Strain with emulsifying activity and anti-plant disease function and application

Abstract

The application discloses a strain with high emulsification activity and anti-plant disease function, and the strain is Bacillus subtilis (CY-57) which is preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC NO.28223. Bacillus subtilis The strain can produce a biological surfactant at a high yield, has excellent emulsification performance on a plurality of hydrocarbon compounds such as cyclohexane, n-hexadecane, liquid paraffin, kerosene and diesel oil, and can significantly inhibit the growth and invasion of a plurality of plant pathogenic bacteria (such as tomato early blight, wheat root rot, wheat scab, soybean root rot and rice seedling disease). The application further discloses a fermentation culture process of the strain, emulsification activity and application in biological control of plant diseases, and application of the strain and metabolites, so that the strain has ecological safety and industrial practicability, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a strain with both emulsifying activity and anti-plant disease function and its application. Background Technology

[0002] Biosurfactants, as amphiphilic compounds produced by microbial metabolism, possess core advantages such as strong biodegradability, low toxicity, and good environmental compatibility, demonstrating enormous application potential in various fields including industrial emulsification, oil extraction, and daily chemical cleaning. However, the emulsifiers widely used in the industrial sector are still mainly chemically synthesized products. These products generally suffer from prominent problems such as insufficient emulsification stability, difficulty in natural degradation, and the potential for environmental pollution with long-term use. Furthermore, most reported biosurfactant-producing bacteria focus only on developing single emulsification functions, lacking multifunctional synergistic properties, thus limiting their application scenarios and failing to meet the complex needs of multiple fields. Meanwhile, plant diseases are a key factor restricting crop yield improvement and quality optimization. Among them, soil-borne diseases (such as wilt and bacterial wilt) and foliar diseases (such as powdery mildew and downy mildew) caused by fungi and bacteria cause serious economic losses to agricultural production due to their wide distribution, rapid spread, and severe damage. Currently, plant disease control still relies primarily on chemical fungicides. However, this single-method approach not only easily induces drug resistance in pathogens, leading to a gradual decline in efficacy, but also fails to meet the auxiliary needs of agricultural production. More importantly, existing biosurfactant products mostly focus on single interfacial activities such as emulsification and wetting, lacking the combined capabilities for plant disease control. This forces farmers to apply fungicides and surfactants separately, increasing production costs and labor intensity, which is inconsistent with the trend towards more efficient and simplified agricultural production.

[0003] Bacillus subtilis, a Gram-positive beneficial microorganism with endophytic, stress-resistant spores, has become one of the core application strains in the field of biopesticides due to its advantages such as high environmental safety, strong stress resistance, mature fermentation technology, and ease of industrial production. Its mechanism of plant disease control is well-defined, primarily through the direct inhibition of pathogen growth by producing antimicrobial substances such as bacitracin and iturobrine, or indirectly through competition for nutrients and living space, and the induction of systemic resistance in plants. Simultaneously, some Bacillus subtilis strains can also metabolize and produce biosurfactants such as lipopeptides and polysaccharides. These substances possess excellent emulsifying, dispersing, and wetting properties, and leave no environmental residues, making them of significant potential value in pesticide formulation optimization and soil microecological improvement. However, existing Bacillus subtilis strains still have significant limitations: most strains focus on single-function development, either concentrating on disease control but lacking efficient emulsifying activity, failing to meet the interfacial activity requirements in pesticide formulations or industrial production; or acting only as biosurfactant-producing bacteria, exhibiting weak disease control effects and a narrow antimicrobial spectrum. Furthermore, some strains suffer from insufficient emulsifying stability and weak colonization ability in natural environments (soil, plant surface), making them difficult to adapt to complex industrial production and agricultural field scenarios, severely limiting their commercial application. Therefore, screening for a Bacillus subtilis strain that combines highly efficient emulsifying activity with broad-spectrum resistance to plant diseases, and developing corresponding application technologies to achieve "dual functionality"—serving as a highly efficient biosurfactant in the industrial field and a broad-spectrum biological fungicide in the agricultural field—not only significantly expands the application boundaries of microbial resources but also reduces industrial application costs through multifunctional integration, meeting the development needs of green industry and ecological agriculture, and possessing significant economic and social value. Summary of the Invention

[0004] The present invention aims to overcome the shortcomings of the prior art and provide a strain and its application that have both emulsifying activity and anti-plant disease function.

[0005] This invention discloses a bacterial strain possessing both emulsifying activity and plant disease resistance, wherein the strain is Bacillus subtilis (… Bacillus subtilis CY-57, this bacterium is deposited at the China General Microbiological Culture Collection Center, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, the deposit date is August 23, 2023, and the deposit number is CGMCC NO.: 28223.

[0006] As a further improvement of the present invention, the 16S rRNA of the Bacillus subtilis strain is shown in SEQ ID NO. 1.

[0007] The present invention relates to the application of a strain of bacteria that combines the functions of producing highly efficient biosurfactants and resisting plant diseases in the emulsification of hydrocarbon substances.

[0008] As a further improvement of the present invention, the hydrocarbon substance includes any one of n-hexane, n-hexadecane, benzene, toluene, kerosene, diesel oil and liquid paraffin.

[0009] The present invention relates to the application of a strain with both emulsifying activity and anti-plant disease function in inhibiting diseases caused by *Fusarium solani*, *Fusarium graminearum*, *Fusarium oxysporum*, *Echinococcus membranaceus*, *Alternaria alternata*, *Syndromea pyriformis*, *Fusarium*, or *Fusarium equisetifolium*.

[0010] The present invention relates to the application of a strain of plant with both emulsifying activity and anti-plant disease function in the prevention and control of plant diseases.

[0011] As a further improvement of the present invention, the plant diseases include early blight of Solanaceae plants, wheat root rot, wheat scab, soybean root rot, rice bakanae disease, cucurbit vine blight, root rot and leaf blight, and pear leaf spot.

[0012] The present invention relates to a Bacillus subtilis strain possessing both emulsifying activity and anti-plant disease function, and its application has the following beneficial effects or advantages: 1. The strain of this invention has both emulsifying activity and resistance to multiple plant diseases as its dual core functions, achieving "one strain with two functions". This breaks through the limitations of the single function of existing strains, expands the application scenarios, and reduces the application cost in the industrial and agricultural fields. 2. The biosurfactants produced by the strain have excellent emulsifying properties, strong stability, and are biodegradable, solving the problem of environmental pollution caused by chemical emulsifiers; they have significant anti-plant disease effects, can replace traditional chemical fungicides, reduce pesticide residues, and are in line with the development trend of green agriculture and environmental protection industry. 3. The strain grows under mild conditions, the fermentation process is simple, no complicated equipment is required, the product extraction and purification process is simple, it is easy to carry out large-scale industrial production, the production cost is low, and it has significant economic value and ecological benefits. Attached Figure Description

[0013] Figure 1 Phylogenetic tree of Bacillus subtilis CY-57; Figures 2-3 The emulsification of different hydrocarbons by Bacillus subtilis CY-57 fermentation broth; Figures 4 to 11 The inhibitory effects of Bacillus subtilis CY-57 on different plant pathogens. Detailed Implementation Example 1

[0014] This embodiment provides a Bacillus subtilis strain with both emulsifying activity and anti-plant disease function, and a screening method thereof.

[0015] (1) Sample enrichment culture: Oil-water samples obtained from oil wells in Daqing Oilfield were inoculated into 100 mL of enrichment medium (LB medium) at a volume ratio of 5% and cultured with shaking at 37℃ and 130 rpm for 5 days. Then, the inoculum was transferred to fresh LB medium at a volume ratio of 5% and cultured with shaking at 37℃ and 130 rpm. The transfer was repeated every 5 days, and the process was repeated 3 times to obtain the enriched culture solution. The enriched culture solution was inoculated into fermentation medium containing 50 g / L crude oil at a volume ratio of 5% and cultured with shaking at 37℃ and 130 rpm for 7 days until the crude oil was completely emulsified. The fermentation medium containing 50 g / L crude oil consisted of: 50 g / L crude oil, 2.13 g / L sodium nitrate, 1.34 g / L ammonium chloride, 0.80 g / L dipotassium hydrogen phosphate, 9.0 g / L sodium dihydrogen phosphate, 0.41 g / L magnesium sulfate, 0.2 g / L yeast, and 0.202 mL / L manganese chloride (0.05 mol / L). The pH was adjusted to approximately 6.8, and the sterilization temperature was 121℃ for 30 minutes.

[0016] (2) Strains isolation and screening: The culture finally obtained in (1) was serially diluted to 10. -8 Take 10 -5 Up to 10 -8 0.1 mL of each diluted solution was spread onto pre-prepared Petri dishes containing LB solid medium and incubated at 37°C for 24 hours. Single colonies were then picked and their emulsification index (E) was determined using liquid paraffin (v:v = 1:1). 24 The emulsifying agent is used to repeatedly streak and purify single bacteria to obtain pure strains for identification and preservation.

[0017] (3) Fermentation production: After the single colony obtained in step (2) is cultured into seed liquid in LB medium, it is inoculated into fermentation medium containing 20.0 g / L sucrose at a volume ratio of 5%. The fermentation conditions are 37 ℃, shaking speed of 130 rpm, and time of 24 h to obtain fermentation liquid.

[0018] The fermentation medium containing 20 g / L sucrose includes the following raw material components at final concentrations: 20 g / L sucrose, 2.13 g / L sodium nitrate, 1.34 g / L ammonium chloride, 0.80 g / L dipotassium hydrogen phosphate, 9.0 g / L sodium dihydrogen phosphate, 0.41 g / L magnesium sulfate, 0.2 g / L yeast, and 0.202 mL / L manganese chloride (0.05 mol / L). Validation of the emulsifying properties of fermentation products: Take 20 mL of fermentation broth, centrifuge at 5000 rpm for 10 min to remove bacterial cells, and determine the E6 content using liquid paraffin as the emulsifying agent. 24 index; Where E24 The method for determining the emulsification index is as follows: The fermentation broth is centrifuged to remove the bacterial cells. The fermentation supernatant is mixed with liquid paraffin at a volume ratio of 1:1 and vortexed for 3 minutes to thoroughly mix and form an oil-water emulsion. The height of the emulsion layer is measured after 24 hours, and the emulsification index (E) is calculated. 24 = (Emulsion layer height / Total liquid height) × 100%.

[0019] (4) Taxonomic identification of the strain: DNA extraction and PCR amplification of the strain were entrusted to Sangon Biotech (Shanghai) Co., Ltd. for sequencing (sequencing primers were universal bacterial primers 27F and 1492R). The obtained 16S rRNA gene sequence (sequence length 1458bp) was obtained. The sequence was submitted to the NCBI GenBank database for homology comparison. The results showed that it had 100% homology with Bacillus subtilis strain (accession number AL009126). The strain was identified as Bacillus subtilis and named CY-57. This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 23, 2023, with accession number CGMCC NO.28223. Example 2

[0020] Physiological and biochemical properties of strain CY-57 were determined: The morphology and Gram staining of strain CY-57 were observed under a microscope, and its ability to hydrolyze proteins, starches, cellulose, and lignin was tested. It was also tested for nitrogenase production, dissolution of organic and inorganic phosphorus, potassium degradation, siderophore production, and ammonification.

[0021] Determination of protease production capacity of the strain: The test strain was inoculated on skim milk powder medium (15g skim milk powder, 1000mL deionized water, 15-20g agar, pH 7.0-7.2, sterilized at 108℃ for 20min) and incubated at 28℃ for 7 days. The plates were observed and the appearance of a clear zone indicated a positive result.

[0022] Determination of the ability of the strain to produce starch hydrolase: The test strain was inoculated on amylase medium (3g beef extract, 10g peptone, 20g soluble starch, 0.5g NaCl, 20g agar, 1000ml distilled water, pH adjusted to 7.0-7.2, sterilized at 121℃ for 30min) and incubated at 28℃ for 7 days. Lugol's iodine solution was added to the area around the colonies on the starch plate, and the formation of a clear zone was considered a positive result.

[0023] Determination of the cellulase production capacity of the strain: The test strain was inoculated onto cellulose Congo red medium (NaNO3 1.0g, Na2HPO4 1.2g, KH2PO4 0.9g, MgSO4 0.5g, KCl 0.5g, yeast extract 0.5g, acid-hydrolyzed casein 0.5g, Congo red 0.2g, cellulose powder 5.0g, agar 15.0g, distilled water 1000mL, pH 7.0-7.5, sterilized at 121℃ for 30min) and incubated at 28℃ for 7 days. The plates were observed, and the absence of a clear zone indicated a negative result.

[0024] Assay for the ligninase production capacity of the strain: The test strain was inoculated on aniline blue-PDA medium (200g peeled potato, 20g glucose, 20g agar, 1000mL distilled water, sterilized and then 0.1 g / L aniline blue was added through a sterile filter) and incubated at 28℃ for 7 days. The plates were observed. The absence of a fading transparent zone was considered negative.

[0025] Nitrogen fixation capacity determination of the strain: The test strain was inoculated on Assab medium (mannitol 10g, CaCO3 5g, KH2PO4 0.2g, MgSO4•7H2O 0.2g, NaCl 0.2g, CaSO4•2H2O 0.1g, distilled water 1000ml, agar 18g, pH 7.0-7.5, sterilized at 121℃ for 30min) and incubated at 28℃ for 7 days. The plates were observed. The appearance of a clear zone was a positive result, indicating that the presence of colonies meant that the strain had nitrogen fixation capacity.

[0026] Determination of the ability of bacterial strains to dissolve organophosphates: The test strains were inoculated onto organophosphate bacteria culture medium (10g glucose, 0.5g (NH4)2SO4, 0.3g MgSO4·7H2O, 0.03g MnSO4·4H2O, 0.3g KCl, 0.03g FeSO4·7H2O, 0.3g NaCl, 5.0g CaCO3, 0.2g lecithin, 1000mL distilled water, pH 7.0-7.5, sterilized at 121℃ for 30min) and incubated at 28℃ for 7 days. The plates were then observed. The absence of a clear zone indicated a negative result.

[0027] Determination of the ability of the strain to solubilize inorganic phosphorus: The test strain was inoculated onto an inorganic phosphorus bacterial culture medium (10g glucose, 0.5g (NH4)2SO4, 0.3g MgSO4·7H2O, 0.03g MnSO4·4H2O, 0.3g KCl, 0.03g FeSO4·7H2O, 0.3g NaCl, 10.0g Ca3(PO4)2, 1000mL distilled water, pH 7.0-7.5, sterilized at 121℃ for 30min) and incubated at 28℃ for 7 days. The plates were then observed. The absence of a clear zone indicated a negative result.

[0028] Determination of potassium degradation capacity of bacterial strains: The test strains were inoculated onto silicate bacterial culture medium (5g sucrose, 0.5g MgSO4, 0.1g CaCO3, 2g Na2HPO4, 0.005g FeCl3, 1.0g glass powder, 4g potassium feldspar, 15g agar, 1000ml water, pH 7.0, sterilized at 121℃ for 30min) and incubated at 28℃ for 7 days. The plates were observed, and those without the appearance of smooth, transparent, oil droplet-like colonies that produce capsules were considered negative.

[0029] Test for siderophore production capacity of the strain: The test strain was inoculated on CAS medium (Crazin S (CAS) 60.5mg, cetyltrimethylammonium bromide (HDTMA) 72.9mg, FeCl3·6H2O 2.645mg, peptone 4.5g, glucose 9g, beef extract powder 2.7g, NaCl 4.5g, agar 20g, distilled water 1000mL, pH 6.8, sterilized at 115℃ for 20min) and incubated at 28℃ for 7 days. The appearance of an orange transparent zone on the plate indicated a positive result.

[0030] Ammonification capacity determination of the strain: The strain was inoculated into LB liquid medium (10g tryptone, 5g yeast extract, 10g NaCl, 1000ml distilled water, pH 7.0, sterilized at 121℃ for 30min) in test tubes with red litmus paper and cultured in a shaker at 37℃. After one week, the color change of the litmus paper was observed, and the degree of ammonification capacity was judged according to the degree of blue color change of the litmus paper.

[0031]

[0032] Note: "-" indicates no ability; "+" indicates a weak ability with resistance below 30% or functional clear ring diameter ≤ 1cm; "++" indicates that the ability is average, with resistance ≥30% and ≤60% or functional clear ring diameter ≥2cm and ≤3cm; "+++" indicates that the ability is good, with resistance ≥60% or functional clear ring diameter ≥3cm. Example 3

[0033] Bacillus subtilis strain CY-57 was obtained using the method described in Example 1. Fermentation of this strain yielded a fermentation broth containing biosurfactants. The phylogenetic tree of this strain is shown below. Figure 1 As shown.

[0034] (1) Preparation of culture medium The seed culture medium consists of 5 g / L yeast extract, 10 g / L peptone, and 10 g / L sodium chloride, with the pH adjusted to 7.0 using 0.5 mol / L sodium hydroxide, and dispensed into 500 mL Erlenmeyer flasks (200 mL per flask). The fermentation medium consisted of: 20.0 g / L sucrose, 2.13 g / L sodium nitrate, 1.34 g / L ammonium chloride, 0.80 g / L dipotassium hydrogen phosphate, 9.0 g / L sodium dihydrogen phosphate, 0.41 g / L magnesium sulfate, 0.2 g / L yeast, and 0.202 mL / L manganese chloride (0.05 mol / L). The pH was adjusted to 6.8 with 0.5 mol / L sodium hydroxide, and the medium was dispensed into 5L Erlenmeyer flasks (3L capacity). The prepared medium was then sterilized at high temperature and stored for later use. (2) Preparation steps Seed culture: One loopful of Bacillus subtilis CY-57 seeds was picked from a slant culture medium stored at 4℃ and inoculated into seed culture medium. The medium was then incubated in a shaker at 37℃ and 130 r / min for 24 h to obtain the seed culture (OD). 600 The value is 1.95). Fermentation culture: The seed culture was inoculated into the fermentation broth culture medium at an inoculation rate of 5% to 10% (v / v) under aseptic conditions. The culture parameters were set to 37℃ and 130r / min. The culture was ended after 24h. Product separation: The fermentation broth was placed in a high-speed centrifuge and centrifuged at 5000 r / min for 20 min. The supernatant was collected to obtain the fermentation broth containing biosurfactants. Example 4

[0035] The fermentation broth containing biosurfactants obtained in Example 3 was used in the emulsification of hydrocarbon substances, wherein the hydrocarbon substances are any one of cyclohexane, n-hexadecane, benzene, toluene, kerosene, diesel oil and liquid paraffin.

[0036] The testing method is as follows: (1) Add 20 mL of biosurfactant and 20 mL of hydrocarbon (volume ratio 1:1) to a 100 mL dry centrifuge tube respectively. Then place the mixture in a high shear dispersion emulsifier and shear at 10000 r / min for 2 min to fully mix and form an oil / water emulsion. Then distribute it evenly into two 50 mL stoppered test tubes.

[0037] (2) After standing at room temperature for 24 hours, if Figure 2 As shown, the height of the emulsion layer and the total liquid height were measured, and the emulsification index E was calculated. 24 (E) 24 =Emulsion layer height / total height × 100%), continue to stand for 96 hours to observe emulsion stability, emulsion state as follows Figure 3 As shown.

[0038]

[0039] The results showed that the biosurfactant of the present invention had an emulsification index E of 7 hydrocarbons.24 All are ≥70%, and the emulsification efficiency of hexadecane, kerosene, and diesel oil is 96h. 96 The emulsification stability remains above 70%. Of the seven hydrocarbons, excluding cyclohexane and benzene, the other five exhibit emulsification stability greater than 90% after 96 hours. Hexadecane, kerosene, and diesel oil show emulsification stability greater than 97%, meeting the requirements for industrial emulsification applications. Example 5

[0040] The ability of strain CY-57 obtained in Example 1 to inhibit *Helicobacter pylori*, *Fusarium graminearum*, *Fusarium oxysporum*, *Cyclocarya paliurus*, *Alternaria alternata*, *Syndromea pyrifolia*, *Fusarium*, and *Fusarium equisetifolium*. Tested pathogens:

[0041] The test method for the flat plate confrontation experiment is as follows: (1) The plant pathogens were evenly spread onto PDA plates and cultured at 28°C for 7 days to obtain expanded pathogen strains; (3) Use a punch with a diameter of 8 mm to punch holes in the PDA plate cultured in (1) to obtain pathogenic fungal cakes, and then place the fungal cakes in the center of another uninoculated PDA plate (mycelial side down). (3) CY-57 was inoculated symmetrically around the perimeter of the PDA plate from (2). Simultaneously, a control group was formed by inoculating the pathogen but not CY-57. All plates were then incubated at 28°C. Once the mycelia of the control group completely covered the plate, photographs were taken and the inhibition rate was calculated. The inhibition rate images are shown below. Figures 4-11 As shown, the inhibition rate is calculated using the following formula: I = [(D1-D2) / (D1)] × 100% In the formula: I: inhibition rate, % D1: Colony diameter in blank control, mm; D2: Diameter of bacterial colonies on experimental plate, mm.

[0042]

[0043] The results showed that Bacillus subtilis CY-57 had a significant inhibitory effect on all eight pathogens, with an inhibition rate of 68-80% in the plate confrontation experiment. It can effectively block the reproduction and infection of pathogens, thereby controlling the corresponding plant diseases. It has broad application prospects in the field of green agricultural control in the future.

[0044] The strain CY-57 obtained in Example 1 was applied to the prevention and control of plant diseases, including early blight of Solanaceae plants, wheat root rot, wheat scab, soybean root rot, rice seedling blight, cucurbit vine blight, plant root rot and leaf blight, and pear leaf spot.

Claims

1. A bacterial strain possessing both emulsifying activity and resistance to plant diseases, characterized in that, The strain is Bacillus subtilis ( Bacillus subtilis CY-57, this bacterium is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on August 23, 2023, with accession number CGMCC NO.28223.

2. The bacterial strain possessing both emulsifying activity and plant disease resistance as described in claim 1, characterized in that, The 16S rRNA of the Bacillus subtilis strain is shown in SEQ ID NO.

1.

3. The application of a strain with both emulsifying activity and plant disease resistance as described in claim 1 in the emulsification of hydrocarbon substances.

4. The application of a strain with both emulsifying activity and plant disease resistance as described in claim 3 in the emulsification of hydrocarbon substances, characterized in that... The hydrocarbons include any one of n-hexane, n-hexadecane, benzene, toluene, kerosene, diesel, and liquid paraffin.

5. The application of a strain with both emulsifying activity and anti-plant disease function as described in claim 1 in inhibiting diseases caused by *Fusarium solani*, *Fusarium graminearum*, *Fusarium oxysporum*, *Eriocaulon cucumeris*, *Alternaria alternata*, *Syndromea pyriformis*, *Fusarium*, or *Fusarium equisetifolium*.

6. The application of a strain with both emulsifying activity and anti-plant disease function as described in claim 1 in the prevention and control of plant diseases.

7. The application of a strain with both emulsifying activity and anti-plant disease function as described in claim 6 in the prevention and control of plant diseases, characterized in that... The plant diseases mentioned include early blight of Solanaceae plants, wheat root rot, wheat scab, soybean root rot, rice seedling blight, cucurbit vine blight, plant root rot and leaf blight, and pear leaf spot.

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