CN118773090A - Peribacillus frigoritolerans NMI04 and its microbial agent and application - Google Patents

Abstract

The invention relates to Peribacillus frigoritolerans NMI strain 04, a microbial agent and application thereof, and belongs to the technical field of microorganisms. Peribacillus frigoritolerans NMI04 is preserved in China general microbiological culture Collection center (CGMCC) No.31153, and the preservation date is 2024, 07 and 03, and the address of a preservation institution: the korean district North Star, beijing city, part No. 1, no. 3. The Peribacillus frigoritolerans NMI of the invention can promote crop growth, realize weight reduction and synergy, control the occurrence of soil-borne bacteria and bacterial diseases, reduce chemical pesticide application, reduce pesticide residues and promote sustainable development of agricultural economy.

Description

Peribacillus frigoritolerans NMI04 and its fungal agent and application

Technical Field

The invention relates to the technical field of microorganisms, and in particular to a strain of Peribacillus frigoritolerans NMI04, a bacterial agent and an application thereof.

Background Art

Biological control refers to the use of one or more antagonistic microorganisms to reduce the number of pathogens or weaken the pathogenic activity of pathogens, thereby reducing the severity of the corresponding diseases caused by pathogens. Since antagonistic microorganisms are safe and harmless to humans and animals, have little environmental pollution and no residue, they can help maintain ecological balance, and many antagonistic microorganisms are beneficial bacteria that can repair the physical and chemical structure of the soil, promote the healthy growth of crops, and maintain the diversity of soil organisms. Compared with traditional chemical control measures, biological control can effectively overcome a series of disadvantages brought by it, with a long-lasting control effect, and is green and safe, with strong killing specificity for plant diseases and insect pests. In particular, with the enhancement of people's environmental awareness, the emphasis on food safety issues, and the requirements for ecological environment construction, protection of biodiversity and sustainable agricultural development, biological control of soil-borne diseases has become a hot spot in current research and development. Screening effective biocontrol bacteria, developing broad-spectrum, safe and effective microbial fungicides, and controlling the occurrence of soil-borne fungal diseases are urgent needs to increase the yield of cash crops, reduce losses, and promote sustainable development of the agricultural economy.

Compared with chemical fertilizers, microbial fertilizers have the following advantages: improve soil structure; increase soil fertility and promote plant absorption of nutrients; stimulate and regulate plant growth, increase yield and improve quality; produce disease resistance and stress resistance, indirectly promote plant growth; reduce agricultural residues and protect the environment. Microbial fertilizers can effectively replace some of the functions of chemical fertilizers and pesticides, and reduce the input of chemical fertilizers and pesticides. At the same time, some microorganisms have the ability to decompose heavy metals or other harmful substances in the soil. Living bacteria can reduce or inhibit the absorption of heavy metals and pesticides by crops, thereby achieving the purpose of purifying and repairing the soil. Microbial fertilizers are an inevitable choice to reduce the use of chemical fertilizers and pesticides and reduce environmental pollution.

The harm of plant pathogens to plants includes root diseases, stem, leaf and fruit diseases. Some of these pathogens mainly infect the roots of plants, causing root rot and wilting, affecting the plant's ability to absorb water and nutrients. Bacillus is a type of aerobic or facultative anaerobic rod-shaped, spore-forming bacteria. The most important feature of Bacillus is that it can form endospores inside the cell, which makes it highly resistant to radiation, dryness, ultraviolet rays, radiation, heat and chemicals, allowing these bacteria to survive under adverse conditions. Therefore, in addition to being mainly distributed in the soil, plant surfaces and water bodies, this type of bacteria also exists in many high and low temperature, high salt, saline-alkali and high radiation areas. There are many species of Bacillus with special functions, which have wide application value in industry, agriculture and scientific research.

Peribacillus frigoritolerans has not been found in the current agricultural production of biological pesticides.

Summary of the invention

In view of the problem that Peribacillus frigoritolerans has not been found in existing biological pesticides, the present invention provides a strain of Peribacillus frigoritolerans NMI04 and a bacterial agent and application to solve the above problem.

In a first aspect, the present invention provides a strain of Peribacillus frigoritolerans NMI04, wherein the Peribacillus frigoritolerans NMI04 is deposited in the General Microbiological Center of China Microorganism Culture Collection Committee, with a deposit number of CGMCC No.31153, a deposit date of July 3, 2024, and an address of the deposit institution: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

Furthermore, the 16S rDNA sequence of Peribacillus frigoritolerans NMI04 is shown in SEQ ID NO.1.

In a second aspect, the present invention provides a bacterial agent containing Peribacillus frigoritolerans NMI04, wherein the bacterial agent includes a liquid bacterial agent and a solid bacterial agent.

A method for preparing the above-mentioned bacterial agent, wherein the preparation method of the liquid bacterial agent is: inoculating Peribacillus frigoritolerans NMI04 into LB culture medium, culturing at 35°C and 180rpm for 24 hours to obtain a bacterial solution containing Peribacillus frigoritolerans NMI04; adjusting the concentration of Peribacillus frigoritolerans NMI04 in the bacterial solution to 3×10 ⁹ cfu/mL to obtain a Peribacillus frigoritolerans NMI04 liquid bacterial agent.

A method for preparing the above-mentioned bacterial agent, the preparation method of the solid bacterial agent is: adding diatomaceous earth with a volume ratio of 10% to the Peribacillus frigoritolerans NMI04 liquid bacterial agent, spray drying at 120°C, to prepare the Peribacillus frigoritolerans NMI04 solid bacterial agent.

Preferably, the LB medium comprises the following components: 10 g/L tryptone, 5 g/L NaCl, 10 g/L yeast extract, sterile water, pH=7.2.

In a third aspect, the present invention provides a use of Peribacillus frigoritolerans NMI04 in nitrogen fixation and phosphorus solubilization.

In a fourth aspect, the present invention provides a use of Peribacillus frigoritolerans NMI04 in inhibiting Fusarium oxysporum cucumerinoides, Rhizoctonia solani, Solanum graminearum var. graminearum, Fusarium oxysporum cubanense and Ralstonia solanacearum.

In a fifth aspect, the present invention provides a use of Peribacillus frigoritolerans NMI04 in preventing and controlling cucumber wilt, potato black mole, wheat take-all disease, banana wilt and tomato bacterial wilt.

In a sixth aspect, the present invention provides a use of Peribacillus frigoritolerans NMI04 in promoting plant growth, wherein the plant is Peribacillus frigoritolerans.

Furthermore, the application includes using a liquid bacterial agent containing Peribacillus frigoritolerans NMI04 to perform root irrigation treatment on plants.

The beneficial effects of the present invention are:

The Peribacillus frigoritolerans NMI04 provided by the present invention has the advantages of broad-spectrum, safety and effective sterilization. When applied to agricultural production, it can control the occurrence of soil-borne fungal and bacterial diseases (such as cucumber wilt, wheat take-all disease, banana wilt, potato black mole disease, tomato bacterial wilt, etc.), reduce the application of chemical pesticides, and reduce agricultural residues. It can also promote crop growth, achieve weight loss and efficiency improvement, and promote the sustainable development of the agricultural economy.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

FIG. 1 is a diagram showing the nitrogen fixation effect of Peribacillus frigoritolerans NMI04 in Example 1 of the present invention.

FIG. 2 is a diagram showing the effect of the phosphate solubilization function of Peribacillus frigoritolerans NMI04 in Example 1 of the present invention.

FIG. 3 is a diagram showing the potassium-dissolving effect of Peribacillus frigoritolerans NMI04 in Example 1 of the present invention.

FIG. 4 is a graph showing the growth of Peribacillus frigoritolerans NMI04 in NA medium in Example 1 of the present invention.

FIG5 is a Gram staining image of Peribacillus frigoritolerans NMI04 in Example 1 of the present invention.

FIG. 6 is a spore staining diagram of Peribacillus frigoritolerans NMI04 in Example 1 of the present invention.

FIG. 7 is a diagram showing the plate confrontation results of Peribacillus frigoritolerans NMI04 in Example 2 of the present invention.

FIG8 is a comparison diagram of the disease conditions of cucumber seedlings in Example 4 of the present invention.

FIG. 9 is a comparative diagram of the growth promotion of green stalk vegetable in Example 4 of the present invention.

DETAILED DESCRIPTION

In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

Example 1

Isolation, Screening and Characterization of Peribacillus frigoritolerans NMI04

(1) Sampling: On April 10, 2024, soil was collected from a tomato greenhouse in Hohhot, Inner Mongolia (111.8548°E, 40.8297°N) and placed in a ziplock bag for future use.

(2) Separation:

Collect 5 g of rhizosphere soil from step (1), add it to a conical flask containing 45 mL of sterile pure water, and place it in a water bath at 100°C for 10 minutes. After standing, take the upper solution and dilute it into samples of different concentration gradients. Use a spreader to spread it on a nutrient agar (NA) plate culture medium, and invert and culture it at 35°C for 24 hours.

(3) Purification: Select single colonies with different morphologies, purify the strains of each single colony by streaking on NA plates three times, screen the strains according to the different colony morphologies and determine their numbers, store them in test tube slants, and store them in a 4°C refrigerator as test bacteria for later tests. At the same time, place them in 50% sterile glycerol tubes and store them at -20°C for future use.

(4) Identification of nitrogen fixation, phosphorus solubilization and potassium solubilization functions

The different strains after separation and purification were inoculated into LB medium, cultured in a shaker at 35°C and 180rpm for 20h, and the bacterial solution was diluted to a ^10-6 gradient. 100μL of the dilution was taken and spread on the identification medium plate. The nitrogen fixation function was identified using the Ashubes medium plate, the phosphorus solubilization function was identified using the inorganic phosphorus bacteria medium plate, and the potassium solubilization function was identified using the Alexander Borov medium plate. If colonies can grow normally on the plate after coating, it indicates that the strain has the corresponding function screened by this plate. The nitrogen fixation function effect diagram of strain NMI04 is shown in Figure 1, the phosphorus solubilization function effect diagram is shown in Figure 2, and the potassium solubilization function effect diagram is shown in Figure 3. According to Figures 1 to 3, it can be judged that strain NMI04 has the functions of nitrogen fixation and phosphorus solubilization, but no potassium solubilization function.

The components and contents of the above three identification culture medium plates are as follows:

Ashurst medium: potassium dihydrogen phosphate 0.2 g/L, magnesium sulfate 0.2 g/L, sodium chloride 0.2 g/L, calcium carbonate 5.0 g/L, mannitol 10.0 g/L, calcium sulfate 0.1 g/L, agar 15.0 g/L, pH = 7.0 ± 0.1.

Inorganic phosphorus bacterial culture medium: glucose 10.0g/L, ammonium sulfate 0.5g/L, sodium chloride 0.3g/L, magnesium sulfate 0.3g/L, manganese sulfate 0.03g/L, potassium sulfate 0.3g/L, ferrous sulfate 0.03g/L, calcium phosphate 5.0g/L, agar 15.0g/L, pH=7.0~7.5.

Alexandrov medium: sucrose 5.0 g/L, magnesium sulfate 0.5 g/L, disodium hydrogen phosphate 2.0 g/L, ferric chloride 0.005 g/L, calcium carbonate 0.1 g/L, soil minerals 1.0 g/L, agar 18.0 g/L, pH = 7.2 ± 0.2.

(5) Morphological identification

The strain NMI04 was inoculated into LB medium and cultured at 35°C and 180 rpm for 24 h. After dilution to 10 ^-7 gradient, 100 μL was spread on NA plate and cultured at 35°C for 48 h. The color, shape and size of the single colony were continuously observed. As shown in Figure 4, the strain NMI04 grew well on NA medium. The colonies were white and slightly yellow, round, shiny on the surface and smooth on the edges.

(6) Gram stain was used for staining. The results of optical microscope observation showed that the bacteria were long rod-shaped and had spores, as shown in Figure 5. Then spore staining was performed, and the results showed that the spores were oval, as shown in Figure 6.

(7) Physiological and biochemical identification

The strain NMI04 was subjected to physiological and biochemical identification. The test items and results are shown in Table 1.

Table 1 - Physiological and biochemical identification results of strain NMI04

(8) Molecular identification

A single colony of strain NMI04 was streaked on a NA plate, and after culturing at 35°C for 12 hours, the cultured NA plate was sent to Sangon Biotech (Shanghai) Co., Ltd. for strain identification. The 16S rDNA sequence is shown in SEQ ID NO.1.

The sequence obtained was submitted to Gen-Bank for homology comparison, and the strain was preliminarily determined to be Peribacillus frigoritolerans . Peribacillus frigoritolerans NMI04 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number CGMCC No.31153 and the deposit date July 3, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

Example 2

Flat plate standoff test

Peribacillus frigoritolerans NMI04 was subjected to a plate confrontation test. The pathogens used in the test included the cucumber wilt pathogen ( Fusarium oxysporum f.sp. cucumerinum ), the potato black spot pathogen ( Rhizoctonia solani ), the wheat take-all pathogen ( Gaeumannomyces graminis ), the banana wilt pathogen ( Fusarium oxysporum f.sp.cubense ) and the tomato bacterial wilt pathogen ( Ralstonia solanacearum ).

The specific method is as follows:

A. Inoculate 6 mm diameter bacterial cakes of cucumber wilt pathogen, potato black mole pathogen, wheat take-all pathogen and banana wilt pathogen in the center of the PDA plate, and use a pipette to take 8 μL of the culture solution of the test strain, inoculate it 2 cm away from the pathogen, and culture it at room temperature for 7-10 days.

B. Spread 100 μL of the shaken bacterial wilt pathogen on the NA plate, use a pipette to take 8 μL of the culture medium of the test strain, inoculate it in the center of the plate, and culture it at room temperature for 7 to 10 days.

Observe the antibacterial conditions in each plate to determine whether the tested strain has the corresponding antibacterial effect. Among them, the plate confrontation test results of strain NMI04 are shown in the figure, which shows that Peribacillus frigoritolerans NMI04 has a significant inhibitory effect on Fusarium oxysporum, Rhizoctonia solani, Aspergillus graminearum, Fusarium oxysporum, and Tomato bacterial wilt pathogens.

Example 3

Preparation of Peribacillus frigoritolerans NMI04 inoculant

Liquid bacterial agent: Activate Peribacillus frigoritolerans NMI04 on NA plate medium, take a loop of the activated Peribacillus frigoritolerans NMI04 and inoculate it into LB medium, and culture it at 35°C and 180rpm for 24h to obtain Peribacillus frigoritolerans NMI04 bacterial solution. The components of LB medium are as follows: 10g/L tryptone, 5g/L NaCl, 10g/L yeast extract, sterile water, pH=7.2. The bacterial concentration of Peribacillus frigoritolerans NMI04 bacterial solution is adjusted to 3×10 ⁹ cfu/mL to obtain Peribacillus frigoritolerans NMI04 liquid bacterial agent.

Solid inoculant: 10% diatomaceous earth was added by volume to the liquid inoculant of Peribacillus frigoritolerans NMI04, and the mixture was spray-dried at 120°C to obtain the solid inoculant of Peribacillus frigoritolerans NMI04.

Example 4

Experiment on the biocontrol ability of Peribacillus frigoritolerans NMI04

Ability to prevent cucumber wilt

Taking cucumber wilt as the control target, the Peribacillus frigoritolerans NMI04 liquid microbial agent prepared in Example 3 was used to carry out a biocontrol test, and a horizontal comparison was made with a Bacillus subtilis AC1 strain with biocontrol function (purchased from the China Agricultural Microbiological Culture Collection Management Center, with a collection number of ACCC 19742). Three parallel treatments were set up, namely the Peribacillus frigoritolerans NMI04 group, the Bacillus subtilis AC1 group and the control group (CK group), and each group was treated with 10 replicates. The Peribacillus frigoritolerans NMI04 liquid microbial agent prepared in Example 3 was applied to the Peribacillus frigoritolerans NMI04 group, the Bacillus subtilis AC1 group was applied to the Bacillus subtilis AC1 microbial agent dilution, and the control group was not applied with any biocontrol product. The specific application method is: Peribacillus frigoritolerans NMI04 liquid inoculant and Bacillus subtilis AC1 are both made into 200-fold dilutions (bacterial concentration is 1.5×10 ⁷ cfu/mL), 100 mL is taken and poured on the roots of 4-week-old cucumber seedlings (the control group is poured with clean water), and after 24 hours of pretreatment, the cucumber wilt pathogen (Fusarium oxysporum cucumber-specific type) with a turbidity value of 1.0 MCF is inoculated by root irrigation method, and the disease index is continuously recorded and the phenotype is observed. The specific results are shown in Table 2, and the disease map of the cucumber seedlings is shown in Figure 8.

Disease level: Level 0, no symptoms; Level 1, cotyledons yellowing but not wilting; Level 2, cotyledons wilting; Level 3, cotyledons and true leaves wilting or plants stunted; Level 4, death.

Disease index = ∑ (number of diseased plants at each level × representative value at each level) / (total number of plants surveyed × highest representative value) × 100.

Control effect (%) = [(control disease index - treatment disease index) / control disease index] × 100.

Table 2- Control results of Peribacillus frigoritolerans NMI04 on cucumber wilt

As shown in Figure 8 and Table 2, almost all plants in the control group showed symptoms of cucumber wilt, with obvious dwarfing, most leaves turning yellow and withering, or even completely dying; most plants in the Bacillus subtilis AC1 group had yellowing and withering old leaves, but no obvious dwarfing; half of the plants in the Peribacillus frigoritolerans NMI04 group showed no symptoms and grew well, while the rest of the plants had yellowing and withering old leaves, but no obvious dwarfing. Both Bacillus subtilis AC1 and Peribacillus frigoritolerans NMI04 had significant control effects on cucumber wilt, but the control effect of Peribacillus frigoritolerans NMI04 was better than that of Bacillus subtilis AC1.

Example 5

Growth-promoting effect of Peribacillus frigoritolerans NMI04 on Brassica rapa

Green stalked cabbage was used as the test object, and the Peribacillus frigoritolerans NMI04 liquid bacterial agent prepared in Example 3 was used to carry out a growth promotion test, and a Bacillus subtilis AC2 with a growth promotion function (purchased from the China Agricultural Microbial Culture Collection Management Center, with a collection number of ACCC 19743) was compared horizontally, and three parallel treatments were set, namely, the Peribacillus frigoritolerans NMI04 group, the Bacillus subtilis AC2 group and the control group (CK group), and each group was treated with 10 replicates. The Peribacillus frigoritolerans NMI04 liquid bacterial agent prepared in Example 3 was made into a 100-fold dilution (bacterial concentration was 3×10 ⁷ cfu/mL), and 100 mL of the dilution was taken for root irrigation treatment; the Bacillus subtilis AC2 group was applied with the Bacillus subtilis AC2 bacterial agent, and the application method was the same as that of the Peribacillus frigoritolerans NMI04 group; the control group was applied with 100 mL of clean water, and no growth promotion product was applied. The green stalk vegetable was transplanted after the third true leaf unfolded. The first root irrigation treatment was carried out on the second day after transplanting, and then the root irrigation treatment was carried out every 7 days, for a total of 3 treatments. 7 days after the third root irrigation treatment, the plant height of the green stalk vegetable was measured with a ruler, the stem diameter was measured with a vernier caliper, and the fresh weight of the aboveground part was weighed with a balance. The results are shown in Table 3 and Figure 9.

Table 3- Growth promotion results of Peribacillus frigoritolerans NMI04 on Chinese cabbage

The test results showed that the plant height, stem diameter and single plant weight of the group treated with Peribacillus frigoritolerans NMI04 solution were significantly better than those of the control group, with plant height increased by 6.8%, stem diameter increased by 25.9%, and fresh weight increased by 38.9%; compared with the group treated with Bacillus subtilis AC2, plant height increased by 4.7%, stem diameter increased by 18.2%, and fresh weight increased by 21.7%. This shows that Peribacillus frigoritolerans NMI04 has a good growth-promoting effect on green cabbage.

Although the present invention has been described in detail with reference to the accompanying drawings and in combination with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention.

Claims (10)

1. The strain Peribacillus frigoritolerans NMI is characterized in that Peribacillus frigoritolerans NMI is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.31153 and the preservation date of 2024, 07, 03 and the address of a preservation institution: the korean district North Star, beijing city, part No. 1, no. 3.

2. The Peribacillus frigoritolerans NMI04 of claim 1, wherein the 16S rDNA sequence of Peribacillus frigoritolerans NMI04 is set forth in SEQ ID No. 1.

3. A microbial agent comprising Peribacillus frigoritolerans NMI a 04 as claimed in claim 1, wherein said microbial agent comprises a liquid microbial agent and a solid microbial agent.

4. The microbial inoculant of claim 3, wherein the liquid microbial inoculant is prepared by the following steps: inoculating Peribacillus frigoritolerans NMI to LB culture medium, culturing at 35deg.C and 180rpm for 24 hr to obtain Peribacillus frigoritolerans NMI-containing bacterial liquid; the Peribacillus frigoritolerans NMI concentration in the bacterial liquid is regulated to be 3 multiplied by 10 ⁹ cfu/mL, and the Peribacillus frigoritoleransNMI liquid bacterial agent is prepared.

5. The microbial inoculum of claim 3, wherein the preparation method of the solid microbial inoculum comprises the following steps: inoculating Peribacillus frigoritolerans NMI to LB culture medium, culturing at 35deg.C and 180rpm for 24 hr to obtain Peribacillus frigoritolerans NMI-containing bacterial liquid; adjusting Peribacillus frigoritolerans NMI concentration in the bacterial liquid to 3X 10 ⁹ cfu/mL to obtain Peribacillus frigoritoleransNMI liquid bacterial agent; adding 10% diatomite into Peribacillus frigoritolerans NMI04 liquid microbial inoculum, and spray drying at 120deg.C to obtain Peribacillus frigoritolerans NMI04 solid microbial inoculum.

6. Use of Peribacillus frigoritolerans NMI according to claim 1 for nitrogen fixation and phosphorus removal.

7. Use of Peribacillus frigoritolerans NMI of claim 1 for inhibiting fusarium oxysporum cucumber specialization, rhizoctonia solani, apotheca graminea Gu Bianchong, fusarium oxysporum copal specialization and solanacearum.

8. Use of Peribacillus frigoritolerans NMI of claim 1 for controlling plant diseases including cucumber fusarium wilt, potato black nevus, wheat take-all, banana fusarium wilt and tomato bacterial wilt.

9. Use of Peribacillus frigoritolerans NMI according to claim 1 for promoting plant growth, wherein the plant is broccoli.

10. The use according to claim 9, comprising rooting of plants with a liquid microbial inoculum containing Peribacillus frigoritolerans NMI a 04.