrogen fertilizer had no significant difference from the control treatment. The effect of microbial agents on the growth of young trees was manifested in that inoculation of microbial agent 2 could significantly increase the root biomass of apple saplings under each fertilization treatment (P<0.05). After inoculation of microbial agent 2 combined with nitrate nitrogen fertilizer, the aboveground and root biomass of apple saplings were significantly higher than the control treatment (CK-C).

[0073] 2. Changes in physical and chemical properties of soil in the root zone of young apple trees under inoculation with microbial agents and different fertilization treatments

[0074] As can be seen from Table 3, compared with no fertilization, the application of ammonium nitrogen fertilizer significantly increased the contents of soil alkaline nitrogen, ammonium nitrogen and nitrate nitrogen, but reduced the pH and available potassium content; the application of nitrate nitrogen fertilizer can significantly increase the soil pH, nitrate nitrogen and available potassium content. Compared with no application of microbial agents, the application of microbial agents increased the contents of soil alkaline nitrogen and ammonium nitrogen to varying degrees (P>0.05), and reduced the soil pH, available potassium and total carbon content (P<0.05). Between the two microbial agents, inoculation of microbial agent 2 was more helpful in stabilizing the soil pH, available potassium, total nitrogen and total carbon content. Compared with the control (CK-C), the soil nitrate nitrogen, available potassium content and pH value increased significantly after inoculation of microbial agent 2 combined with nitrate nitrogen fertilizer (P<0.05).

[0075] Table 3 Effects of different treatments on the physical and chemical properties of soil in the apple root zone

[0076]

[0077] Note: 1N: NH4-N; 2N: NO3-N; CK: no fertilization; A: inoculation with bacterial agent A; B: inoculation with bacterial agent B; C: no inoculation. The data in the table are average values. Different capital letters, Roman characters and lowercase letters represent significant differences among different fertilizations, different bacterial agents and all treatments (P<0.05).

[0078] 3. DGGE analysis of soil bacterial communities in the apple root zone under inoculation and different fertilization treatments

[0079] Denaturing gradient gel electrophoresis results of soil bacteria in apple root zone showed that ( Figure 4 ), fertilization and inoculation of microorganisms had a greater impact on the structure and composition of soil bacterial populations. The clustering results showed that the treatment without fertilization was clustered into one category, and the treatment with nitrate nitrogen fertilizer was clustered into another category ( Figure 5 ). The clustering results showed that soil fertilization treatment had a more significant effect on soil bacterial composition than microbial agents. From the perspective of strip brightness, in general, inoculation with microbial agents and fertilization treatments increased the brightness of strips 3, 4, 5, 6, and 7 in the soil, while the type of microbial agents affected the composition of soil bacterial community structure under fertilization treatment to varying degrees. Compared with the control (CK-C), the inoculation with microbial agent 2 and nitrate nitrogen treatment significantly increased the brightness of strips 3, 5, and 7 in the soil.

[0080] The results of soil bacterial diversity index analysis showed (Table 4) that compared with the unfertilized control, the application of ammonium nitrogen fertilizer reduced the soil bacterial diversity (H), richness (S), uniformity (J) and dominance (D) indexes, and the application of nitrate nitrogen fertilizer increased the soil bacterial H and S indexes, but reduced the J index. Under different fertilization treatments, inoculation of microbial agent 1 can increase soil bacterial diversity indexes to varying degrees, while microbial agent 2 mainly increases S diversity. Compared with the control (CK-C), the combination of inoculation of microbial agent 2 and nitrate nitrogen fertilizer is conducive to the increase of soil bacterial population diversity (H) and richness (S).

[0081] Table 4 Diversity index of soil bacterial community in potted apple saplings under different treatments

[0082]

[0083] 4. Correlation analysis between soil properties, bacterial diversity and fruit tree biomass

[0084] The results of correlation analysis showed (Table 5) that the content of ammonium nitrogen and nitrate nitrogen in the soil was extremely significantly positively correlated with the aboveground biomass of the plants (P<0.01), and extremely significantly negatively correlated with the plant root system and the bacterial diversity index (H, J and D) in the soil (P<0.01), while the soil pH showed the opposite. The above results suggest that higher nitrogen content in the soil has an inhibitory effect on plant root growth and bacterial population diversity. Since the nitrate nitrogen content is closely related to factors such as soil pH (P<0.01), the change in soil pH after fertilization and bacterial agent application may be an important factor affecting soil nutrient supply, microbial population distribution and apple plant growth.

[0085] Table 5 Correlation analysis between soil physicochemical parameters and bacterial diversity and plant biomass

[0086]

[0087] Note: * represents P<0.05, ** represents P<0.01

[0088] The types of inoculated agents and the types of nitrogen applied had different effects on the physical and chemical properties of soil under apple saplings, the distribution of soil microbial populations, and the biomass of fruit trees. Nitrate nitrogen fertilizer increased soil nitrogen content and pH, and agent 2 helped maintain soil pH, available potassium, total nitrogen, and total carbon content. Correlation analysis results showed that pH, nitrate nitrogen, and ammonium nitrogen content were important factors affecting soil nutrient supply, bacterial population diversity, and apple sapling growth. The combination of agent 2 (H7-5, H9-7, and H11-4) and nitrate nitrogen fertilizer affected the diversity characteristics of soil bacterial populations, and correspondingly improved soil pH and potassium supply, thereby promoting the growth of apple saplings.

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[0110] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. The application of heterotrophic nitrifying bacteria agent combined with nitrogen fertilizer in improving soil habitat and promoting the growth of young apple trees, characterized in that: The heterotrophic nitrifying bacteria agent is composed of pure cultured bacterial strains H7-5, H9-7 and H11-4; the H7-5 is Microbacterium, the H9-7 is Pseudomonas, and the H11-4 is Arthrobacter; Adjust the OD of each strain 620 The values ​​were 0.4 respectively. After equal amounts were mixed, a bacterial agent mixture was prepared. 30 mL of the mixed bacterial solution was applied to the cultivation soil of each apple plant. Nitrate nitrogen fertilizer was applied to the cultivation soil. 0.5 g of pure nitrogen was applied to each young tree each time. The bacterial solution and nitrogen were applied to the cultivation soil once every 15 days, for a total of 4 times. A 1.5L plastic pot was used as the culture container. The position of the plastic pot was changed regularly. The plants were harvested after 90 days of growth from the application of the bacterial agent and fertilizer. The aboveground parts and roots of the plants were harvested separately. The plant samples were dried at 70℃ for 48h and weighed. The soil samples were stored in a -80℃ low-temperature refrigerator for subsequent bacterial diversity analysis. The heterotrophic nitrifying bacteria agent combined with nitrate nitrogen fertilizer increases the content of nitrate nitrogen and available potassium in the soil, improves the pH value of the soil, and promotes the growth of young apple trees; the heterotrophic nitrifying bacteria agent combined with nitrate nitrogen fertilizer increases the soil bacterial richness and Shannon diversity index.

2. The use according to claim 1, characterized in that: The preparation method of the heterotrophic nitrifying bacteria agent is as follows: sterilize the improved separation medium at 121° C. for 30 minutes, inoculate the bacterial strains H7-5, H9-7 and H11-4 into the improved separation medium respectively, and culture at 25° C. for 15 days.

3. The use according to claim 2, characterized in that: The components of the improved separation medium are: (NH4)2SO4, 2g, K2HPO4, 0.75g, NaH2PO4, 0.25g, MnSO4·4H2O, 0.01g, MgSO4·7H2O, 0.03g, CaCO3, 5g and 1% soil extract.

4. The use according to claim 3, characterized in that: The soil extract is prepared by taking 400 g of air-dried soil from an apple orchard, filtering it through a 20-mesh sieve, adding 1 L of tap water, sterilizing it at 121° C. for 1 h, taking it out and settling it at room temperature for 6-7 h, centrifuging it, and taking the supernatant for later use.

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