A Bacillus strain and its application in inhibiting bacteria and degrading cellulose and lignin

Abstract

The invention discloses a strain of Bacillus and its application in inhibiting bacteria and degrading cellulose and lignin, and the deposit number of the strain is CGMCC No. 30495. The strain of Bacillus is used to degrade cellulose and lignin, and has antagonistic or inhibitory properties against soil-borne pathogens of soybean, including Fusarium oxysporum, Sclerotinia sclerotiorum, Phytophthora and other soil-borne pathogens. After being made into a bacterial agent, the crops can be protected from pathogens, which is very beneficial to the healthy growth of crops and disease prevention and control.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, to microbial fermentation and its application, and in particular to a bacillus strain and its application in inhibiting bacteria and degrading cellulose and lignin. Background Art

[0002] China is one of the largest agricultural producers in the world, with a vast area of ​​crop planting. According to data from the past few years, China's annual straw production is roughly over 1 billion tons. At the same time, China's straw resource waste problem is very prominent, and it is usually burned or disposed of at will and cannot be effectively utilized. Straw contains rich nutrients, which can improve soil fertility, maintain soil moisture, and improve soil structure. Returning straw to the field can produce a series of positive effects while utilizing straw resources. It is an important measure to promote the transformation of agricultural production methods and improve the level of sustainable development of agriculture, and help achieve efficient, green and sustainable development of agriculture. In recent years, the Chinese government has introduced a series of policies and measures to promote the implementation of returning straw to the field to cope with environmental problems and resource waste in agricultural production.

[0003] However, returning straw to the field also faces some challenges and problems. On the one hand, cellulose and lignin are difficult to degrade. Straw is mainly composed of cellulose, lignin, etc., which are the main components of plant cell walls and have high resistance to degradation. In the process of returning to the field, these difficult-to-degrade organic substances will gradually decompose, but at a slow rate, resulting in a longer residual time of straw in the soil. Refractory organic substances may also have a certain inhibitory effect on the activity of soil microorganisms and affect the biological cycle process in the soil. On the other hand, pathogens carried in straw may spread through the soil, infect the roots of crops in the soil, and cause diseases in underground parts, such as root rot and root-knot nematode disease. Moreover, pathogens in straw may survive in the soil for a period of time. Long-term straw return to the field may lead to the accumulation of pathogens in the soil and increase the risk of disease.

[0004] Soybean (Glycine max (L.) Merr.) is one of the important crops and is widely planted around the world. It is a food crop rich in protein, oil and carbohydrates, and also produces a large amount of straw resources. The above-mentioned problems also exist in the return of soybean straw to the field, especially in high-latitude areas due to the low and long winter temperatures. Not only is the straw degradation slow, but soil-borne pathogens accumulate, which ultimately affects soybean yield and quality. Soil-borne pathogens refer to pathogenic microorganisms that spread through spores or hyphae in the soil. They invade the soybean root system and cause various diseases. Common soybean soil-borne pathogens include root rot bacteria, sclerotinia bacteria and damping-off bacteria.

[0005] Phytophthora root rot of soybeans is a disease caused by the Phytophthora root rot fungus invading the soybean roots and destroying the root tissue, causing the plants to turn yellow, wilt, and even die. The disease is more serious in high humidity environments, especially when the soil is over-irrigated.

[0006] Fusarium root rot is caused by Fusarium oxysporum and occurs primarily in moist and warm conditions. The disease first infects the taproot of soybeans, then spreads to the lateral roots and stems, causing root damage, growth stunting, and leaves gradually turning yellow. In severe cases, the plant may die.

[0007] Sclerotinia sclerotiorum is a common disease of soybeans. It is most severe in high humidity and cool conditions. Sclerotinia sclerotiorum infects the stems, leaves and flowers of soybeans, forming black sclerotia, which lead to tissue rot and the production of white hyphae. Sclerotinia sclerotiorum can cause soft rot and dryness of the stems and leaves, weaken the plant, and may lead to plant death in severe cases.

[0008] Traditional methods for the prevention and control of soil-borne soybean pathogens include reasonable field management, planting disease-resistant varieties, and the use of chemical pesticides. However, these methods have certain limitations, such as high cost, environmental pollution, and pesticide resistance, and these methods are not sustainable.

[0009] In short, soybean is an important crop, but the difficulty of rapid straw degradation and the accumulation of soil-borne pathogens pose a serious threat to its planting and yield. Therefore, the key to solving the problem of returning soybean straw to the field is to effectively destroy the plant lignin and cellulose structure, release valuable chemicals and inhibit the growth of soybean pathogenic fungi. Therefore, it has become an important research direction to find antagonistic bacteria that can efficiently degrade cellulose and lignin in straw and have biological control effects from the returned soil and apply them to straw degradation and disease control in farmland. Through the research and application of biological microbial agent technology, the infection of soybean soil-borne pathogens can be effectively controlled while returning straw to the field, and the nutrients of straw can be fully utilized and the soil ecological environment can be improved to increase soybean yield and quality.

[0010] In the future, we need to further study the interaction mechanism between soybean soil-borne pathogens and antagonistic bacteria to develop more efficient and low-cost biological control methods to better solve the problems of low degradation and utilization rate and fungal diseases caused by returning straw to the field. In the short term, we can give full play to the advantages of returning straw to the field, improve soil fertility, maintain soil moisture, and improve soil structure. In the long term, we can increase the content of beneficial bacteria in farmland, realize green cycle with bacterial cultivation, gradually reduce dependence on chemical fertilizers and pesticides, and contribute to the green and sustainable development of soybean planting industry and even agricultural system. Summary of the invention

[0011] The technical problem to be solved by the present invention is to provide a bacillus strain and its application in inhibiting bacteria and degrading cellulose and lignin in view of the deficiencies of the prior art.

[0012] A strain of Bacillus, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.30495.

[0013] The bacillus is used in degrading cellulose and lignin and inhibiting soybean soil-borne pathogens, and the soybean soil-borne pathogens include Fusarium oxysporum, Sclerotium sclerotium and Phytophthora.

[0014] The bacillus cells, sterile fermentation liquid, or a mixture of the cells and the sterile fermentation liquid are used in degrading cellulose and lignin and inhibiting soybean soil-borne pathogens.

[0015] A biocontrol agent, the components of which include the bacillus cells, or the fermentation liquid of the bacillus, or a mixture of the bacillus cells and the fermentation liquid.

[0016] The biocontrol agent is applied after being processed into an agriculturally acceptable preparation.

[0017] The biocontrol agent is characterized in that it is used to degrade cellulose, lignin and inhibit soybean soil-borne pathogens.

[0018] The Bacillus screened out by the present invention can efficiently degrade cellulose and lignin; in addition, pathogen antagonism experiments were carried out, mainly targeting common soil-borne diseases of soybean, an important national oil crop; the purpose is to provide a multifunctional straw degrading strain, and at the same time provide a high-efficiency potential strain for improving soil quality, biological control and the development of multifunctional microbial agents.

[0019] Experiments have shown that this strain can efficiently and quickly degrade the difficult-to-degrade cellulose and lignin in straw, shortening the straw decomposition cycle; moreover, this strain can antagonize a variety of soybean soil-borne pathogens, and after being made into a microbial agent, it can weaken the invasion of pathogens caused by returning straw to the field; moreover, this strain has a low production cost and a short cycle, and has good prospects for agricultural application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The cellulose degradation effects of the 17 purified strains are shown;

[0021] Figure 2 The lignin degradation effects of CK and the 7 strains screened were shown;

[0022] Figure 3 is the phylogenetic tree of strain 0S14;

[0023] Figure 4 Colony morphology of strain 0S14;

[0024] Figure 5 Cellulose degradation effect of strain 0S14

[0025] Figure 6 The lignin degradation effect of strain 0S14

[0026] Figure 7 Antagonistic effect of strain OS14 on Fusarium oxysporum; A: CK; B: treatment of strain OS14

[0027] Figure 8 The antagonistic effect of strain 0S14 on Phytophthora; A: CK; B: strain 0S14 treatment

[0028] Fig. 9 The antagonistic effect of strain OS14 on Sclerotinia sclerotiorum; A: CK; B: strain OS14 treatment; DETAILED DESCRIPTION

[0029] The present invention is described in detail below in conjunction with specific embodiments.

[0030] Example 1

[0031] This example describes the isolation, purification process and identification results of the strain.

[0032] 1. Source of soil samples:

[0033] Soil samples at a height of 0-20 cm from the ground were collected from a farm in Yangling, Shaanxi Province that has been practicing straw return for a long time. The five-point sampling method was used and the samples were mixed evenly and then packed into sterile bags and refrigerated for later use.

[0034] 2. Culture medium formula:

[0035] 1) CMC-Na screening medium: 5 g sodium carboxymethyl cellulose, 5 g yeast powder, 0.5 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 16 g agar, 1000 mL distilled water, sterilize at 121°C for 20 min.

[0036] 2) CMC-Na screening and identification medium: 5g sodium carboxymethyl cellulose, 5g yeast powder, 0.5g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 16g agar, 0.2g Congo red, 1000mL distilled water, sterilize at 121℃ for 20min.

[0037] 3) LB medium: 10 g tryptone, 5 g yeast powder, 10 g NaCl, 15 g agar powder, 1000 mL distilled water, pH 7.2-7.4, sterilize at 121°C for 20 min.

[0038] 4) Aniline blue medium: 5g yeast powder, 10g tryptone, 10g NaCl, 0.1g aniline blue, 15g agar, 1000mL distilled water, sterilize at 121℃ for 20min.

[0039] 3. Strain separation and purification:

[0040] Take 3 mg of the original soil and add it to 300 ml of sterile water. Add sterile magnetic beads and grind it on a shaker at 180 rpm for 2 hours to prepare a suspension. Prepare carboxymethyl cellulose culture medium and sterilize it in an autoclave at 121°C for 20 minutes. Dilute the sample in a gradient to 10 -4 , 10 -5 , 10 -6 , make 5 parallels for each gradient, take 200ul of each dilution sample and spread it on the CMC-Na screening plate, put the plate in a 28℃ constant temperature incubator and culture it upside down for 3 days until a single clone appears, pick out single colonies with different colors, sizes and shapes, and use LB medium for continuous streaking for purification.

[0041] The cellulose degradation ability of the 397 purified bacterial strains was identified. Each single colony was selected and cultured on a cellulose screening and identification medium. 17 strains with strong cellulose degradation ability were screened out (see Table 1). The degradation effects are shown in Table 1. Figure 1 The selected strains were inoculated into aniline blue medium and the degradation effect was as follows. Figure 2 , screened out strains that can degrade both cellulose and lignin, and plated the purified strains for three times to screen out strains with obvious antagonism to soybean soil-borne pathogens.

[0042] Table 1 Degradation effect of the screened strains on cellulose

[0043]

[0044] The strain OS14 was isolated and screened on LB medium and placed in a cryovial containing 50% glycerol (1:1) and stored at -80°C. Figure 3 The streaking results are for the strains.

[0045] 1. 16S rDNA sequence identification

[0046] The strain was cultured in a liquid medium at 140 rpm for 2 days, and then DNA was extracted and 16s rDNA sequencing was performed. The sequencing result is shown in SEQ ID NO: 1.

[0047] The sequencing results were compared with the NCBI (National Center for Biotechnology Information) database by BLAST sequence comparison, and the base sequences of related strain genes with high homology were downloaded. The phylogenetic tree was constructed using the neighbor-joining method using MEGA software. Figure 3 As shown, strain 0S14 has a similarity of 99% with Bacillus halotolerans strain DSM 8802, belongs to the genus Bacillus sp. and is identified as Bacillus 0S14 strain.

[0048] The deposit information is as follows.

[0049] Classification and nomenclature: Bacillus sp.

[0050] Storage time: May 6, 2024

[0051] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0052] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0053] Deposit number: CGMCC No.30495.

[0054] Example 2

[0055] This example describes the test of the cellulose degradation ability of strain OS14.

[0056] Prepare cellulose screening and identification culture medium, sterilize at 121℃ for 20min, pick up single colonies of purified strains with bamboo sticks and inoculate them on Congo red sodium carboxymethyl cellulose culture medium plates for inverted culture, set up 5 replicates for each strain, and culture at a constant temperature of 28℃ for 3 days, which will form a transparent circle that degrades cellulose. Measure the diameter of the transparent circle (D) and the diameter of the colony (d) of the strain on the plate and keep a record, calculate the ratio of D / d, which represents the strength of cellulose degradation ability, and the higher the ratio, the stronger the degradation ability of the strain. The experimental results show that strain 0S14 has good cellulose degradation performance. Figure 4 , the D / d ratio is as high as 4.59.

[0057] Example 3

[0058] This example describes the strain OS14 lignin degradation ability test.

[0059] Aniline blue decolorization method is used to screen lignin-degrading bacteria, because the degradation ability of microorganisms is directly related to the production capacity of ligninase. Prepare aniline blue culture medium and sterilize it in a sterilizer at 121℃ for 20 minutes. Pick out the single bacteria obtained by screening and inoculate them on aniline blue culture medium and incubate them upside down in a constant temperature incubator at 28℃. Observe the presence and size of the decolorization circle on the plate. The larger the decolorization circle, the stronger the ability of the strain to degrade lignin. Figure 5 The results showed that strain 0S14 has efficient lignin degradation activity.

[0060] Example 4

[0061] This example describes an in-dish antibacterial standoff test of the OS14 strain.

[0062] The tested fungi of soil-borne pathogens of soybean were: Fusarium oxysporum M3-3XU strain; Phytophthora sojae; Sclerotinia sclerotiorum (Lib.) de Bary.

[0063] Prepare PDA culture medium and sterilize it in a sterilizer at 121℃ for 20min. Use a hole puncher to punch holes in the edge area of ​​the colony of soybean soil-borne pathogenic fungi to make a 5mm diameter cake. Make a cross mark on the back of the PDA plate, inoculate the pathogen cake at the center point, and inoculate the single colony of the strain at equidistant points (2.5cm) around the center point. Inoculate 4 points per dish and replicate 5 times for each strain. At the same time, set up a PDA plate control that only inoculates the pathogen cake. Place the inoculated plate in a 28℃ constant temperature incubator for dark culture, and observe the growth after 6 days. Measure the diameter of the pathogen control colony and the diameter of the screening colony pointed by the pathogen colony and calculate the inhibition rate.

[0064] Inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter × 100%.

[0065] The size of the inhibition rate represents the strength of its antibacterial ability. Table 2 shows the inhibition rate of the strain against three soybean soil-borne pathogens. Figure 7 , the antibacterial effect on soybean phytophthora is shown in Figure 8 , the antibacterial effect on Sclerotinia sclerotiorum is shown in Fig. 9 The experimental results showed that strain OS14 had high antibacterial activity against soil-borne soybean pathogens such as root rot, phytophthora, and sclerotinia.

[0066] Table 2 Antibacterial effect of strain OS14 on soybean soil-borne pathogens

[0067]

[0068] It should be understood that those skilled in the art can make improvements or changes based on the above description, and all these improvements and changes should fall within the scope of protection of the appended claims of the present invention.

Claims

1. A strain of Bacillus ( Bacillus sp. ) 0S14, characterized in that, It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No. 30495.

2. The use of the Bacillus sp. OS14 according to claim 1 in degrading cellulose, lignin and inhibiting soil-borne pathogens of soybean, wherein the soil-borne pathogens of soybean are Fusarium oxysporum, Sclerotinia sclerotiorum or Phytophthora.

3. The application of the bacterium of Bacillus OS14 according to claim 1, the sterile fermentation liquid, or the mixture of the bacterium and the sterile fermentation liquid in degrading cellulose, lignin and inhibiting soybean soil-borne pathogens, wherein the soybean soil-borne pathogens are Fusarium oxysporum, Sclerotinia sclerotiorum or Phytophthora.

4. A biocontrol agent, characterized in that: The components of the biocontrol agent include the bacillus OS14 cells of claim 1, or a mixture of the bacillus OS14 cells of claim 1 and a fermentation broth.

5. The biocontrol agent according to claim 4, characterized in that The biocontrol agent is processed into an agriculturally acceptable formulation and then applied.

6. The biocontrol agent according to claim 4, characterized in that The biocontrol agent is used for degrading cellulose and lignin and inhibiting soil-borne pathogens of soybean, wherein the soil-borne pathogens of soybean are Fusarium oxysporum, Sclerotinia sclerotiorum or Phytophthora.

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