Microflora for in-situ enrichment of caenorhabditis elegans in red soil and application of flora

Abstract

The invention discloses a flora for in-situ enrichment of nematode in red soil and application of the flora. The flora comprises one or more of bacteria A, bacteria B, bacteria C and bacteria D, the bacteria A are Cupriavidus necator and a bacterial strain YTR-C1, the bacteria B are Sinorhizobium sp. And a bacterial strain YTR-S1, the bacteria C are Mesorhizobium amorphae and a bacterial strain YTR-M3, the bacteria D are Ralstonia pickettii and a bacterial strain YTR-R242, the strains are combined for use, the concentration ratio of the strains is (1-10): (1-10): (1-10): (1-10), and the number of the caenorhabditis in red soil can be increased, so that the growth of crops is promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of soil animal breeding, and particularly relates to a bacterial flora for in-situ enrichment of Pristina nematodes in red soil and application. BACKGROUND

[0002] Phosphorus is one of the essential nutrients for plant growth, which is not only a component of many important organic compounds in plants, but also participates in various physiological processes in plants in various ways. More than 95% of phosphorus in soil is ineffective phosphorus, which is difficult to be absorbed and utilized by plants. In agricultural production, high water-soluble phosphorus fertilizer is often applied to meet the demand of plants for phosphorus. However, after the phosphorus fertilizer is applied to soil, it is easy to form insoluble phosphate, which is quickly adsorbed and fixed by soil minerals or held by microorganisms. Southeast hilly region is the main production area of tropical and subtropical economic forests, economic crops and food (rice) in China, and plays an important role in regional sustainable development. Red soil is the main low-yield soil in this region. Due to the presence of a large amount of free iron and aluminum oxides, phosphorus is more easily fixed, resulting in a serious phosphorus deficiency. The utilization rate of phosphorus in red soil is only 10%-25% in the current season. The problem of soil phosphorus deficiency and productivity decline caused by red soil acidification has seriously threatened the stability of the ecological system and the efficient use of resources.

[0003] Nematodes are one of the main functional groups of soil animals and the most abundant metazoans in soil. Soil nematodes are abundant in nutrition and widely distributed in various soil habitats, and the proportion of bacterivorous nematodes in rhizosphere soil is as high as about 90%. Nematodes have the characteristics of short generation cycle and sensitive response to environmental changes, and are often used as an indicator of changes in farmland management measures. The ecological index of nematodes can provide unique information about the soil ecosystem. Nematodes occupy a key central position in the soil food web, and through selective predation, they not only change the biomass, diversity and community composition of microorganisms, but also directly affect the survival adaptability and health of nematodes. Nematode-microorganism interaction regulates the energy transfer and carbon, nitrogen and phosphorus nutrient transformation and storage processes in the soil ecosystem, which plays an important role in maintaining the structural stability and functional diversity of the soil food web. Soil nematodes are dominated by free-living bacterivorous nematodes, and bacterivorous nematodes release the nutrients held in microorganisms by predating soil microorganisms, thereby promoting plant growth. In order to alleviate the phosphorus stress of red soil and improve the biological availability of phosphorus, indigenous bacterivorous Pristina nematodes can be enriched in situ in red soil, and the culture conditions of Pristina nematodes can be optimized. The Pristina nematodes are applied to low-yield dry red soil, so as to improve the phosphatase activity and effective phosphorus pool content of red soil, and realize the yield and quality increase of crops. SUMMARY

[0004] In view of the technical problems and actual needs of the prior art in developing a rapid and efficient in-situ reproduction method of nematodes, the present application provides a bacterial flora for in-situ enrichment of Pristina nematodes in red soil and application.

[0005] To achieve the above object, the application adopts the following technical scheme: a bacterial flora for in-situ enrichment of Allonyx nematodes in soil, which comprises one or more of bacteria A, bacteria B, bacteria C and bacteria D, wherein the concentration ratio of the bacteria A, bacteria B, bacteria C and bacteria D is (1-10):(1-10):(1-10):(1-10); wherein, The bacteria A is Cupriavidus necator strain YTR-C1, which was preserved in the China General Microbiological Culture Collection Center on May 16, 2023, and the preservation number is CGMCC27358. The bacteria B is Sinorhizobium sp. strain YTR-S1, which was preserved in the China General Microbiological Culture Collection Center on September 18, 2020, and the preservation number is CGMCC20681. The bacteria C is Mesorhizobium amorphae strain YTR-M3, which was preserved in the China General Microbiological Culture Collection Center on April 9, 2019, and the preservation number is CGMCC17532; and the bacteria D is Ralstonia pickettii strain YTR-R242, which was preserved in the China General Microbiological Culture Collection Center on October 21, 2024, and the preservation number is CGMCC32269.

[0006] Preferably, in the strain combination, the abundance of one strain is higher than the sum of the other three strains.

[0007] Preferably, the effective viable bacterial count of the bacteria A, bacteria B, bacteria C and bacteria D is 104-108 per gram of soil.

[0008] Preferably, the strain group is inoculated in red soil and composted pig manure substrate.

[0009] The application provides the application of the bacterial flora for in-situ enrichment of Allonyx nematodes in soil, which is used for planting corn and rapeseed.

[0010] Preferably, the mass ratio of the composted pig manure to the red soil is (3-7):1.

[0011] The application provides an adaptive culture medium for nematode enrichment, which comprises red soil to be inoculated with nematodes, composted pig manure and the bacterial flora. Beneficial effects

[0012] The application simplifies operation, improves the in-situ growth environment of nematodes, and improves the reproduction capacity of the Aphelenchoides nematodes. Adding the culture medium rich in nematodes to the farmland red soil can promote the activation of the red soil phosphorus, increase the soil phosphatase activity and the phosphorus availability, and improve the yield and quality of corn. Compared with the prior art, the application has the following advantages: (1) The in-situ enrichment of the farmland red soil indigenous dominant nematodes can enhance the survival rate and reproduction capacity of the nematodes in the red soil by adding microbial agents and agricultural organic waste, and improve the total number of bacterivorous nematodes and the number of the Aphelenchoides nematodes. The Aphelenchoides nematodes cultured by the technical scheme of the application grow rapidly and have enhanced reproduction capacity, and the in-situ enrichment method of the nematodes has low production cost, simple technology, and is convenient for large-scale promotion.

[0013] (2) The Aphelenchoides nematodes are applied to the red soil, increase the alkaline and acid phosphatase activity of the red soil, promote the activation and release of the red soil phosphorus, and improve the effective phosphorus content of the red soil.

[0014] (3) The Aphelenchoides nematodes are applied to the red soil, increase the absorption and utilization of the nutrients in the red soil by corn, improve the growth condition of corn, significantly improve the yield, aboveground and root biomass of corn, and improve the soluble sugar content of leaves, the soluble protein content of leaves, and the indole acetic acid content of leaves. The Aphelenchoides nematodes cultured by the in-situ enrichment method of the application have obvious yield-increasing and quality-improving effects after application, and are suitable for large-area use in crop production areas. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The figure shows the change of the number of nematodes in different proportion adaptive culture medium; Figure 2 The figure shows the Aphelenchoides nematodes in the red soil; Figure 3 The figure shows the reproduction of nematodes under different isolated and enriched bacterial single colony feeding conditions; Figure 4 The figure shows the change of the phosphatase activity and the effective phosphorus content of the corn rhizosphere red soil under high, medium and low nematode dosing conditions. DETAILED DESCRIPTION

[0016] The application will be further explained in combination with the examples. The following examples are only used to more clearly illustrate the technical scheme of the application, and should not and cannot limit the protection scope of the application. EXAMPLES

[0017] Nematodes were isolated using a modified shallow-disc-sucrose centrifugation-flotation continuous extraction method, and their counting was performed using a Motic SMZ-168 stereomicroscope. The counted nematode aqueous solution was completely poured into 50 mL centrifuge tubes, allowed to stand for approximately 12 h, and then the supernatant was aspirated, retaining 9 mL of the nematode aqueous solution. This solution was then incubated in a 60°C water bath for 5 min. After cooling, 1 mL of formalin solution was added to each sample to complete preservation. 150-200 well-preserved nematodes were aspirated from a small plastic dish and placed on a glass slide, sealed with a coverslip, and identified to their genus level under an optical microscope (Olympus BX50).

[0018] The nematodes cultured in this invention are *Cephalophyllum* nematodes, belonging to the order Micrococcus, suborder Padnaeidae, and family Cephalophyllaceae. For example... Figure 2 As shown, the biological characteristics of *Headleaf* nematodes are as follows: The worm is medium-sized, curving slightly ventrally after heat treatment. The cuticle has fine annular rings, with 2-4 lateral bands extending to the tail tip. The labial region is slightly constricted, lacking a head protrusion; the labial protrusion is low and rounded. The anterior and posterior parts of the esophagus are cylindrical, with a narrow isthmus; the posterior esophageal bulb is oval, with a distinct butterfly-shaped ossification flap. A neural ring encircles the posterior part or isthmus of the esophagus. It possesses cervical papillae. The lateral caudal glands are located near the middle of the tail. Females have a single, protruding gonad; the ovary folds back twice; it possesses a spermatheca and a posterior vulvar uterine sac; the vulva is flat or protruding; the tail is conical, with a blunt or pointed tip. Males have a wedge-shaped gubernaculum, a conical tail that arches ventrally, and a short, hook-like tip at the end. Example

[0019] Four bacterial species were isolated and enriched from in situ red soil. Bacterium A was a member of the genus *Copper-loving Bacteria*. Cupriavidus necator The bacterial strain YTR-C1 was deposited on May 16, 2023, at the China General Microbiological Culture Collection Center (CGMCC27358). Bacterium B belongs to the genus *Rhizobium sinense*. Sinorhizobium sp. The strain YTR-S1 was deposited on September 18, 2020, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC20681. Bacterium C is an intermediate rhizobium. Mesorhizobium amorphae Strain YTR-M3 was deposited on April 9, 2019, at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC17532. Bacterium D belongs to the genus *Rowstoneella*. Ralstonia pickettii, strain YTR-R242, deposited at the China General Microbiological Culture Collection Center on October 21, 2024, with the accession number CGMCC32269. The above-mentioned bacteria have been disclosed in patent documents as follows: Sinorhizobium sp., patent number: ZL 202011189260.X; Mesorhizobium amorphae, patent number: ZL 201911119326.5; Cupriavidus necator, patent number: ZL 202310872862.2; Ralstonia pickettii, application number: 202411659347.7. The effect of bacteria on the growth of nematodes is analyzed by feeding experiments, and the steps are as follows: 1. Red soil was collected from a long-term positioning test in the field, built in the Jiangxi Province Yichun Agroecosystem National Field Research Station (116°55¢E, 28°13¢N). The test area belongs to the subtropical monsoon climate, with an average annual temperature of 17.8°C and an average annual precipitation of 1795 mm, with a frost-free period of 262 days.

[0020] 2. The red soil was collected using the cutting ring method, mixed uniformly, and then evenly divided into three parts. 5 g of red soil was taken for each part to extract DNA from the red soil and perform high-throughput sequencing. Separately enriched bacteria A, bacteria B, bacteria C, and bacteria D were added to 50 g of sterilized red soil at a concentration of 10 6 individuals per gram of soil, in triplicate. At the same time, a control group was set up by adding an equal amount of sterile water. In each treatment, 45 g of in-situ red soil and 5 g of sterilized composted pig manure were added. The nematode (adult) was released at a rate of 300 individuals per 100 grams of soil. The indoor incubation conditions were soil moisture of 60%-70% of the field water holding capacity, dark incubation for 12 days, and incubation temperature of 12-28°C.

[0021] 3. After 12 days of incubation, the nematodes were isolated using the improved shallow plate-sucrose centrifugal flotation continuous extraction method, and counted using a Motic SMZ-168 body microscope. As shown in Figure 3 , bacteria A, bacteria B, bacteria C, and bacteria D all increased the number of nematodes, with the total number of nematodes increasing by 6.2, 1.5, 6.3, and 6.5 times, respectively, the number of bacterivorous nematodes increasing by 65.3, 20.2, 66.5, and 68.4 times, respectively, and the number of species increasing by 195.3, 59.5, 195.8, and 204.7 times, respectively.

[0022] 4. DNA was extracted from the red soil according to the operation steps provided by the kit Mo-Bio Power Soil DNA Extraction Kit (Mo-Bio Laboratories, Inc., CA, USA), and the quality and quantity of the DNA were detected by the Nano Drop spectrophotometry (Nano Drop Technologies, Wilmington, DE, USA). The extracted DNA was stored in dry ice and then sent to Ling'en Company for high-throughput sequencing. The primers for sequencing were 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 907R (5'-CCGTCAATTCCTTTGAGTTT-3'), which were used to amplify the V4 ~ V5 region of the bacterial 16S rRNA gene. The information of the bacterial species and abundance in the red soil showed that the abundance ratio of bacteria A, bacteria B, bacteria C and bacteria D was about 6:1:5:4. Example

[0023] The red soil-agricultural organic waste-bacterial agent mixture under the optimal ratio was obtained as the adaptive culture medium for nematodes in the natural field environment, and the nematodes stored in the adaptive culture medium were obtained by the following steps: The composition of the adaptive culture medium was the red soil to be inoculated with nematodes, matured pig manure and bacterial agent.

[0024] 1) The red soil in the test plot was collected and passed through a 2 mm sieve for nematode inoculation. The pig manure was produced in a local pig farm, and the fresh pig manure was collected, watered to a moisture content of 80%, and mixed uniformly. 10% of the red soil was added to the pig manure, and the mixture was stored in a fermentation box (length 70 cm, width 60 cm, height 50 cm) for aeration. The heating temperature was 40°C in the early stage, 70°C in the middle stage, and 90°C in the late stage, and the whole cycle lasted for 30 days. The material was mixed every 2 days in the early stage, and the turning frequency was accelerated in the late stage to accelerate water evaporation and maturation. The application amount of matured pig manure was 5 tons / hm 2 (dry basis). After the pig manure was matured, the matured pig manure and the red soil collected in situ in the test plot were mixed uniformly at a mass ratio of 3:1, 5:1 and 7:1, and filled into plastic containers with holes at the bottom. When filling, 5 cm thick culture medium was filled each time, and the atomized water was sprayed uniformly into the soil with a watering can to adjust the soil moisture to 60%-70% of the field water holding capacity.

[0025] 2) Sterile water containing nematodes was inoculated by spraying at a rate of 300 individuals per 100 g of soil. The microbial inoculants were bacteria A, bacteria B, bacteria C, and bacteria D, with concentration ratios of T1:6:1:5:4, T2:4:4:4:4, T3:10:2:2:2, T4:2:10:2:2, T5:2:2:10:2, T6:2:2:2:10, with 10 4 , 10 6 , 10 8 , 10 7 , 10 4 , and 10 6 , respectively, providing a direct food source for bacterivorous nematodes. A control group T0 was also set up without the addition of microbial inoculants. The concentration of the bacteria was mainly based on the original concentration ratio of different bacteria in red soil, with the same concentration ratio and one dominant bacteria (one bacteria with a higher abundance than the sum of the other three bacteria, to observe the dominant effect of this bacteria). The effective number of viable bacteria was mainly based on the concentration of bacteria in soil, which was about 10 8 , 10 4 , and 10 6 , respectively, with low concentration (10 8 , slightly lower than natural abundance (10 6 , and slightly higher than natural abundance (10 8 .

[0026] During the in-situ enrichment culture of nematodes, the moisture of the culture medium was maintained stable, and the adaptive culture medium was cultured in a natural environment for 10-12 days in the dark, with a temperature of 12-28°C.

[0027] 2. After the nematodes were cultured in the adaptive medium, a modified shallow plate-sucrose centrifugal floatation continuous extraction method was used for nematode separation, and a Motic SMZ-168 body microscope was used for counting. The counted nematode water solution was completely poured into a 50 mL centrifuge tube, and after standing for about 12 h, the upper liquid was sucked out, 9 mL of nematode water solution was retained, and water bath was carried out in a water bath at a temperature of 60°C for 5 min. After cooling, 1 mL of formalin solution was added to each sample to complete preservation; 150-200 preserved nematodes were taken from the plastic dish and placed on a glass slide, covered with a cover glass, sealed, and identified to the genus under an optical microscope (Olympus BX50). The results showed that after the in-situ enrichment of nematodes in the adaptive medium, the total number of nematodes and bacterivorous nematodes increased significantly, especially the number of Aphelenchus nematodes increased extremely significantly. The results of the number of nematodes are shown in Table 1: compared with the control group, all the treatment groups with added bacteria can increase the number of nematodes. Among them, the mature pig manure and the red soil collected in-situ in the test plot were mixed in a mass ratio of 5:1, and the concentration of the microbial inoculant was 10 6The total number of nematodes, bacterivorous nematodes and cephalobus nematodes were the highest under the condition of 1000 bacteria A, 1000 bacteria B, 1000 bacteria C and 1000 bacteria D per 100 grams of dry soil, with a concentration ratio of T4. Under this condition, the total number of nematodes can increase by 9.2 times (0.03-4.38 times higher than other treatments), the number of bacterivorous nematodes can increase by 115.6 times (0.12-5.19 times higher than other treatments), and the number of cephalobus nematodes can increase by 192.3 times (0.12-5.49 times higher than other treatments) after in-situ enrichment culture. EMBODIMENT

[0028] Field planting test of corn and rape 1. Test treatment: Two treatments were set in this experiment, high, medium and low dosage (100 strips / 100 grams of dry soil, 500 strips / gram of soil and 1000 strips / 100 grams of soil) of cephalobus nematodes were applied, and the control treatment (CK) without applying nematodes. The test plot area was 20 m 2 (5 m × 4 m), and each treatment was repeated 3 times.

[0029] 2. Variety and planting method: The corn seeds were Su Yu 24, which had the excellent characteristics of high disease resistance, drought resistance and high temperature resistance. The seeding rate was generally 20 kg / hm 2 , the plant spacing was 20 cm × 40 cm, the seeding depth was 10-15 cm, and the planting density was 68,000 plants / hm 2 . The rape seeds were Qingyou 8. The seeding rate was 4 kg / hm 2 , the plant spacing was 15 cm × 30 cm, the seeding depth was 3-4 cm, and the planting density was about 90,000 plants / hm 2 .

[0030] 3. Nutrient management: In corn planting, the application rates of urea, calcium magnesium phosphate fertilizer and potassium fertilizer were 152.72 kg N / hm 2 , 116.20 kg P2O5 / hm 2 and 166.32 kg K2O / hm 2 respectively. The pig manure was completely composted by high-temperature composting technology, and the water content of the composted pig manure was less than 50%. The pig manure application amount was 5 tons / hm 2 (dry basis) to replace 60% of chemical fertilizer. Phosphorus and potassium were all base fertilizers, 70% of nitrogen was base fertilizer, and 30% was topdressing at the small trumpet stage. The same amount of chemical fertilizer was applied for rape.

[0031] 4. Application of nematodes: According to the inoculation density of 100 strips of bacterivorous cephalobus nematodes per 100 grams of dry soil, 500 strips per gram of soil and 1000 strips per 100 grams of soil, the adaptive substrate was applied to the test plot together with the nematodes, and the red soil 0-30 cm layer was artificially tilled.

[0032] 5. Field management: During the planting of corn and rape, adjust the soil moisture to 60%-70% of the field water holding capacity. During the critical periods of crop growth such as seedling stage and flowering stage, irrigate in time according to the water requirement of crops to ensure normal growth. Irrigate in time when drought occurs during the growth period of corn, and drain water in time when heavy rain occurs. In addition to not spraying pesticides, manual weeding, fertilizer application and other field management refer to the local routine.

[0033] 6. Field test was carried out to the maturity of corn and rape, and rhizosphere soil was collected to determine the activity of alkaline phosphatase and the content of available phosphorus in red soil of each treatment, and the results are shown in Figure 4 The activity of acid and alkaline phosphatase, the content of available phosphorus and the yield of corn in the treatment of adding medium amount of D. cephalota were the highest, which increased by 64.7%, 30.6%, 39.8% and 10.2% 50.5% respectively compared with the control CK.

[0034] 7. Field test was carried out to the maturity of corn and rape, and corn plants and rape seeds were harvested to determine the biomass of corn aboveground and root, soluble sugar, soluble protein in leaves and indole acetic acid in leaves. The results showed that: compared with high and low dosage conditions, the corn plant height, straw dry weight, soluble sugar, soluble protein and indole acetic acid content in plant leaves added with medium amount of D. cephalota reached the highest. Under this condition, the corn straw dry weight was 29.5% higher than CK, and the root dry weight was 18.7% higher than CK; the soluble sugar, soluble protein and indole acetic acid content in plant leaves added with D. cephalota were 33.8%, 24.5% and 29.9% higher than CK respectively. The yield of rape seeds increased by 50.5% compared with the control CK.

[0035] The four kinds of bacteria are fed to nematodes in turn under laboratory conditions, and the effects of the four kinds of bacteria on the growth of nematodes are analyzed. The nematodes used here are bacterivorous nematodes; (2) the red soil is weighed from the test plot, agricultural organic waste and different concentrations of bacterial inoculants are added, and through optimization test, the optimal ratio of the mixture of red soil-agricultural organic waste-bacterial inoculant is selected as the adaptive culture medium of nematodes in the natural environment of the field, and the nematodes preserved in the adaptive culture medium are obtained; (3) the adaptive culture medium rich in bacterivorous nematodes is backfilled into the soil of the test plot according to high, medium and low dosages. The soil of the test plot is managed in the field, the soil moisture is maintained stable, and the growth conditions and yield changes of corn and rape in the field under different nematode dosages are detected. The in-situ growth environment of nematodes is improved, and the reproduction capacity and yield of the Pristina nematodes are improved. Based on the long-term positioning test of biological introduction of the Chinese Academy of Sciences Yingtan Red Soil Ecological Experimental Station, the total number of soil nematodes and the number of Pristina nematodes in the red soil are increased. Through field tests, it is found that the addition of Pristina nematodes significantly increases the alkaline and acid phosphatase activity and effective phosphorus content in the red soil, and improves the growth conditions and quality of corn and rape.

[0036] Finally, the above only some specific embodiments of the present application, not limited to the scope of the present application. It should be noted that for those skilled in the art, without departing from the principles of the present application can be made to the technical solutions of the present application several improvements and refinements, should be considered as the present application defined within the scope of protection.

Claims

1. Use of a bacterial population for in situ enrichment of a Pristionchus nematode in soil, characterized in that: The bacterial community comprises bacteria A, bacteria B, bacteria C and bacteria D, and the concentration ratio of the bacteria A, bacteria B, bacteria C and bacteria D is (1-10):(1-10):(1-10):(1-10); wherein, The bacteria A is Cupriavidus necator Strain YTR-C1 was deposited with the China General Microbiological Culture Collection Center on May 16, 2023, and the deposit number is CGMCC NO. 27358. The bacteria B is Sinorhizobium sp. Strain YTR-S1 was deposited with the China General Microbiological Culture Collection Center on September 18, 2020, and was assigned accession number CGMCC NO. 20681. The bacteria C is Mesorhizobium amorphae Strain YTR-M3 was deposited with China General Microbiological Culture Collection Center on April 9, 2019, and the deposit number is CGMCC NO. 17532. The bacteria D is Ralstonia pickettii Strain YTR-R242 was deposited with the China General Microbiological Culture Collection Center on October 21, 2024, and was assigned accession number CGMCC NO. 32269.

2. Use of the bacterial flora according to claim 1 for in situ enrichment of the soil of Pristinus nematodes, characterized in that: The abundance of one kind of bacteria in the strain combination is higher than the total of the other three kinds of bacteria.

3. Use of the bacterial flora according to claim 1 for in situ enrichment of Allonylon nematodes in soil, characterized in that: The effective viable cell number of the bacteria A, bacteria B, bacteria C, and bacteria D is 10 4 -10 8 cells / gram of soil.

4. Use of the bacterial flora according to claim 1 for in situ enrichment of Allonylon nematodes in soil, characterized in that: The bacterial community is inoculated into red soil and mature pig manure substrate.

5. Use of the bacterial flora according to claim 1 for in situ enrichment of Pristinus nematodes in soil, characterized in that: The nematodes are used to promote the growth of corn and rapeseed.

6. Use of the bacterial flora according to claim 4 for in situ enrichment of Pristinus nematodes in soil, characterized in that: The mass ratio of mature pig manure to red soil is (3-7):1.

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