Biological complex microbial inoculant for pitaya growth
By screening and preparing marine bacterial complex liquid from the soil around the roots of mangrove plants, the problem of unclear growth promotion mechanism of dragon fruit is solved, the promotion of dragon fruit growth and improvement of soil environment is achieved, and the growth and yield of dragon fruit is improved.
Patent Information
- Application Number
- CN202510320966.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-18
- Publication Date
- 2025-07-11
AI Technical Summary
In the prior art, the mechanism of promoting the growth of dragon fruit by plant proliferation bacteria is unclear, which affects its effective application and promotion in dragon fruit planting. A complex bacteria agent targeted to promote the growth of dragon fruit is urgently needed.
By screening and preparing marine bacterial complex solutions from the root soil of mangrove plants, including Neoascomyces, Cyperus prosthetic spp., Cyperus cerevisiae, Candolleomyces incanus, Streptococcus, Streptococcus vermis, Streptococcus vermis, Streptococcus vermis, Rhesus purpura, Lactococcus vermis, S. coris, L. coris, S. s., Diaportthe subclavata, Diaportthe cf.nobilis, Streptococcus hydride, Streptococcus vermis, Penicillium vermis, etc., the growth of these bacteria promotes substances and symbiotic relationships, improves the soil microecological environment, and enhances the absorption capacity and stress resistance of plants.
Promote the development of the root system and the growth of the above ground of the dragon fruit, improve soil fertility, enhance plants' absorption capacity of nutrients, improve photosynthetic rate and chlorophyll content, enhance their resistance to environmental stress, degrade toxic substances in the soil, and improve plants' reversal tolerance.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of pitaya cultivation, and specifically relates to a biological compound bacterium agent for the growth of pitaya. Background Art
[0002] Pitaya (Hylocereus undulatus Britt), as a tropical fruit, is highly favored in domestic and foreign markets due to its unique taste and rich nutritional value. In recent years, with the continuous progress of cultivation techniques and the increasing consumer demand, the planting area and yield of pitaya have both increased rapidly. However, how to further improve the quality and yield of pitaya to meet the market demand for high-quality fruits has become an important issue faced by the current pitaya cultivation industry.
[0003] Studies have shown that by screening growth-promoting strains and culturing a compound bacterium solution for spraying on pitaya plants, it can not only promote the growth of pitaya but also play a certain role in pest control. Plant growth-promoting bacteria (PGPB) are generally classified as bacteria in the plant rhizosphere and have a close relationship with the roots of plants. When inoculated on the seeds, tuberous roots, roots, tubers or soil of plants, they can promote plant growth. PGPB can be divided into plant growth-promoting rhizobacteria (PGPR) and plant growth-promoting endophytes (PGPE). Among them, PGPR is the main research object, including various bacteria such as Bacillus, Pseudomonas, Flavobacteria, Azotobacter, etc. However, due to limitations such as insufficient samples and environmental factors such as climate, the growth-promoting mechanism of plant growth-promoting bacteria on pitaya has not been fully elucidated, and the uncertainty of this mechanism affects the effective application and promotion of PGPB in pitaya cultivation, especially the actual effect in promoting pitaya growth. Therefore, there is an urgent need for a compound bacterium agent that specifically promotes the growth of pitaya, and this compound bacterium agent should be based on in-depth research and screening to ensure that the selected strains can effectively promote the growth of pitaya. Summary of the Invention
[0004] In order to solve the above problems, the object of the present invention is to provide a biological compound bacterium agent for the growth of pitaya, which can promote the growth of pitaya and increase the yield.
[0005] In order to achieve the above object, the technical solution of the present invention is as follows:
[0006] A biological compound bacterium agent for pitaya growth, comprising a marine bacteria compound solution; the marine bacteria compound solution includes but is not limited to Neofusicoccum, Curvularia pseudobrachyspora, Rigidoporus vinctus, Cortinarius, Candolleomyces incanus, Streptomyces, Streptomyces albidoflavus, Leuconostoc mesenteroides, Rhodococcus rhodochrous, Lactococcus garvieae, Diaporthe eres, Diaporthe subclavata, Diaporthe cf. nobilis, Streptomyces hydrogenans, Streptomyces pratensis, Streptomyces sp., and Penicillium janthinellum.
[0007] Furthermore, the marine bacteria compound solution is prepared from a rhizosphere soil sample of mangrove plants; the mangrove plants include Kandelia obovata, Avicennia marina, Excoecaria agallocha, Clerodendrum inerme, and Rhizophora stylosa.
[0008] Furthermore, the preparation method includes the following steps:
[0009] Step 1: Collect the rhizosphere soil sample of mangrove plants and perform pretreatment;
[0010] Step 2: Dissolve the pretreated rhizosphere soil sample in sterile water at a preset ratio, and then perform gradient dilution;
[0011] Step 3: Coat the suspension of each gradient onto solid plates of sterilized nutrient-rich medium Marine Agar and oligotrophic medium Difco R2A agar using a sterile spreader. Coat 5 plates for each gradient and each medium, and place the plates in an incubator at 28°C for cultivation;
[0012] Step 4: Select colonies with different morphologies on each plate for isolation and purification;
[0013] Step 5: Inoculate the purified single colonies onto plates of Marine Agar medium and cultivate at 28°C until the bacteria stop growing. Then, mix the obtained bacteria in a sterile environment to prepare the marine bacteria compound solution.
[0014] Furthermore, the pretreatment includes the following steps: air-dry the collected rhizosphere soil sample of mangrove plants; after two weeks, divide it into several equal parts of sieved soil samples using the quartering method.
[0015] Furthermore, in Step 2, the preset ratio is 0.01 g / mL.
[0016] Furthermore, the gradients include dilutions of 10 times, 100 times, 1000 times, and 10000 times.
[0017] Furthermore, after the pretreatment of the soil sample, physical and chemical property determination is also performed. The determination method includes the following steps:
[0018] The pre-treated soil samples were used to determine the fresh weight of the soil by the gravimetric method;
[0019] After drying at 105 °C for 12 hours, the dry weight of the soil was measured and the water content was calculated;
[0020] The pre-treated soil samples were dissolved in 1M potassium chloride solution and measured by a pH meter;
[0021] The organic carbon and total nitrogen contents in the soil were determined by the combustion method, and after combustion, they were measured using a CHNS - analyzer system;
[0022] The available phosphorus in the soil was extracted by the Mehlich - 3 method and determined by the molybdate blue colorimetric method at a wavelength of 880 nm;
[0023] The available nitrogen in the soil was determined using a FIAstar 5000 Analyzer;
[0024] A TTF - 100 type soil aggregate analyzer was used to determine the soil mass of soil water - stable aggregates at diameters of 0.038 mm, 0.25 mm, 1 mm, 2 mm, and 4 mm.
[0025] Furthermore, when determining the organic carbon by the combustion method, the combustion condition was 450 °C; when determining the total nitrogen content, the combustion condition was 1250 °C.
[0026] Furthermore, it also includes Step Six: sequencing and identification of the bacterial 16S rRNA gene. Extract the DNA of the bacteria obtained after separation and purification in Step Five, and then use the universal primers F27 and R1492 of bacteria to perform PCR amplification on the 16S rRNA gene; detection of the PCR amplification product; perform 16S rRNA gene sequencing using the PCR amplification product with a highlighted target band; use bioinformatics tools to align the 16S rRNA gene sequences obtained by sequencing to determine the types of bacteria in the sample.
[0027] Furthermore, the detection of the PCR amplification product includes the following steps:
[0028] Prepare agarose gel: Dissolve agarose in TAE buffer, add GoldView nucleic acid dye, and pour it into the gel - making tank to cool and solidify;
[0029] Electrophoresis: Add the PCR amplification product and DNA marker into the gel wells together, and perform electrophoresis in an electrophoresis apparatus under the conditions of a voltage of 110 V and a current of 110 mA;
[0030] Observe the results: Use a UV gel imager to observe the electrophoresis results and confirm the size and purity of the PCR amplification product.
[0031] The above - mentioned solution has the following beneficial effects:
[0032] 1. In this solution, through the marine bacterial complex solution screened and prepared from the rhizosphere soil of mangrove plants, a rich microbial community is introduced to the roots of pitaya. These microorganisms can secrete various growth - promoting substances, such as plant hormones, organic acids, etc., which directly act on the pitaya root system and promote its growth and development. By improving the soil micro - ecological environment, increasing the number of beneficial soil microorganisms, and improving soil fertility, the root development and above - ground growth of pitaya are promoted.
[0033] 2. This solution utilizes the symbiotic relationship between the microorganisms in the marine bacterial complex solution and pitaya to form a biofilm in the rhizosphere area, providing a larger absorption surface and enhancing the plant's nutrient absorption capacity. In addition, the microorganisms can also secrete growth hormones, such as indole - 3 - acetic acid and gibberellin, etc., to promote plant growth and development. They can also produce some beneficial substances, such as amino acids and vitamins, etc., to provide additional nutrient supply for plants. In addition, pot experiments have proved that the growth - promoting and yield - increasing effects of the marine bacterial complex solution may be related to the combined action of plant photosynthetic rate, chlorophyll content, and enzyme activity. Microorganisms can also enhance the plant's stress resistance by inducing the production of stress - resistant substances in plants. They can activate the plant's defense system, promote the production of antioxidant enzymes and antiviral substances in plants, etc., to enhance the plant's resistance to environmental stress. In addition, microorganisms can degrade toxic substances in the soil, reduce the degree of soil toxicity, and improve the plant's stress tolerance. Brief Description of the Drawings
[0034] Figure 1 It is a schematic diagram of the preparation process of an embodiment of a biological compound bactericide for pitaya growth according to the present invention.
[0035] Figure 2 It is a schematic diagram of Neokalmusia sp (1_TSS20230915 - 0771 - 00116).
[0036] Figure 3 It is a schematic diagram of Curvularia pseudobrachyspora (3_TSS20230915 - 0771 - 00116).
[0037] Figure 4 It is a schematic diagram of Duportella tristicula (5_TSS20230915 - 0771 - 00116).
[0038] Figure 5 It is a schematic diagram of Coprinellus sp (6_TSS20230915 - 0771 - 00116).
[0039] Figure 6 Schematic diagram of 7_TSS20230915-0771-00116 Candolleomyces incanus.
[0040] Figure 7 Schematic diagram of 8_TSS20230915-0771-00116 Streptomyces sp.
[0041] Figure 8 Schematic diagram of 9_TSS20230915-0771-00116 Streptomyces albidoflavus.
[0042] Figure 9 Schematic diagram of 10_TSS20230915-0771-00116 Leuconostoc mesenteroides.
[0043] Figure 10 Schematic diagram of WT Penicillium janthinellum on TYA medium.
[0044] Figure 11 Schematic diagram of WT Penicillium janthinellum on PDA medium.
[0045] Figure 12 Schematic diagram of EC-6 Penicillium janthinellum on PDA medium.
[0046] Figure 13 Schematic diagram of EC-6 Penicillium janthinellum on TYA medium.
[0047] Figure 14 Schematic diagram of UC-8 Penicillium janthinellum on PDA medium.
[0048] Figure 15 Schematic diagram of UC-8 Penicillium janthinellum on TYA medium. Detailed implementation manners
[0049] The following is a further detailed description through specific implementation manners:
[0050] The examples are basically as shown in the appendix Figure 1Shown: A biological composite bacterium agent for pitaya growth, including a marine bacteria composite solution; the marine bacteria composite solution is prepared from a mangrove plant rhizosphere soil sample. In this embodiment, the mangrove plants include Kandelia candel, Avicennia marina, Excoecaria agallocha, Clerodendrum inerme, and Rhizophora stylosa.
[0051] Specifically, the preparation method includes the following steps:
[0052] Step 1: Collect the mangrove plant rhizosphere soil sample and perform pretreatment;
[0053] Specifically, after mixing the plant rhizosphere soil and fine roots, they are respectively put into clean self-sealing bags and quickly taken back to the laboratory. Gently separate the plant rhizosphere soil from the fine roots, and at the same time collect the soil attached to the roots, which is often rich in microorganisms and root exudates. Then quickly put the collected soil sample into an incubator with ice packs and take it back to the laboratory, and transfer it to the refrigerator for storage as soon as possible to maintain the original state of the sample. Immediately perform air-drying treatment after bringing it back to the laboratory; two weeks later, divide it into several equal parts of sieved soil samples by the quartering method.
[0054] In this embodiment, after the pretreatment of the soil sample, the physical and chemical properties are also measured. The measurement method includes the following steps:
[0055] Measure the fresh weight of the soil by the gravimetric method for the pretreated soil sample;
[0056] After drying at 105 °C for 12 hours, measure the dry weight of the soil and calculate the water content;
[0057] Dissolve the pretreated soil sample in 1M potassium chloride solution and measure it with a pH meter;
[0058] Measure the organic carbon and total nitrogen contents in the soil by the combustion method respectively, and measure them with a CHNS-analyzer system after combustion; when measuring the organic carbon, the combustion condition is 450 °C; when measuring the total nitrogen content, the combustion condition is 1250 °C.
[0059] Extract the soil available phosphorus by the Mehlich-3 method and measure it by the molybdate blue colorimetric method at a wavelength of 880 nm;
[0060] Measure the soil available nitrogen with a FIAstar 5000 Analyzer;
[0061] Use a TTF-100 type soil aggregate analyzer to measure the soil mass of soil water-stable aggregates at diameters of 0.038 mm, 0.25 mm, 1 mm, 2 mm, and 4 mm.
[0062] Step 2: Dissolve the pretreated rhizosphere soil sample in sterile water at a preset ratio, and then perform gradient dilution;
[0063] Specifically, the pretreated rhizosphere soil samples were dissolved in sterile water at a ratio of 0.01 g / mL, and then gradient diluted by 10-fold, 100-fold, 1000-fold, and 10000-fold respectively.
[0064] Step 3: Coat the suspension of each gradient onto the solid plates of sterilized rich nutrient medium Marine Agar and oligotrophic medium Difco R2A agar with a sterile spreader. Coat 5 plates for each gradient and each medium. Place the plates in an incubator at 28 °C for cultivation; observe the growth of colonies on the plates with a stereomicroscope every day, and record the characteristics such as the color, light transmittance, viscosity, and morphology of the colonies.
[0065] Step 4: Select colonies with different morphologies on each plate for isolation and purification;
[0066] Step 5: Inoculate the purified single colonies onto the Marine Agar medium plates and culture them at 28 °C. After the bacteria stop growing, mix the obtained bacteria in a sterile environment to prepare a marine bacteria complex solution.
[0067] Specifically, select single colonies with different morphologies and good growth on the plates for isolation.
[0068] Use a sterile tool (such as an inoculation loop or toothpick) to transfer the single colony or suspected single colony to a new Marine Agar medium plate for streak isolation to ensure obtaining a pure culture.
[0069] Repeat the streak isolation step until completely single colonies are obtained.
[0070] Mix the isolated and purified single colonies to make a marine bacteria complex solution.
[0071] It also includes Step 6: Sequencing and identification of the bacterial 16S rRNA gene. Extract the bacterial DNA of the isolated and purified single colonies above, and then use the universal primers F27 and R1492 of bacteria to perform PCR amplification on the 16S rRNA gene; detect the PCR amplification products; perform 16S rRNA gene sequencing on the PCR amplification products with highlighted target bands; use bioinformatics tools to align the 16S rRNA gene sequences obtained by sequencing to determine the types of bacteria in the samples.
[0072] Specifically, in this example, molecular biology methods (such as 16S rRNA gene sequencing) are used to identify the isolated and purified strains to clarify their taxonomic status. The 16S rRNA gene is widely used in the taxonomic identification and phylogenetic research of bacteria because of its moderate sequence length, rich information, and high conservation.
[0073] Bacterial DNA extraction
[0074] Reagents and tools: Use a bacterial genomic DNA extraction kit produced by a certain company.
[0075] Operation steps: Extract bacterial DNA according to the instructions provided by the kit, ensuring that the operating environment is sterile to avoid DNA contamination.
[0076] 16S rRNA gene PCR amplification
[0077] Primer design: Use universal bacterial primers F27 and R1492, synthesized by a certain Sangon Biotech Co., Ltd.
[0078] F27: 5'-AGAGTTTGATCATGGCTCAG-3'
[0079] R1492: 5'-TAGGGTTACCTTGTTACGACTT-3'
[0080] PCR reaction system:
[0081] ddH2O: 9.5 μl
[0082] PrimeSTAR Max: 12.5 μl (containing Taq enzyme, dNTPs, buffer, etc.)
[0083] Primer F27: 1 μl
[0084] Primer R1492: 1 μl
[0085] Template DNA: 1 μl (extracted bacterial DNA)
[0086] Total volume: 25 μl
[0087] PCR amplification program:
[0088] Initial denaturation at 95°C for 10 min
[0089] Denaturation at 94°C for 30 sec
[0090] Annealing at 55°C for 30 sec (33 cycles in total)
[0091] Extension at 72°C for 90 sec
[0092] Final extension at 72°C for 10 min
[0093] Incubation at 4°C for 59 min (or until taken out)
[0094] Detection of PCR amplification products
[0095] Agarose gel electrophoresis:
[0096] Preparation of 1% agarose gel: Weigh 0.5 g of agarose into a conical flask, add 50 mL of 1×TAE buffer, heat to dissolve, add 5 μL of GoldView nucleic acid dye, and pour into a gel casting tank to cool and solidify.
[0097] Electrophoresis: Place the gel block into the electrophoresis apparatus, add 1×TAE buffer, load the samples (5 μL of DNA marker and 5 μL of PCR amplification product), and perform electrophoresis for 30 min under the conditions of 110 V and 110 mA.
[0098] Observation: Use a UV gel imager to observe the electrophoresis results and confirm whether there is a bright target band of about 1500 bp.
[0099] 16S rRNA gene sequencing
[0100] Sample preparation: Hand over the PCR amplification product with a bright target band of about 1500 bp to a certain Sangon Biotech Co., Ltd. for sequencing.
[0101] Sequence alignment and taxonomic identification
[0102] Use the EzBioCloud platform to align the 16S rRNA gene sequences obtained by sequencing.
[0103] Based on the alignment results, determine the taxonomic status of the bacteria for taxonomic identification.
[0104] The classification results are shown in Table 1.
[0105] Table 1 Identification results
[0106]
[0107]
[0108]
[0109]
[0110]
[0111]
[0112]
[0113] Experimental verification
[0114] Purpose of the experiment: This experiment aims to explore the effects of different concentrations of marine bacterial complex on the growth of pitaya, and evaluate the potential of the marine bacterial complex prepared by this scheme in promoting the growth of pitaya by comparing it with the control groups treated with urea and water.
[0115] Materials:
[0116] Pitaya seedlings from Guangxi;
[0117] Treatment solutions:
[0118] Experimental groups: The marine bacterial complex diluted 100 times, 300 times, and 500 times
[0119] Control groups: Urea diluted 100 times, 300 times, 500 times and water
[0120] Cultivation environment: Laboratory potted plants and greenhouse
[0121] Measurement indicators: Fresh weight, dry weight, plant height, stem diameter, root length, photosynthesis, leaf chlorophyll content, root soil enzyme activity;
[0122] Experimental design
[0123] Set up 20 treatment groups, each group including experimental groups (irrigation and spraying with 3 concentrations of the complex bacterial solution) and control groups (irrigation and spraying with 3 concentrations of urea and water treatment), and each treatment is repeated 3 times. Randomly allocate pitaya seedlings to each treatment group to ensure consistent initial growth conditions.
[0124] Irrigate and spray the pitaya seedlings according to the designed concentrations to ensure that the treatment solutions evenly cover the roots and leaves.
[0125] The control groups are treated with urea or water according to the corresponding concentrations.
[0126] Measurement of growth indicators:
[0127] At 14 days after treatment, randomly select pitaya plants from each treatment group for sampling.
[0128] Measure growth indicators such as fresh weight, dry weight (which needs to be dried to a constant weight first), plant height, stem diameter, and root length. Use a photosynthesis measuring instrument to detect the photosynthesis rate of the plants.
[0129] Measure the leaf chlorophyll content (such as using a SPAD meter).
[0130] Collect root soil samples and measure soil enzyme activity (such as urease, phosphatase, etc.).
[0131] Experimental results
[0132] Table 2 Growth indicator table
[0133]
[0134] Table 3 Photosynthesis and Chlorophyll Content
[0135] Treatment group <![CDATA[Photosynthetic rate (μmol CO2 / m 2 / s)]]> Chlorophyll content (SPAD value) 100 - fold marine bacterial complex solution 15.2±0.8 52.3±2.1 300 - fold marine bacterial complex solution 14.5±0.7 50.8±1.9 500 - fold marine bacterial complex solution 13.8±0.6 49.5±1.8 100 - fold urea 12.9±0.5 47.2±1.6 300 - fold urea 12.1±0.4 45.8±1.4 500 - fold urea 11.5±0.4 44.1±1.3 Fresh water 10.8±0.3 42.5±1.2
[0136] Table 4 Soil Enzyme Activity
[0137] Treatment group Urease activity (U / g soil) Phosphatase activity (U / g soil) 100 - fold marine bacterial complex solution 25.6±1.2 18.9±0.8 300 - fold marine bacterial complex solution 23.8±1.1 17.5±0.7 500 - fold marine bacterial complex solution 22.1±1.0 16.2±0.6 100 - fold urea 20.4±0.9 15.1±0.5 300 - fold urea 19.2±0.8 14.3±0.4 500 - fold urea 18.1±0.7 13.5±0.3 Fresh water 16.8±0.6 12.7±0.4
[0138] As can be seen from Table 2, Table 3 and Table 4, the treatment group with the marine bacterial complex showed a trend of being superior to the urea treatment group and the water control group in growth indexes such as the fresh weight, dry weight, plant height, stem diameter and root length of pitaya.
[0139] The treatment with the complex bacterial solution improves the photosynthesis efficiency and leaf chlorophyll content of pitaya, and promotes the accumulation of photosynthetic products.
[0140] The increase in the soil enzyme activity of the root system indicates that the complex bacterial solution has a positive impact on the soil microbial community, thereby promoting the growth of pitaya.
[0141] In summary, the raw material of the marine bacterial complex prepared by this scheme comes from marine source plants, which has more possibilities and stronger advantages than land plants. On the basis of measuring the growth-promoting effect of the marine bacterial complex on pitaya in indoor potted plants, this study further conducted experiments on the growth-promoting effect of this bacterial solution on pitaya in the field, and it has good growth-promoting effects both indoors and outdoors.
[0142] The growth-promoting and yield-increasing experiments indoors and in the field can prove that the marine bacterial complex can promote the growth of the roots, leaves, fruits, etc. of pitaya, and promote the increase of photosynthetic rate and chlorophyll. The increase in the plant photosynthetic rate and chlorophyll content may be related to plant growth and yield. Long-term photosynthesis may help to improve the accumulation of substances, thereby improving plant productivity, proving that the marine bacterial complex has a growth-promoting effect on pitaya.
[0143] The above are only the embodiments of the present invention, and common knowledge such as specific structures and / or characteristics well known in the scheme are not described in detail here. It should be noted that for those skilled in the art, without departing from the structure of the present invention, several deformations and improvements can still be made, and these should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent. The protection scope required by this application should be based on the content of its claims, and the specific implementation manners described in the specification can be used to explain the content of the claims.
Claims
1. A biological composite bactericide for the growth of pitaya, characterized in that: It includes a marine bacterial complex solution; the marine bacterial complex solution includes, but is not limited to, Neoascochyta, Curvularia lunata, Rigidoporus vinctus, Cortinarius, Candolleomyces incanus, Streptomyces, Streptomyces albidoflavus, Leuconostoc mesenteroides, Rhodococcus rhodochrous, Lactococcus garvieae, Parasphaeria, Acrocalymma vagum, Diaporthe subclavata, Diaporthe cf. nobilis, Streptomyces hydrogenans, Streptomyces pratensis, Streptomyces sp., and Penicillium janthinellum.
2. The biological compound bacterial agent for pitaya growth according to claim 1, wherein: The marine bacterial complex solution is prepared from a rhizosphere soil sample of mangrove plants; the mangrove plants include Kandelia obovata, Avicennia marina, Excoecaria agallocha, Clerodendrum inerme, and Rhizophora stylosa.
3. The biological compound bacterial agent for pitaya growth according to claim 2, wherein: The preparation method includes the following steps: Step 1: Collect a rhizosphere soil sample of mangrove plants and perform pretreatment. Step 2: Dissolve the pretreated rhizosphere soil sample in sterile water at a preset ratio, and then perform gradient dilution. Step 3: Coat the suspension of each gradient onto solid plates of sterilized rich-nutrient medium Marine Agar and oligotrophic medium Difco R2A agar using a sterile spreader. Coat 5 plates for each gradient for each medium, and place the plates in an incubator at 28°C for cultivation. Step 4: Select colonies with different morphologies on each plate for isolation and purification. Step 5: Inoculate the purified single colonies onto plates of Marine Agar medium and culture at 28°C until the bacteria stop growing. Then, mix the obtained bacteria in a sterile environment to prepare the marine bacterial complex solution.
4. The biological compound bactericide for pitaya growth according to claim 3, wherein: The pretreatment includes the following steps: Air-dry the collected rhizosphere soil sample of mangrove plants. After two weeks, divide it into several equal parts of sieved soil samples using the quartering method.
5. The biological composite bacterial agent for pitaya growth according to claim 4, characterized in that: In Step 2, the preset ratio is 0.01 g / mL.
6. The biological composite bacterial agent for pitaya growth according to claim 5, wherein: The gradients include dilution by 10 times, 100 times, 1000 times, and 10000 times.
7. The biological composite bacterium agent for pitaya growth according to claim 6, wherein: After the pretreatment of the soil sample, physical and chemical property determination is also carried out. The determination method includes the following steps: Determine the fresh weight of the soil sample through the gravimetric method for the pretreated soil sample. After drying at 105°C for 12 hours, measure the dry weight of the soil and calculate the water content. Dissolve the pretreated soil sample in 1M potassium chloride solution and measure it using a pH meter. Determine the organic carbon and total nitrogen contents in the soil through the combustion method. After combustion, use a CHNS-analyzer system for determination. Extract soil available phosphorus through the Mehlich-3 method and determine it by the molybdate blue ratio method at a wavelength of 880 nm. Determine soil available nitrogen using a FIAstar 5000 Analyzer. Use a TTF-100 type soil aggregate analyzer to determine the soil mass of soil water-stable aggregates at diameters of 0.038 mm, 0.25 mm, 1 mm, 2 mm, and 4 mm.
8. The biological composite bacterial agent for pitaya growth according to claim 7, wherein: When determining organic carbon by the combustion method, the combustion condition is 450°C; when determining the total nitrogen content, the combustion condition is 1250°C.
9. The biological composite bacterial agent for pitaya growth according to claim 8, wherein: It also includes Step Six: sequencing and identification of the bacterial 16S rRNA gene. Extract the DNA of the bacteria obtained after separation and purification in Step Five, and then use the universal primers F27 and R1492 of bacteria to perform PCR amplification on the 16S rRNA gene; detect the PCR amplification product; perform 16S rRNA gene sequencing on the PCR amplification product with the highlighted target band; use bioinformatics tools to align the sequenced 16S rRNA gene sequence to determine the types of bacteria in the sample.
10. The biological composite bacterial agent for pitaya growth according to claim 9, characterized in that: The detection of the PCR amplification product includes the following steps: Prepare the agarose gel: dissolve the agarose in the TAE buffer, add the GoldView nucleic acid dye, and then pour it into the gel-making tank to cool and solidify. Electrophoresis: add the PCR amplification product and the DNA marker into the gel wells together, and perform electrophoresis in the electrophoresis apparatus under the conditions of 110V voltage and 110mA current. Observe the results: use the ultraviolet gel imager to observe the electrophoresis results and confirm the size and purity of the PCR amplification product.