Coxsackii strain, microbial agent and application of coxsackii strain and microbial agent

Abstract

The invention belongs to the technical field of agricultural microorganisms, and discloses a coxsackii strain which is named as coxsackii P1-4 and preserved in China Center for Type Culture Collection on September 20, 2023, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 20231759. The invention further discloses a microbial agent containing the bacterial strain, application of the microbial agent in promoting rice growth and / or improving soil health and application of the microbial agent in reducing the content of cadmium in soil and / or reducing the content of cadmium in rice. The coxakella P1-4 strain and the microbial inoculum thereof provided by the invention can synchronously and remarkably improve the key nutrient indexes of soil, can remarkably improve the contents of nutrients such as NH4-N, NO3-N, organic phosphorus and organic matters in the soil, and comprehensively improve the fertility and biological activity of the soil.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbiology technology, and in particular relates to a strain of Cossackie, microbial agents and their applications. Background Technology

[0002] The sustainable development of modern agriculture faces two key challenges: first, soil degradation and environmental pollution caused by the long-term excessive use of chemical fertilizers; and second, the threat of soil heavy metal cadmium (Cd) pollution to crop safety and ecosystem health.

[0003] The over-reliance on chemical fertilizers has triggered a series of environmental problems. Long-term, excessive application has led to soil compaction, acidification, and salinization, damaging soil structure and microbial activity. Low utilization rates of nutrients such as nitrogen and phosphorus not only result in resource waste, but unabsorbed nutrients also exacerbate eutrophication pollution through leaching into water bodies. Furthermore, fertilizer production is energy-intensive, and its application may be accompanied by greenhouse gas emissions and heavy metal accumulation in the soil, severely hindering the green transformation of agriculture.

[0004] Developing inoculants using beneficial microorganisms is an important way to reduce reliance on chemical fertilizers. Existing microbial fertilizers can improve nutrient utilization and promote crop growth through nitrogen fixation, phosphorus solubilization, and growth hormone secretion, and also have advantages such as environmental friendliness and improved soil ecology. However, these products still have significant limitations in practical applications: most strains have relatively single functions, mainly promoting growth or nutrient activation; they lack stability under different field conditions and their effects fluctuate greatly; and their ability to remediate complex stresses such as heavy metal pollution in soil is weak, limiting their comprehensive application effects and promotional value.

[0005] On the other hand, soil cadmium pollution is becoming increasingly serious, threatening agricultural product safety and human health. Existing remediation technologies each have their shortcomings: physical methods (such as soil replacement, soil washing, deep tillage, and electroremediation) require significant human, material, and financial resources, are difficult to implement, and the remediated soil is prone to re-contamination, making large-scale application difficult. Chemical passivation (such as applying lime) can reduce cadmium activity, but its effectiveness is unstable and it is difficult to consistently control crop cadmium levels below safe thresholds. Microbial remediation, while environmentally friendly, has limited adsorption and fixation efficiency for cadmium by existing microbial agents, and its effectiveness in soils with varying degrees of pollution is uncertain. In particular, there is a lack of multifunctional bacterial strains that simultaneously promote efficient growth and stabilize and passivate cadmium.

[0006] Therefore, current agricultural practices urgently need a type of microbial resource that integrates multiple functions: one that can efficiently activate soil nutrients, promote crop growth, and increase yield, while simultaneously addressing heavy metal pollution by steadily reducing the available cadmium content in the soil and controlling cadmium migration to crops, thus achieving the dual goals of increased yield and safety. Developing such multifunctional strains with significant synergistic effects and strong environmental adaptability, along with their application technologies, is of great significance for promoting innovation in green inputs and ensuring food security. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the background art above, and to provide a strain of Cossackievirus, a microbial agent and its application.

[0008] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows: A strain of Cossackie bacterium was named Cossackie bacterium ( Kosakonia sacchari P1-4 were deposited on September 20, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO:M 20231759.

[0009] Based on a general inventive concept, the present invention also provides a microbial inoculant, comprising the aforementioned Cossackella (…). Kosakonia sacchari P1-4 or including Cossackella (as described above) Kosakonia sacchari Fermentation broth of P1-4.

[0010] Based on a general inventive concept, the present invention also provides the above-mentioned Cossackella ( Kosakonia saccharin Application of P1-4 or other microbial agents as described above in promoting rice growth and / or improving soil health.

[0011] In the above-mentioned applications, preferably, promoting rice growth includes increasing the biomass, root length, plant height, and / or yield of rice roots, stems, leaves, panicles, and panicles.

[0012] In the above-mentioned applications, preferably, the improvement of soil health includes increasing one or more of NH4-N, NO3-N, organic phosphorus, organic matter, and pH in the soil.

[0013] The above-described application, preferably, involves using the Cossackella (… Kosakonia sacchari P1-4 or the microbial agent described herein may be irrigated into the soil.

[0014] In the above-described application, preferably, the dosage of the microbial agent is 5-15 L / m³. 2 The OD600 value of the microbial agent is 0.8-1.1.

[0015] Based on a general inventive concept, the present invention also provides a Cossackella as described above ( Kosakonia saccharin Application of P1-4 or other microbial agents as described above in reducing soil cadmium content and / or reducing rice cadmium content.

[0016] The above-mentioned application, preferably, involves applying the Cossackella ( ) during the rice tillering stage. Kosakonia sacchari P1-4 or the aforementioned microbial inoculants.

[0017] In the above-described application, preferably, the dosage of the microbial agent is 5-15 L / m³. 2 Further preferred is 8-12 L / m 2 A further preferred value is 10L / m 2 The OD600 value of the microbial agent is 0.8-1.1, more preferably 1.0.

[0018] The present invention also provides the application of Cossackella strain P1-4 or the microbial agent in reducing the cadmium content in rice.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The Cossackella P1-4 strain and its agent provided by the present invention can simultaneously and significantly improve the key nutrient indicators of soil. It can significantly increase the content of nutrients such as NH4-N, NO3-N, organic phosphorus and organic matter in the soil, comprehensively improve soil fertility and biological activity, and provide a biological solution for solving the problems of soil compaction, acidification and soil fertility decline caused by long-term application of chemical fertilizers.

[0020] (2) Applying the Cossackella P1-4 strain and its inoculant provided by the present invention can significantly promote the root development and plant growth of rice. Specifically, it can effectively increase the biomass of rice roots, stems, leaves and panicles, significantly increase root length and plant height, and ultimately achieve a significant increase in rice yield, providing efficient microbial assistance for food security and increased production.

[0021] (3) The Cossackella P1-4 strain and its agent provided by the present invention have excellent cadmium (Cd) fixation ability, which can increase soil pH and change soil physicochemical properties, thereby effectively reducing the bioavailability and mobility of cadmium.

[0022] (4) The Cossackella P1-4 strain and its agent provided by the present invention can not only significantly reduce the content of available cadmium in the soil, but also extend the effect to the edible parts of crops, significantly reduce the accumulation of cadmium in rice, and ensure the safety of rice products.

[0023] Biological Preservation Instructions The Cossackella involved in this invention ( Kosakonia sacchariP1-4 were deposited on September 20, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO:M20231759. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This invention is based on the strain Sacchari ( Kosakonia sacchari Tolerance of P1-4 to 4 mmol / L heavy metal Cd; Figure 2 The cadmium content and pH value of the supernatant at different times and in different treatment groups are shown (A represents cadmium content, B represents pH value). Detailed Implementation

[0026] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0027] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0028] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0029] Example 1: The types and components of the culture media mentioned below are as follows: LB liquid medium: Weigh 10.0 g sodium chloride, 5.0 g yeast extract, and 10.0 g tryptone, and bring the volume to 1000 mL with ultrapure water. Adjust the pH to 7.0, dispense into Erlenmeyer flasks, seal, and autoclave at 121°C for 30 minutes. Use after cooling.

[0030] LB solid medium: Weigh 10.0 g sodium chloride, 5.0 g yeast extract, 10.0 g tryptone and 15 g agar, and make up to 1000 mL with ultrapure water. Adjust the pH to 7.0, dispense into Erlenmeyer flasks, seal and autoclave at 121°C for 30 minutes. Cool to 50°C and pour into plates.

[0031] Phosphate-solubilizing bacteria screening medium: Weigh 5 g of tricalcium phosphate [Ca3(PO4)2], 10 g of glucose, 0.25 g of MgSO4·7H2O, 5 g of MgCl2·6H2O, 0.1 g of (NH4)2SO4, and 0.2 g of KCl. Make up to 1000 mL with ultrapure water, adjust the pH to 7.1-7.5, add 15 g of agar, and autoclave at 121℃ for 30 minutes. Cool to 50℃ and pour into plates.

[0032] Nitrogen-fixing bacteria selection medium (Ashby medium): Weigh 0.2 g KH2PO4, 10 g mannitol, 0.2 mL NaCl, 0.2 g MgSO4·7H2O, 5 g CaCO3, and 0.1 g CaSO4·2H2O. Make up to 1000 mL with ultrapure water, adjust the pH to 7.0, add 15 g agar, and autoclave at 121°C for 30 minutes. Cool to 50°C and pour into plates.

[0033] This embodiment provides the isolation, screening, and analysis of Cossackie bacteria.

[0034] 1. Separation and screening: At the sewage outlet of the Xiangjiang River, select vigorous Miscanthus sinensis, pull it out along with its roots, shake off the loose soil on the surface of the roots, collect the soil tightly attached to the surface of the roots (rhizosphere soil), put it into a pre-prepared sterile bag, seal it, and quickly bring it back to the laboratory for storage in a 4°C refrigerator.

[0035] Weigh 5g of soil and place it in a 45mL sterile Erlenmeyer flask containing glass beads. Shake for 20 minutes to thoroughly mix the soil sample with the water. Then let it stand. Take 1mL of the supernatant and dilute it to 1×10⁻⁶. 4 1×10 5 and 1×10 6After dilution, 100 μL of the diluted supernatant was spread onto LB agar plates and incubated upside down in a constant temperature incubator (28℃). The growth of the strains on the plates was observed periodically. After 5 days of growth, strains with different morphological characteristics (color, size, shape, etc.) were selected from the plates using an inoculation loop and streaked onto LB agar plates. Nine bacterial strains were screened and named: P1-1, P1-2, P1-3, P1-4, P1-5, P1-6, P1-7, P1-8, and P1-9. After activation of the single bacteria, 1.5 mL of the bacterial solution was aliquoted into 2 mL centrifuge tubes at a bacterial suspension to sterile glycerol ratio of 1:1 and stored at -80℃ for later use.

[0036] 2. Functional analysis of the strain (1) Qualitative analysis of the function of producing glutenin (IAA) The isolated bacteria were inoculated into LB liquid medium containing L-tryptophan (100 mg / L) and cultured in a shaker (28 ℃, 180 rpm) for 36 hours. 50 μL of the cultured bacterial suspension, plus 50 μL of Salkowski's colorimetric solution, was placed in the wells of a white ceramic plate. The positive control consisted of 50 μL IAA (50 mg / L) + 50 μL Salkowski's colorimetric solution. After standing in the dark at a constant temperature (28 ℃) for 30 minutes, the color changes were observed.

[0037] (2) Qualitative analysis of phosphorus solubility The isolated bacteria were inoculated onto phosphate-solubilizing bacterial selection medium. After 3 days, the presence of phosphate-solubilizing zones on the plates was observed, and the strain and the size (diameter, mm) of the phosphate-solubilizing zones were recorded.

[0038] (3) Qualitative analysis of nitrogen fixation function The isolated bacteria were inoculated onto a nitrogen-fixing bacteria selection medium, and their growth was observed after 3 days.

[0039] (4) Analysis of the siderophore function of growth-promoting bacteria The isolated bacteria were inoculated onto CAS plates and placed in an incubator (28℃) for inverted culture. After 5 days, the presence of an orange-yellow halo around the inoculated bacteria was observed, and the size (diameter, mm) of the bacteria and the halo was recorded.

[0040] The results are shown in Table 1 below.

[0041] Table 1. Siderophore production capacity and growth-promoting capacity of growth-promoting bacteria

[0042] The ability of bacteria to produce siderophores is represented by the formula HD / CD, where CD is the diameter of the bacterial strain and HD is the diameter of the yellow halo. Analysis of the strain's ability to promote growth: "+" indicates the presence of this function, and "-" indicates the absence of this function.

[0043] As shown in Table 1, the isolated and screened strain P1-4 has good growth-promoting ability and siderophore production ability.

[0044] (5) Cd resistance analysis The isolated and screened strain P1-4 was inoculated into LB liquid medium and cultured in a shaker (28℃, 180rpm) for 36 hours. 100μL of the bacterial suspension was spread onto LB solid medium containing 4 mmol / L Cd and incubated upside down in a 28℃ incubator. Growth was observed over 5 days.

[0045] Table 2. Tolerance of the isolated bacteria to different concentrations of heavy metal Cd

[0046] Tolerance to the heavy metal cadmium: "+" indicates the presence of colonies, "++" indicates a large number of colonies, and "-" indicates the absence of colonies.

[0047] The results are as follows Figure 1 As shown in Table 2, the isolated and screened bacteria have a certain resistance to Cd and are cadmium-fixing bacteria.

[0048] 3. Identification of strains The previously isolated and screened P1-4 strain was inoculated into LB liquid medium for activation. After centrifugation using a high-speed refrigerated centrifuge, the bacterial cells were collected, and bacterial DNA was extracted using a bacterial DNA extraction kit (TaKaRa 16S rDNA Bacterial Identification PCR Kit, Takara) according to the manufacturer's instructions. The amplified DNA products were sent to Hunan Saisiwei Biotechnology Co., Ltd. for sequencing and identification. The bacterial gene sequence was then compared online with the NCBI database to identify the species of the isolated and screened bacteria. The determined 16S rDNA sequence is shown in SEQ ID NO: 1, and the specific sequence is as follows:

[0049] The results showed that the bacteria isolated and screened were strains of the genus Cossackella, with the Latin name [missing information]. Kosakonia saccharin It was named Cossackie ( Kosakonia sacchari P1-4 were deposited on September 20, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO:M20231759.

[0050] Example 2: This embodiment provides the effect of microbial inoculants on the growth and development of rice.

[0051] 1. Cossackie ( Kosakonia sacchari P1-4 was inoculated into LB liquid medium (pH 7.0) and cultured in a constant temperature shaker (28℃, 200 rpm) for 12 hours. The bacterial culture was then removed and its OD600 value was measured. The culture was then centrifuged at 6000 rpm for 3 minutes, the supernatant LB was discarded, and sterile water was added proportionally to adjust the OD600 value to 1.0. The viable cell count was approximately 1 × 10⁻⁶. 8 CFU / mL, shake well to obtain microbial inoculum.

[0052] 2. Select plump Huanghuazhan rice seeds, disinfect them successively with alcohol and sodium hypochlorite, and then wash them with distilled water. Soak the seeds in a 28℃ constant temperature incubator for 4 days to promote germination. Then, use hydroponics to cultivate the seeds in plastic pots (daytime 26-28℃ / nighttime 20-22℃, relative humidity 60%-70%, light intensity 300-500 μmol / L). m -2 s -1 The light exposure time is 12-14 hours / day. After the rice seedlings have grown to the three-leaf stage, they are transplanted into soil-grown pots (the soil used in the pots is experimental soil from the Ningxiang Rice Experimental Field, and the soil nutrient content is shown in Table 3 below).

[0053] Table 3 Soil Nutrient Content

[0054] 3. Apply the microbial agent during the rice tillering stage at concentrations of 0, 5, 10, and 15 L / m², respectively. 2 The dosage was irrigated into the rice soil, with 3 replicates for each treatment.

[0055] 4. The average results of rice dry matter weight, root length and plant height measured from the time of management and cultivation to rice maturity are shown in Table 4 below; the average results of soil nutrient content measured from rice are shown in Table 5 below.

[0056] Table 4 Results of rice dry weight, root length and plant height measurements

[0057] As shown in Table 4, Cossackella ( Kosakonia sacchari P1-4 significantly promoted the growth of rice.

[0058] Table 5 Nutrient content in paddy soil

[0059] As shown in Table 5, Cossackella ( Kosakonia sacchari P1-4 significantly increased the content of nutrients such as NH4-N, NO3-N, organic phosphorus and organic matter in the soil, improved soil fertility and soil health.

[0060] Example 3: This embodiment provides the effect of microbial agents on cadmium in soil.

[0061] Purchased Cossacella strains from Ningbo Mingzhou Biotechnology Co., Ltd. Kosakonia sp. (Item No.: BMZ148293), used as the control group (CK), and the Cossacella of the present invention ( Kosakonia sacchari P1-4 was used as the experimental group (P1-4) to compare the cadmium fixation efficiency of the CK group and the P1-4 group.

[0062] The CK group and P1-4 group bacteria were inoculated at a 5% inoculum into LB liquid medium (pH=6.0) containing a final cadmium concentration of 2.0 mg / L. Samples were taken and centrifuged at 3h, 6h, 12h, 24h, 48h, 72h, 96h, 120h and 144h after inoculation to determine the residual cadmium content and pH value in the supernatant. Each treatment was designed with 3 replicates.

[0063] Fixation efficiency = (initial environmental cadmium concentration - environmental cadmium concentration at the time of measurement) / initial environmental cadmium concentration × 100%.

[0064] The results are shown in Tables 6 and 7. Figure 2 As shown, when the cadmium concentration in the environment was 2.0 mg / L, the fixation efficiency of P1-4 reached its maximum of 98.67% after 144 hours, and the pH remained stable at 7.71. When the cadmium concentration in the CK group was 2.0 mg / L, apart from endocytosis and exocytosis to absorb a small amount of cadmium, it did not play a role in cadmium fixation, and the pH remained essentially unchanged at 6.35.

[0065] Table 6. Cadmium content in the environment (mg / L)

[0066] Table 7 pH in the environment

[0067] This indicates that the Cossackella of the present invention ( Kosakonia sacchari P1-4 has a good cadmium fixation ability, which can effectively reduce the cadmium content in the environment and significantly increase the pH value in the environment.

[0068] Example 4: This embodiment provides the effect of microbial inoculants on the cadmium content in brown rice.

[0069] 1. Cossackie ( Kosakonia sacchari P1-4 was inoculated into LB medium (pH 7.0) and cultured in a constant temperature shaker (28℃, 200 rpm) for 12 hours. The bacterial culture was then removed to detect its OD. 600 Centrifuge at 6000 rpm for 3 minutes, discard the upper LB layer, and add sterile water in proportion to reduce OD. 600 The value was adjusted to 1.0 (1.0 × 10). 8 (CFU / ml), shake well to obtain microbial inoculum.

[0070] 2. Select plump Huanghuazhan rice seeds, disinfect them with 3% sodium hypochlorite for 30 minutes, and then wash them with distilled water. Soak the seeds in a 28℃ constant temperature incubator for 24 hours, changing the water every 4 hours. After draining the water, place the seeds in a germination chamber, maintaining a temperature between 28-30℃ and a humidity of 80%, for 4 days to germinate. Then, use hydroponics, sowing the seeds in plastic pots containing Kimura B nutrient solution (purchased from Beijing Coollab Technology Co., Ltd.) for cultivation (temperature 26-28℃ during the day / 20-22℃ at night, relative humidity 60%-70%, light intensity 300-500 μmol). m -2 s -1 (Light duration 12-14 hours / day). Kimura B nutrient solution has a pH of 5.6-5.8, and 1L of Kimura B nutrient solution can supply 30 rice plants to grow to the three-leaf stage.

[0071] 3. Once the rice seedlings have grown to the three-leaf stage, they are transplanted into cadmium-contaminated fields. The cadmium-contaminated soil used in this experiment is the experimental soil from the Ningxiang rice experimental field, with a total cadmium content of 0.6 mg / kg and an available cadmium content of 0.3 mg / kg, classifying it as moderately cadmium-contaminated farmland soil.

[0072] 4. Apply the aforementioned prepared microbial inoculant during the rice tillering stage at a rate of 0, 5, 10, or 15 L / m². 2The appropriate dosage was irrigated into the rice soil, with three replicates for each treatment. The rice was then cultivated under normal management until maturity, and the cadmium content in the brown rice was determined. The average content is shown in Table 8.

[0073] Table 8. Cadmium content in brown rice from different treatments

[0074] The results showed that the present invention contains Cossackella ( Kosakonia sacchari) The microbial inoculant P1-4 can significantly reduce the cadmium content in rice, ensuring food safety. When the amount of microbial inoculant applied is 10 L / m³... 2 At that time, the cadmium content of the rice met the requirements of GB2762-2022 "National Food Safety Standard - Limits of Contaminants in Food" (cadmium in rice ≤ 0.2 mg / kg). Although the application rate was 15 L / m³. 2 At this time, the cadmium reduction effect is better, but the production cost is increased, so it is set at 10L / m³. 2 The application rate is more suitable.

[0075] As can be seen from Examples 3 and 4, the Cossackella strains screened using this invention (…) Kosakonia sacchari) P1-4 and its inoculants have extremely high cadmium fixation efficiency in the environment, and can stably increase the environmental pH, reduce the cadmium content in rice, and thus promote rice growth.

[0076] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of Cossackella, characterized in that, Named Cossackie ( Kosakonia sacchari P1-4 were deposited on September 20, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO:M 20231759.

2. A microbial inoculant, characterized in that, Including Cossackella as described in claim 1 ( Kosakonia sacchari P1-4 or includes Cossacchari bacteria as described in claim 1. Kosakonia sacchari Fermentation broth of P1-4.

3. A Cossackella as described in claim 1 ( Kosakonia sacchari The application of P1-4 or the microbial agents as described in claim 2 in promoting rice growth and / or improving soil health.

4. The application as described in claim 3, characterized in that, The promotion of rice growth includes increasing the biomass of rice roots, stems, leaves, panicles, root length, plant height, and / or yield.

5. The application as described in claim 3, characterized in that, The improvement of soil health includes increasing one or more of the following in the soil: NH4-N, NO3-N, organic phosphorus, organic matter, and pH.

6. The application as described in claim 3, characterized in that, The Cossac bacteria ( Kosakonia sacchari P1-4 or the microbial agent described herein may be irrigated into the soil.

7. The application as described in claim 6, characterized in that, The dosage of the microbial agent is 5-15 L / m³. 2 The OD600 value of the microbial agent is 0.8-1.

1.

8. A Cossackella as described in claim 1 ( Kosakonia sacchari The application of P1-4 or the microbial agents as described in claim 2 in reducing soil cadmium content and / or reducing rice cadmium content.

9. The application as described in claim 8, characterized in that, Apply the aforementioned Cossacella during the rice tillering stage ( Kosakonia sacchari P1-4 or the aforementioned microbial inoculants.

10. The application as described in claim 9, characterized in that, The application rate of the microbial agent is 5-15 L / m³. 2 The OD600 value of the microbial agent is 0.8-1.1.

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