Bacillus mucilaginosus XC-2 and application thereof in preparation of plant pathogen bacteriostatic agent

Abstract

The invention belongs to the technical field of microorganisms, and discloses a bacillus pumilus XC-2 strain and an application of the bacillus pumilus XC-2 strain in preparation of a plant pathogen bacteriostatic agent, and the preservation number of the bacillus pumilus XC-2 strain is CCTCC NO: M20241670. The strain has the functions of salt resistance, acid and alkali resistance, heat resistance, phytopathogen resistance and the like; the strain can grow under the conditions that the pH is 5.0-9.0, the salt concentration is 11% and the temperature is 20-50 DEG C, the inhibition effect on various plant pathogenic bacteria is good, the inhibition rate on eight plant pathogenic fusarium is higher than 95% or above, and the strain has potential application value of being prepared into a bacteriostatic agent in the aspect of preventing and treating saline-alkali soil plant wilt.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Bacillus marineus XC-2 and application of the strain in preparing a plant pathogenic bacteria antibacterial agent. Background Art

[0002] As an important pathogenic factor in agricultural production, plant pathogenic fungi often cause necrosis, rot or withering of plant roots or organs, posing a serious threat to the agricultural economy and causing immeasurable losses. Therefore, in order to ensure the stability and development of agricultural production, the research and prevention and control of plant pathogenic fungi are particularly important. There are many ways for plant pathogenic fungi to cause disease, such as pathogenic Fusarium invading and blocking vascular tissues, hindering the transportation of water and nutrients, or producing Fusarium toxins to cause plant wilt, etc. At present, there are physical, chemical and biological methods for the prevention and control of plant pathogenic fungi. Physical methods such as land rotation are often used as auxiliary methods; chemical methods are widely used as the main means of preventing and controlling plant diseases. Its significant advantage is that it can effectively prevent and control most plant diseases, with significant and rapid efficacy. However, the use of chemical agents also has potential risks. Long-term use of chemical agents may cause pathogenic fungi to develop drug resistance, thereby reducing the prevention and control effect. In addition, the unreasonable use of chemical agents may also cause soil and water pollution, and even pose a potential threat to human health. The biological method mainly uses living organisms, or extracts active substances that are resistant to plant pathogens from the growth and metabolism of organisms to make plant antibacterial agents. The advantages of the live antibacterial agents that have inhibitory effects on plant pathogens obtained by biological methods are that they are free of pesticide pollution, no residue, safe for humans and animals, have little impact on the ecological environment, and have good control effects, strong specificity, and increase production and income.

[0003] Discovering disease-resistant microorganisms that have inhibitory effects on plant pathogenic fungi from extreme environments can provide a variety of biocontrol strains for plant disease prevention and control in agricultural production. Among them, Bacillus has become a hot spot in the development of live microbial pesticide preparations due to its good stability.

[0004] Zhao Feilong et al. (2017) reported that a strain of Bacillus oceanisediminis DD174 from soybean nodules had an antibacterial activity of 62.48% against cotton wilt (Fusarium oxysporum F.sp.vasinfectum), but did not study the strain's broad-spectrum inhibitory activity against other new plant pathogens; Chen Wen et al. (2022) found that Bacillus oceanisediminis MCCC1A14063 had a good killing effect on root-knot nematodes, but did not study its inhibitory activity against plant pathogens; Qin Dongli et al. (2022) used Bacillus oceanisediminis HL-44 in integrated rice-fish farming, but did not study its inhibitory activity against plant pathogens.

[0005] In summary, the existing literature has not reported that Bacillus oceanisediminis has a broad-spectrum anti-inhibitory activity against other new plant pathogens, nor has its salt-alkali tolerance and enzyme production properties been studied. Therefore, the development of Bacillus oceanisediminis strains that can effectively antagonize a variety of plant pathogenic Fusarium and are salt-alkali tolerant and have high enzyme production activity has important practical significance for the prevention and control of plant wilt disease in saline-alkali land. Summary of the invention

[0006] The invention aims to provide a salt-alkali resistant Bacillus oceanisediminis XC-2, which is deposited in China Center for Type Culture Collection and named as Bacillus oceanisediminis. The deposit number of the Bacillus oceanisediminis is CCTCC NO: M20241670.

[0007] Another object of the present invention is to provide a salt-alkali tolerant strain of Bacillus marine mud XC-2 for use in the preparation of a plant pathogenic bacteria antibacterial agent. In order to achieve the above object, the present invention adopts the following technical measures:

[0008] The applicant isolated a salt-tolerant bacterium from a soil sample in Pucheng County, Weinan City, Shaanxi Province, which has an antibacterial effect on a variety of pathogens and a high antibacterial rate. It was identified as Bacillus oceanisediminis XC-2. The bacterium was deposited in the China Center for Type Culture Collection on July 22, 2024; the deposit address is Wuhan University, Wuhan, China, and the classification name is Bacillus oceanisediminis XC-2, and the deposit number is: CCTCC NO: M20241670.

[0009] The isolated salt-alkali tolerant Bacillus oceanisediminis XC-2 has a colony morphology on PDA solid medium. Figure 1As shown, the colony morphology is milky white, smooth, irregularly shaped, and the colonies are plump and opaque. Physiological and biochemical manifestations are positive for Gram staining, can hydrolyze starch, negative for methyl red test, positive for catalase, no production of hydrogen sulfide, and the ability to decompose gelatin. Bacillus oceanisediminis XC-2 can secrete celluloglucanase, amylase, cellulose, and protease ( Figure 2-5 ), can utilize carbon and nitrogen substrates in the environment, and has the ability to grow rapidly and resist plant pathogens in the environment.

[0010] The protection scope of the present invention includes:

[0011] A compound preparation containing Bacillus marineus XC-2.

[0012] The application of the above-mentioned Bacillus marine mud XC-2 or the composite preparation in inhibiting plant pathogens.

[0013] The use of the above-mentioned Bacillus marine mud XC-2 or the composite preparation in the preparation of plant pathogen inhibitors.

[0014] In the above application, the plant pathogen is Fusarium;

[0015] In the above application, preferably, the Fusarium includes: Fusarium variasi, Fusariumfalciforme, Fusarium ngaiotongaense, Fusarium solani, Fusarium foetens, Fusariumoxysporum F.sp.Cubense and / or Fusarium chlamydos.

[0016] Compared with the prior art, the present invention has the following characteristics:

[0017] The marine mud bacillus XC-2 of the present invention can secrete amylase and cellulase, grow rapidly, and can secrete plant pathogenic fungal cell wall hydrolases (glucanase, protease), and has strong salt and alkali resistance. The salt resistance of the XC-2 provided by the present invention is as high as 11% sodium chloride and can tolerate pH 9.0.

[0018] The marine mud Bacillus XC-2 in the present invention has a good broad-spectrum antibacterial effect on various pathogenic Fusarium fungi that cause plant wilt, and has a high-efficiency inhibition effect, with an average inhibition rate of 95.02%; the comprehensive performance such as salt and alkali resistance is more superior, so it can be used in saline-alkali land agricultural production to develop a live bacteria antibacterial agent that antagonizes plant pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a diagram of the culture characteristics of Bacillus marineus XC-2.

[0020] Figure 2 Schematic diagram of the glucanase production experiment of Bacillus marine mud XC-2.

[0021] Figure 3 Schematic diagram of the amylase production experiment of Bacillus marine mud XC-2.

[0022] Figure 4 Schematic diagram of the cellulase production experiment of Bacillus marine mud XC-2.

[0023] Figure 5 Schematic diagram of the protein enzyme production experiment of Bacillus marine mud XC-2.

[0024] Figure 6 Schematic diagram of the inhibitory effect of Bacillus marineus XC-2 on Fusarium ZZY18.

[0025] Figure 7 Schematic diagram of the inhibitory effect of Bacillus marineus XC-2 on Fusarium ZZY20.

[0026] Figure 8 Schematic diagram of the inhibitory effect of Bacillus marineus XC-2 on Fusarium ZZY21.

[0027] Fig. 9 Schematic diagram of the inhibitory effect of Bacillus marineus XC-2 on Fusarium ZZY26.

[0028] Fig.10 Schematic diagram of the inhibitory effect of Bacillus marineus XC-2 on Fusarium ZZY27.

[0029] Fig.11 Schematic diagram of the inhibitory effect of Bacillus marineus XC-2 on Fusarium H33.

[0030] Fig.12 Schematic diagram of the inhibitory effect of Bacillus marineus XC-2 on Fusarium FS-263.

[0031] Fig.13 Schematic diagram of the inhibitory effect of Bacillus marineus XC-2 on Fusarium XL-1Y-5. DETAILED DESCRIPTION

[0032] The technical solutions described in the present invention, unless otherwise specified, are all conventional technologies; the reagents or materials described, unless otherwise specified, are all from commercial channels.

[0033] Embodiment 1:

[0034] Isolation and identification of Bacillus oceanisediminis XC-2:

[0035] Take 2.5g of soil sample from Pucheng County, Weinan City, Shaanxi Province, and place it in a triangular flask filled with 50mL of sterile water. Incubate at 30℃ and 250r / min for 1h. After the incubation, take 500μL of soil suspension and place it in a PA bottle filled with 4.5ml of sterile water for gradient dilution. -1 , 10 -2 , 10 -3 The dilutions of these dilution degrees were added to LB culture medium for coating, and the growth of the colonies on the plate was observed after culturing for 24h, 48h, and 72h. All the grown colonies were picked out and subjected to plate antibacterial test. Bacteria with high antibacterial rate and broad antibacterial spectrum were selected, and the strains were further screened for salt-alkali resistance, acid resistance, alkali resistance and heat resistance. Finally, a strain of marine mud Bacillus XC-2 was screened out.

[0036] (1) Morphological characteristics

[0037] The colony morphology of the isolated salt-alkali tolerant Bacillus marine mud XC-2 on PDA solid medium is as follows: Figure 1 As shown, the colony morphology is milky white, smooth, irregularly round, and the colonies are plump and opaque.

[0038] (2) Physiological and biochemical experiments

[0039] Physiological and biochemical manifestations include positive Gram staining, negative methyl red test, positive catalase, no production of hydrogen sulfide, gelatin liquefaction ability, and the ability to secrete glucanase, cellulase, amylase, and protease ( Figure 2-5 ).

[0040] (3) Genetic identification of Bacillus marineus XC-2

[0041] Use a bacterial DNA extraction kit to extract the genomic DNA of the strain to be tested. Refer to the instructions for specific steps. Use the extracted bacterial genomic DNA as a template to amplify the bacteria. The primers for amplifying 16S rRNA are:

[0042] 27F:5'-GTTTGATCCTGGCTCAG-3'

[0043] 1492R:5′-TACGGCTACCTTGTTACGACTT-3′;

[0044] The sequence was analyzed by homology comparison with NCBI Blast16SrDNA sequence. The results showed that it had the highest similarity with the 16SrDNA gene sequence of the strain Bacillus oceanisediminis. It belongs to a strain of Bacillus oceanisediminis. The bacterium was sent to the China Center for Type Culture Collection on July 22, 2024. The address is: Wuhan University, Wuhan, China. The classification name is: Bacillus oceanisediminis XC-2, and the collection number is: CCTCC NO: M20241670. .

[0045] Embodiment 2:

[0046] Tolerance test of Bacillus marineus XC-2:

[0047] (1) pH tolerance test:

[0048] The seed liquid of Bacillus marine mud XC-2 was inoculated into LB medium with different initial pH values ​​at a 2% inoculation rate, and cultured at 30°C and 200 r / min. After 16 h of culture, samples were taken to measure the OD 600nm The initial pH gradient of the culture medium was set to pH 3.0, pH 4.0, pH 5.0, pH 6.0, pH 7.0, pH 8.0, pH 9.0, and pH 10.0.

[0049] LB medium: peptone 10 g, yeast extract powder 5 g, NaCl 10 g, distilled water 1000 mL, pH 7.0, sterilization at 121 °C for 30 min.

[0050] Initial OD 600nm The values ​​are counted as zero and the results are shown in the following table.

[0051] pH 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 <![CDATA[OD 600nm ]]> 0.032 0.039 0.308 0.332 0.530 1.272 1.066 0.011

[0052] (2) Salt concentration tolerance test:

[0053] The seed liquid of Bacillus marine mud XC-2 was inoculated into LB medium with different salt concentrations at 2% and cultured at 30°C and 200 r / min. After 16 h of culture, samples were taken to measure OD 600nm The salt concentration gradient of the culture medium was set to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% (1% salt concentration: 1 g NaCl / 100 g water, and so on).

[0054] LB medium: peptone 10 g, yeast extract powder 5 g, NaCl 10 g, distilled water 1000 mL, pH 7.0, high-pressure steam sterilization at 121 °C for 30 min.

[0055] Initial OD 600nm The values ​​are counted as zero and the results are shown in the following table.

[0056] Salt concentration (%) 1 2 3 4 5 6 7 8 <![CDATA[OD 600nm ]]> 3.195 10.34 10.01 9.63 6.74 5.115 4.655 3.99 Salt concentration (%) 9 10 11 12 13 14 15 <![CDATA[OD 600nm ]]> 3.3 0.64 0.195 0.075 0.075 0.084 0.093

[0057] (3) Growth temperature tolerance test:

[0058] The seed liquid of Bacillus marine mud XC-2 (cultured for 24 h) was inoculated into LB medium at 2% and cultured in a shaking incubator at 200 r / min under different temperature conditions. After culturing for 16 h, samples were taken to measure OD 600nm The temperature gradient of the culture medium was set to 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, and 50°C.

[0059] OD 600nm Using deionized water as a control, the results are shown in the following table.

[0060] Temperature(℃) 15 20 25 30 35 40 45 50 <![CDATA[OD 600nm ]]> 0.024 0.268 2.123 0.530 0.367 0.391 0.337 0.170

[0061] Embodiment 3:

[0062] Inhibitory effect of Bacillus marineus XC-2 on plant pathogens:

[0063] The inhibition rate of Bacillus XC-2 against pathogens was determined by the plate inhibition zone method. The Bacillus XC-2 strain was activated in LB medium, and the activated seed solution was diluted 100 times and then applied to the PDA plate, with deionized water applied as the control test group. A hole was punched on the PDA plate full of pathogenic fungi with a gun tip (gun tip diameter = 7.5 mm), and the fungal blocks were picked out and inoculated upside down on the PDA plate coated with Bacillus XC-2 solution and deionized water. The plate was placed at 30°C for 7 days, the fungal colony growth diameter was measured, and the corresponding inhibition rate was calculated to analyze the antibacterial effect.

[0064] The plant pathogens selected in this experiment include Fusarium variasi ZZY18, Fusarium falciformeZZY20, Fusarium ngaiotongaense ZZY21, Fusarium solani ZZY26, Fusarium foetensZZY27, Fusarium oxysporum F.sp.Cubense H33, Fusarium oxysporum FS-152, Fusariumsolani FS-263 and Fusarium chlamydos XL-1Y-5

[0065] PDA medium: 200 g potato, 20 g glucose, 1000 mL distilled water, 15% agar powder, natural pH, sterilization at 115°C for 30 min.

[0066]

[0067] Note: D, diameter of colonies on the control plate (mm); d, diameter of colonies on the experimental plate (mm).

[0068] The average inhibition rate of Bacillus marineus XC-2 on the tested pathogenic Fusarium reached 95.02%. Among them, XC-2 had a very good control effect on pathogens ZZY18, ZZY20, ZZY21, ZZY26, H33, FS263, and XL-1Y-5, with an inhibition rate between 93.60% and 98.84%. The antibacterial effect on pathogenic fungus ZZ27 was also good, reaching 79.20%. Figure 6-13 .

[0069] In summary, Bacillus marine mud XC-2 has a good inhibitory effect on a variety of plant pathogenic Fusarium and has a broad-spectrum inhibitory effect.

[0070] The inhibition rate of Bacillus marineus XC-2 against various plant pathogens is shown in the following table.

[0071]

[0072] It can be seen from the above examples that the present invention provides a strain of Bacillus marineus XC-2, which has high salt tolerance and strong alkali resistance and has a good broad-spectrum control effect on preventing and controlling plant wilt caused by Fusarium.

[0073] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An isolated strain of Bacillus maritima ( Bacillus oceanisediminis ) XC-2, the deposit number of the marine mud Bacillus is: CCTCC M20241670.

2. A composite preparation containing the marine mud Bacillus according to claim 1.

3. Use of the marine mud Bacillus according to claim 1 or the composite preparation according to claim 2 in inhibiting plant pathogens.

4. Use of the marine mud Bacillus according to claim 1 or the composite preparation according to claim 2 in the preparation of plant pathogen inhibitors.

5. The use according to claim 3 or 4, wherein the plant pathogen is Fusarium ( Fusarium ).

6. The use according to claim 5, wherein the Fusarium include: Fusarium variasi , Fusarium falciforme , Fusarium ngaiotongaense , Fusarium solani, Fusarium foetens , Fusarium oxysporum F . sp . Cubense and / or Fusarium chlamydos .

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