Lipopeptide mixture for antagonizing wet rot of radix pseudostellariae in storage period and application of lipopeptide mixture
By using the lipopeptide mixture produced by the Bacillus atrophaeus strain FJAT-25833 to control the wet rot of Panax pseudoginseng, the problem of easy rot of ginseng seeds during storage was solved, and efficient disease prevention and control effects were achieved.
Patent Information
- Application Number
- CN202510699953.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-28
- Publication Date
- 2025-10-10
AI Technical Summary
The existing technology lacks effective methods for preventing and controlling wet rot of Pseudostellaria heterophylla during storage, which causes the ginseng tubers to rot easily during storage, affecting the quality and yield of the ginseng.
A lipopeptide mixture produced by the Bacillus atrophaeus strain FJAT-25833, including C15-17fengycinA, C12fengycinB, C17fengycinB, C16fengycinA2, C18fengycinA2, C17fengycinB2, C12-16surfactinA and C14surfactinA derivatives, was used to prevent and control wet rot by adding the lipopeptide mixture to fine loess and mixing it with ginseng seeds.
It significantly reduces the incidence of wet rot of Pseudostellaria heterophylla and maintains the integrity of ginseng tubers, with a control effect of 85.32%, and has a broad-spectrum inhibitory effect on multiple pathogens.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of microbial technology, and particularly relates to a bacillus strain antagonizing wet rot of Pseudostellaria heterophylla during storage and a lipopeptide mixture produced by the bacillus strain, and application in plant disease control. BACKGROUND
[0002] Pseudostellaria heterophylla belongs to Caryophyllaceae, and is a Chinese medicinal material with root tuber used as medicine, having the effects of benefiting qi and invigorating spleen and generating fluid and moistening lung. The uneven quality of seed tubers and the serious occurrence of diseases are important reasons for the continuous cropping obstacles of Pseudostellaria heterophylla. The reported diseases of seed tubers include root rot, purple stripe and wet rot. The research team found that the disease incidence of seed tubers in different planting areas is usually 10% to 30% in the early investigation. The wet rot of Pseudostellaria heterophylla caused by Apiospora arundinis is a new disease discovered and reported by the team for the first time. The incidence of wet rot of field-dug Pseudostellaria heterophylla seed tubers is generally 2-5% within one month of direct storage, and the incidence is more than 10% when the storage time is 2-3 months. The wet rot of seed tubers is more serious when the seed tubers are infected with bacteria, the storage environment is humid and not ventilated, and the seed tubers are rotted, which is a new threat in the production process of Pseudostellaria heterophylla. Fresh Pseudostellaria heterophylla seed tubers usually need to be stored for 1-3 months from digging to complete planting, and are prone to mildew and rot if stored improperly. However, there is no related report on the prevention and control of wet rot of Pseudostellaria heterophylla seed tubers. SUMMARY
[0003] In order to solve the problem of preventing and controlling the wet rot of Pseudostellaria heterophylla, the inventors provide a bacillus strain antagonizing wet rot of Pseudostellaria heterophylla during storage and a lipopeptide mixture produced by the bacillus strain. The technical scheme is as follows:
[0004] A lipopeptide mixture antagonizing wet rot of Pseudostellaria heterophylla during storage, wherein the lipopeptide mixture is produced by Bacillus atrophaeus strain FJAT-25833; the Bacillus atrophaeus strain FJAT-25833 is preserved in the China Center for Type Culture Collection of Wuhan University on March 31, 2025, and the preservation number is CCTCC NO.M2025668, and the preservation address is Wuhan University, Luojiashan, Banyi Road, Wuchang District, Wuhan City, Hubei Province.
[0005] The lipopeptide mixture produced by the Bacillus atrophaeus strain FJAT-25833 comprises fengycin A, C 15-17 fengycin A, C 12 fengycin A, C 17 fengycin A, C 16 fengycin A2, C 18fengycin A2, C 17 fengycin B2, C 12-16 surfactin A and C 14 surfactin A derivative.
[0006] Further, the fengycin and surfactin content in the lipopeptide mixture is 69.98% and 30.02%, respectively.
[0007] The separation and purification method of the lipopeptide mixture comprises the following steps:
[0008] (1) Activation of the strain FJAT-25833: the Bacillus atrophaeus strain FJAT-25833 is streaked on NA medium with an inoculation loop, and cultured in a constant temperature incubator for 36-60 h, and the culture temperature is set to 25-35°C; the components of the NA medium are as follows: beef extract 0.2-0.5%, peptone 0.2-0.8%, glucose 0.5-1.5%, agar 1.5-2.0%, prepared with water, pH 7.0-7.2, and the percentages in the medium components are weight percentages;
[0009] (2) Preparation of the seed liquid: the single colony of the Bacillus atrophaeus strain FJAT-25833 obtained in the above step is inoculated into a flask containing PDB medium, and the flask is placed in a constant temperature shaker for shaking culture, the rotation speed is 150-200 r / min, the temperature is set to 25-35°C, and the seed liquid is obtained after 20-30 h of culture; the components of the PDB medium are as follows: potato powder 0.2-0.8%, peptone 0.5-1.5%, sodium chloride 0.2-0.8%, glucose 1.0-2.0%, prepared with water, pH 7.0-7.2, and the percentages in the medium components are weight percentages;
[0010] (3) Preparation of the fermentation liquid: the seed liquid of the Bacillus atrophaeus strain FJAT-25833 obtained in the above step is inoculated into PDB medium, the inoculation amount is 0.8-1.2%, the stirring speed is 150-200 r / min, the temperature is set to 25-35°C, and after 36-60 h of culture, the measured bacterial concentration value is 3.0×10 8 -7.0×10 8 CFU / mL, and the required fermentation liquid is obtained;
[0011] Preparation of the lipopeptide mixture: the fermentation liquid obtained in the above step is centrifuged at a rotation speed of 8000-10000 r / min; after centrifugation, the bacterial cells are discarded to obtain the supernatant, 1.5-2.5 mol / L hydrochloric acid is added to the supernatant to a pH <2, and the mixture is placed at 2-8°C for 15-30 h, and then centrifuged to obtain a precipitate; the precipitate is dried by low-temperature vacuum freeze drying to obtain a lipopeptide mixture powder.
[0012] Further, the separation and purification method of the lipopeptide mixture is preferably:
[0013] (1) Activation of the strain: the Bacillus atrophaeus strain FJAT-25833 is streaked on NA medium and cultured in a constant temperature incubator for 48 h, and the culture temperature is set to 30 DEG C; the components of the NA medium are as follows: beef extract 0.3%, peptone 0.5%, glucose 1.0%, agar 1.8%, prepared with water, pH 7.0-7.2, and the percentages in the components of the medium are weight percentages;
[0014] (2) Preparation of the seed liquid: the single colony of the Bacillus atrophaeus strain FJAT-25833 obtained in the above step is inoculated into a flask containing PDB medium, and the flask is placed in a constant temperature shaker for oscillation culture, the rotation speed is 170 r / min, the temperature is set to 30 DEG C, and the seed liquid is obtained after 24 h of culture; the components of the PDB medium are as follows: potato powder 0.5%, peptone 1%, sodium chloride 0.5%, glucose 1.5%, prepared with water, pH 7.0-7.2, and the percentages in the components of the medium are weight percentages;
[0015] (3) Preparation of the fermentation liquid: the seed liquid of the Bacillus atrophaeus strain FJAT-25833 obtained in the above step is inoculated into PDB medium, the inoculation amount is 1%, the stirring speed is 170 r / min, the temperature is set to 30 DEG C, and after 48 h of culture, the measured bacterial concentration value is 5.0*10 8 CFU / mL, and the required fermentation liquid is obtained;
[0016] (4) Preparation of the lipopeptide mixture: the fermentation liquid obtained in the above step is centrifuged at a rotation speed of 9000 r / min; after centrifugation, the bacterial bodies are discarded to obtain the supernatant, 2 mol / L hydrochloric acid is added to the supernatant to pH < 2, and after 24 h of standing at 4 DEG C, the precipitate is obtained by centrifugation; the precipitate is dried by low-temperature vacuum freeze drying, and the lipopeptide mixture powder is obtained.
[0017] Further, the lipopeptide mixture is applied in the prevention and treatment of wet rot of Radix Pseudoginseng; the lipopeptide mixture powder of the Bacillus atrophaeus strain FJAT-25833 is added to fine yellow soil at 0.05-0.5%, and the soil containing the antibacterial lipopeptide is mixed with Radix Pseudoginseng seeds at a mass ratio of 1: (4-6), and the mixture is stored at 2-8 DEG C; the wet rot of Radix Pseudoginseng during storage is prevented and treated with good effect and has a good application prospect in biological control.
[0018] The beneficial effects of the present application are:
[0019] (1) The present invention obtains for the first time the Bacillus atrophaeus strain FJAT-25833 which has an inhibitory effect on Nymphoides australis and can be used to prevent and treat wet rot of Pseudomonas heterophylla seeds during storage.
[0020] (2) The lipopeptide mixture obtained by the present invention has a broad-spectrum antibacterial activity and has a strong inhibitory effect on various pathogens such as the root rot of Pseudostellaria chinensis (Fusarium oxysporum), the root rot of Atractylodes macrocephala (Fusarium oxysporum), the stem rot of Dendrobium officinale (Fusarium seminatum), and the anthracnose of Amomum villosum (Fructus anthracnose and Colletotrichum sojae). BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 Figure 2 shows the inhibitory effect of the Bacillus atrophaeus strain FJAT-25833 on different pathogens described in the specific embodiment, wherein Figure A shows the inhibitory effect on F. pseudoginseng FJAT-32563, Figure B shows the inhibitory effect on F. pseudoginseng FJAT-31620, Figure C shows the inhibitory effect on F. oxysporum FJAT-31169, Figure D shows the inhibitory effect on F. seminatum FJAT-33684, Figure E shows the inhibitory effect on F. amomum fruit anthracnose FJAT-33674, and Figure F shows the inhibitory effect on F. soybean anthracnose FJAT-33675.
[0022] Figure 2 This is a circle map of the genome of strain FJAT-25833 described in the specific embodiment.
[0023] Figure 3 Figure 1 shows the efficacy of the Bacillus atrophaeus strain FJAT-25833 lipopeptide mixture described in the specific embodiment against wet rot of Pseudostellaria heterophylla during storage. Figure A shows the control group's diseased ginseng tubers engulfed and infected by Nymphoma australis hyphae after 90 days of storage. Figure B shows the ginseng tubers treated with the lipopeptide mixture after 90 days of storage, with the epidermis and buds still intact. DETAILED DESCRIPTION
[0024] In order to explain the technical content, structural features, achieved objectives and effects of the technical solution in detail, the following is a detailed description in conjunction with specific embodiments and accompanying drawings.
[0025] Example 1
[0026] Bacillus atrophaeus strain FJAT-25833 was isolated and screened from the rhizosphere soil of Pseudostellaria heterophylla in Zherong County, Fujian Province, and has good biological control effect.
[0027] 1. Isolation of Bacillus strains
[0028] (1)Radix Pseudostellariae harvest, collection of root and tuber surface rhizosphere soil, 10 g of rhizosphere soil in 90 mL of sterile water mixed thoroughly after, in the water bath pot heating 10 min at 80℃, then gradient dilution to 10 -4 ,10 -5 and 10 -6 , each dilution solution was taken 200 μL on the NA plate coated, placed in a 30℃ incubator for 48 h.
[0029] (2) pick up the culture obtained in step (1) of single colony for smear, basic fuchsin staining after microscopic examination, select the strain that can produce spores into the slope culture medium preservation, as the measured strain, then the measured strain streaked on the NA solid plate, and the NA solid plate was placed in a 30℃ incubator for 48 h; wherein the composition of NA liquid medium: peptone 0.5%, beef extract 0.3%, glucose 1.0%, agar 1.8%, prepared with distilled water, pH 7.2, the percentage in the components are all weight ratio.
[0030] (3) with Radix Pseudostellariae wet rot fungus Apiospora arundinis strain FJAT-32563 (NCBI database ITS gene sequence accession number OM920984; BenA gene sequence accession number OM953823), Radix Pseudostellariae root rot fungus Fusarium oxysporum FJAT-31620, Atractylodes root rot fungus Fusarium oxysporum FJAT-31169, Dendrobium stem rot fungus Fusarium semitectum FJAT-33684, cardamom anthracnose fungus Colletotrichum gloeosporioides FJAT-33674 and cardamom anthracnose fungus Colletotrichum truncatum FJAT-33675 and other traditional Chinese medicinal materials pathogenic fungi as indicator. Test strains are preserved in the Bacillus Center of Institute of Resource Environment and Soil Fertilizer, Fujian Academy of Agricultural Sciences.
[0031] (4) using plate confrontation method to screen the Bacillus strains isolated in step (2), the specific operation: using PDA medium (potato powder 0.5%, peptone 1%, sodium chloride 0.5%, glucose 1.5%, agar 1.8%) for the culture of 6 strains of fungi, cultured to 5 d, with a diameter of 0.6 cm puncher in the edge of the fungus cake, placed in the center of PDA plate, 2 cm from the center of the cake straight line inoculation of test Bacillus strains. Inoculated with only fungus cake as a blank control. At 28℃ in the dark for 6 d, each treatment was repeated 3 times, with sterile water as control. 5-7 d when measuring the fungal colony radius and calculating the inhibition rate, select the strain with strong inhibition effect for the next step test. Inhibition rate = (control colony radius - treatment colony radius / control colony radius) x 100%.
[0032] A Bacillus strain with good inhibitory effect on F. heterophylla FJAT-32563, F. heterophylla oxysporum FJAT-31620, F. atractylodes macrocephala FJAT-31169, F. seminatiferum FJAT-33684, F. anthracnose fungus FJAT-33674, and F. sojae fungus FJAT-33675 was obtained. Figure 1 ), and the strain was named Bacillus atrophaeus strain FJAT-25833.
[0033] 2. Identification of strains
[0034] The genomic DNA of strain FJAT-25833 was extracted and sequenced by Nanopore PromethION and Illumina NovaSeq PE150 to obtain the whole genome data of FJAT-25833 strain. The Genbank accession number is SAMN48580704. The genome circle map is shown in Figure 2 The strain's G+C content was 43.11 mol%. Using ChunLab's online Average Nucleotide Identity (ANI) calculator, the ANI value of strain FJAT-25833 compared to the Bacillus atrophaeus model strain NRRLNRS213 was 98.57%, exceeding the 96% threshold for Bacillus species classification. Therefore, strain FJAT-25833 was identified as Bacillus atrophaeus.
[0035] Example 2
[0036] 1. Preparation of Lipopeptide Mixture from Bacillus atrophaeus Strain FJAT-25833
[0037] (1) Activation of Bacillus atrophaeus strain FJAT-25833: Strain FJAT-25833 was streaked onto NA medium using an inoculating loop and incubated in a constant temperature incubator at 30°C for 48 h. NA medium consisted of 0.3% beef extract, 0.5% peptone, 1.0% glucose, and 1.8% agar, prepared in water at a pH of 7.0-7.2. The percentages of the medium components are by weight.
[0038] (2) Preparation of seed solution: A single colony of the Bacillus atrophaeus strain FJAT-25833 obtained in step (1) was inoculated into a triangular flask containing a seed solution culture medium (the bottle volume was 50 mL / 250 mL), and the culture was placed in a constant temperature shaker with a rotation speed of 170 r / min and a temperature set to 30° C. After culturing for 24 h, the seed solution was obtained;
[0039] (3) Preparation of fermentation broth: the seed liquid of Bacillus atrophaeus strain FJAT-25833 obtained in step (2) was inoculated into PDB liquid medium (potato glucose nutrient broth) at an inoculation amount of 1%, a stirring rate of 170 r / min, and a temperature setting of 30°C. After 48 h of culture, the bacterial concentration value was determined to be 5.0 x 10 8 CFU / mL, and the required fermentation broth was obtained.
[0040] In step (2), the seed liquid medium and the fermentation medium in step (3) are both PDB medium, and the components thereof are: potato powder 0.5%, peptone 1%, sodium chloride 0.5%, glucose 1.5%, prepared with water, pH 7.0-7.2; the percentages in the medium components are all weight ratios.
[0041] (4) Preparation of lipopeptide mixture: the fermentation broth obtained in step (3) was centrifuged at a speed of 9000 r / min; after centrifugation, the bacterial bodies were discarded to obtain the supernatant. 2 mol / L hydrochloric acid was added to the supernatant to a pH < 2, and the mixture was placed at 4°C for 24 h. After centrifugation, the precipitate was dried by low-temperature vacuum freeze drying to obtain a lipopeptide mixture powder.
[0042] 2. Inhibition of pathogenic fungi by the lipopeptide mixture of strain FJAT-25833
[0043] (1) Preparation of spore suspension of test pathogenic fungi
[0044] 6 strains of pathogenic fungi were respectively inoculated on PDA plates, and after the mycelium grew, a small amount of mycelium was picked and placed in PDB medium, and cultured at 28°C and 170 r / min on a shaking table for 5 d. Spore suspension was obtained by filtering with double-layer sterile lens paper, and the concentration was adjusted to 1 x 10 7 CFU / mL, and was ready for use.
[0045] (2) Preparation of supernatant of Bacillus metabolic product: Bacillus FJAT-25833 was inoculated on NA plates with a sterile inoculation loop, and cultured at 30°C for 2 d. Single colonies were picked and placed in PDB liquid medium, and cultured at 180 r / min and 30°C on a shaking table for 2 d. The fermentation broth was removed and centrifuged at 8000 r / min and 25°C for 10 min. The supernatant was filtered with a 0.22 μm filter, and was ready for use.
[0046] (3) Preparation of lipopeptide mixture of Bacillus: the prepared lipopeptide mixture of Bacillus atrophaeus strain FJAT-25833 was used to prepare lipopeptide solutions with concentrations of 1, 2.5, 5, 10, 15, and 30 mg / mL, respectively.
[0047] (4) Inhibition zone method to determine the inhibition activity of FJAT-25833 on pathogenic fungi: Take 1 mL of the six kinds of pathogenic fungus spore suspensions prepared in step (1), mix with 99 mL of PDA solid medium cooled to about 50°C, pour into the plate, and the medium is about 1 / 3 height of the plate. After the plate is cooled, place the sterilized Oxford cup (inner diameter 6 mm) on the medium, gently press the Oxford cup to make it seamless with the surface of the medium to prevent the liquid from seeping out. Place 4 in each dish, and mark on the bottom of the dish. Take 100 μL of the supernatant of Bacillus prepared in step (2) and the lipopeptide solution prepared in step (3) and add them to the Oxford cup. Take sterile methanol solution as blank control, repeat 3 times for each treatment. Measure the inhibition zone diameter after 5-7 days of culture at 30°C.
[0048] (5) Inhibition effect of different concentrations of FJAT-25833 lipopeptide mixture on C. araliae: The method for preparing the plate containing pathogenic fungi is the same as step (4). Then take 100 μL of the supernatant of Bacillus prepared in step (2) and the lipopeptide solution prepared in step (3) of different concentrations and add them to the Oxford cup. Take sterile methanol solution as blank control, repeat 3 times for each treatment. Measure the inhibition zone diameter after 6 days of culture at 30°C. Use scanning electron microscope to observe the inhibition effect of different concentrations of FJAT-25833 lipopeptide mixture on C. araliae FJAT-32563. Use a blade to cut the medium block from the edge of the inhibition zone of different treatment groups and the control group and close to the pathogenic fungi side. First, fix the cut medium block sample with 5% glutaraldehyde for more than 4 h; wash the fixed liquid with PBS buffer for 3 times; fix with 1% osmium acid for 2 h; after washing with ultrapure water, dehydrate with ethanol (50%, 70%, 80%, 90%, 100%); replace with propylene oxide for 2 times, dry, spray gold, and observe the mycelial morphology with scanning electron microscope.
[0049] (6) Test results
[0050] The best inhibition effect of Bacillus atrophaeus strain FJAT-25833 mixture (30 mg / mL) on C. araliae FJAT-32563 and C. araliae FJAT-33674 was 19.00 mm and 18.00 mm in diameter of inhibition zone, respectively (Table 1); followed by F. oxysporum f. sp. panacis FJAT-31620, F. oxysporum f. sp. araliae FJAT-31169 and C. araliae FJAT-33674, with 15.67 mm, 16.00 mm and 16.33 mm in diameter of inhibition zone, respectively. It is shown that the lipopeptide mixture of FJAT-25833 has broad-spectrum antibacterial activity.
[0051] Table 1 Inhibition activity of Bacillus FJAT-25833 lipopeptide mixture on various plant pathogenic fungi
[0052]
[0053] Note: The same column data after different lower case letters represent significant differences between treatments (P <0.05)
[0054] The results of the inhibition of Phyllactinia taiwaniana FJAT-32563 by Bacillus atrophaeus strain FJAT-25833 at different concentrations showed that the diameters of the inhibition zone were 19.00 mm (30 mg / mL), 14.50 mm (15 mg / mL), and 9.00 mm (10 mg / mL), respectively; the minimum inhibitory concentration was 2.5 mg / mL; the higher the concentration of the lipopeptide, the better the inhibitory effect; the inhibitory effect of the supernatant was equivalent to that of the 15 mg / mL lipopeptide treatment group; the methanol control group and the water control group had no inhibitory effect.
[0055] Table 2 Inhibitory effect of lipopeptide mixture at different concentrations on Phyllactinia taiwaniana
[0056]
[0057] Note: The same column data after different lower case letters represent significant differences between treatments (P <0.05)
[0058] The results of the scanning electron microscope observation of Phyllactinia taiwaniana treated with the lipopeptide mixture of Bacillus atrophaeus showed that the mycelium of Phyllactinia taiwaniana was deformed, swelled, and folded and curled, and the normal growth of Phyllactinia taiwaniana was affected by the lipopeptide mixture of Bacillus atrophaeus at different concentrations; the higher the concentration of the lipopeptide, the more obvious the inhibitory effect on the mycelium; the mycelium of the methanol control group was smooth, uniform in size and thickness, and had an intact morphological structure.
[0059] Example 3
[0060] 1. Determination of the composition of the lipopeptide mixture of strain FJAT-25833
[0061] (1) Test material
[0062] 30 mg / mL lipopeptide mixture (prepared according to the method of Example 2)
[0063] (2) Test method
[0064] The lipopeptide produced by strain FJAT-25833 was determined by LC-QTOF-MS / MS technology, and the test conditions were as follows:
[0065] Liquid chromatography conditions: The chromatographic column was Agilent ZORBAX Extend-C 18Chromatographic column (2.1 x 150 mm, 1.8-Micron), flow rate 0.3 mL / min; mobile phase A 0.1% formic acid water; mobile phase B methanol; elution program 0, 60% B; 60 min, 100% B; 65 min, 60% B.
[0066] Mass spectrometry conditions: ESI (+ / -), dry gas temperature 350 °C, dry gas flow rate 8 L / min, nebulizer 30 psig, Fragmentor 175 V, Collision Energy 100 V, Skimmer 65 V, scan mode auto MS / MS; ion scan range: 100-3000 m / z.
[0067] (3) Test results
[0068] The lipopeptides produced by strain FJAT-25833 were determined by LC-QTOF-MS / MS technique, and the lipopeptide composition is shown in Table 3. The results show that the lipopeptides produced by strain FJAT-25833 are composed of C 15-17 fengycin A, C 12 fengycin B, C 17 fengycin B, C 16 fengycin A2, C 18 fengycin A2, C 17 fengycin B2, C 12-16 surfactin A and C 14 surfactin A derivative. The contents of fengycin and surfactin in the lipopeptide mixture are 69.98% and 30.02%, respectively.
[0069] Table 3 Lipopeptide composition of strain FJAT-25833
[0070]
[0071] 2. Prevention and treatment effect of lipopeptide mixture of strain FJAT-25833 on wet rot of Radix Pseudoginseng
[0072] (1) Test materials
[0073] The test cultivated variety was Radix Pseudoginseng ZhenShen No. 1, which was a farmer's own variety, and the seedlings with obvious disease symptoms and damaged seedlings were removed. The fine yellow soil was crushed and collected from a new land.
[0074] (2) Preparation of lipopeptide mixture of strain FJAT-25833: the preparation method was the same as that in Example 2.
[0075] (3) Test treatment group and treatment method: 2 treatment groups were set. Lipopeptide treatment group: 1 kg of fine loess was mixed with 2 g of lipopeptide mixture powder, and then mixed with 5 kg of ginseng seed; control group: fine loess seed dressing control. The treated ginseng seeds were packed in breathable woven bags and stored at 4 DEG C for 90 days, and then the incidence of wet rot of the roots of the ginseng seeds in different treatments and the control effect were investigated.
[0076] Control effect (%) = [(incidence of control group - incidence of treatment group) / incidence of control group] * 100
[0077] (5) Test results
[0078] The investigation results of the roots of the ginseng seeds in different treatment groups after 90 days of storage showed that: after 90 days of storage, part of the roots of the ginseng seeds in the control group were wrapped and infected by the mycelium of Arachniotus arundinaceus (A), and most of the roots of the ginseng seeds in the Bacillus atrophaeus FJAT-25833 lipopeptide mixture treatment group still maintained the integrity of the epidermis and bud (B). Figure 3 Figure 3 The incidence of the lipopeptide treatment group was only 1.44%, which was significantly lower than that of the conventional control group of 9.08%, and the control effect was 85.32%.
[0079] In summary, the lipopeptide prepared from Bacillus atrophaeus strain FJAT-25833 has a strong inhibitory effect on the wet rot pathogen of ginseng. The lipopeptide mixture powder is added to the fine loess at 0.05-0.5%, and then the loess containing the antibacterial lipopeptide is mixed with the ginseng seeds at a mass ratio of 1:5, so that the wet rot of ginseng during storage can be prevented.
[0080] It should be noted that although the above embodiments have been described in this paper, the patent protection scope of the present application is not limited thereby. Therefore, based on the innovative idea of the present application, the changes and modifications of the embodiments described in this paper, or the equivalent structure or equivalent process transformation made by using the contents of the present application specification and drawings, directly or indirectly apply the above technical solutions to other related technical fields, are all included in the patent protection scope of the present application.
Claims
1. A lipopeptide mixture for antagonizing wet rot of Pseudostellaria heterophylla during storage, characterized in that: The lipopeptide mixture is produced by Bacillus atrophaeus strain FJAT-25833; the Bacillus atrophaeus strain FJAT-25833 was deposited in the China Center for Type Culture Collection of Wuhan University, China on March 31, 2025, with the deposit number CCTCC NO.M2025668, and the deposit address is Wuhan University, Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.
2. The lipopeptide mixture for antagonizing wet rot of Pseudostellaria heterophylla during storage according to claim 1, characterized in that The lipopeptide mixture is composed of C 15-17 fengycinA, C 12 fengycinB、C 17 fengycinB、C 16 fengycinA2、C 18 fengycinA2、C 17 fengycinB2、C 12-16 surfactin A and C 14 It is composed of surfactinA derivatives.
3. The lipopeptide mixture for antagonizing wet rot of Pseudostellariae Radix according to claim 1 or 2, characterized in that: The separation and purification method of the lipopeptide mixture comprises the following steps: Strain activation: Bacillus atrophaeus strain FJAT-25833 was streaked onto NA medium and cultured in a constant temperature incubator at 25-35°C for 36-60 hours. NA medium consists of: 0.2-0.5% beef extract, 0.2-0.8% peptone, 0.5-1.5% glucose, and 1.5-2.0% agar, prepared in water at a pH of 7.0-7.
2. The percentages of the medium components are by weight. Preparation of seed solution: A single colony of the Bacillus atrophaeus strain FJAT-25833 obtained in the previous step is inoculated into PDB medium and cultured in a constant temperature shaker at a speed of 150-200 rpm and a temperature set at 25-35°C for 20-30 hours to obtain the seed solution. The PDB medium comprises: 0.2-0.8% potato extract powder, 0.5-1.5% peptone, 0.2-0.8% sodium chloride, and 1.0-2.0% glucose, prepared in water to a pH of 7.0-7.
2. The percentages of the medium components are by weight. Preparation of fermentation broth: The seed liquid of Bacillus atrophaeus strain FJAT-25833 obtained in the previous step was inoculated into PDB medium with an inoculum size of 0.8-1.2%, a stirring rate of 150-200 r / min, and a temperature of 25-35°C. After culturing for 36-60 h, the bacterial concentration was measured to be 3.0×10 8 -7.0×10 8 CFU / mL, the desired fermentation broth is obtained; Preparation of the lipopeptide mixture: The fermentation broth obtained in the previous step is centrifuged at a speed of 8000-10000 r / min; after centrifugation, the bacteria are discarded to obtain the supernatant, 1.5-2.5 mol / L hydrochloric acid is added to the supernatant to a pH <2, and the supernatant is allowed to stand at 2-8°C for 15-30 hours, followed by centrifugation to obtain a precipitate; the precipitate is dried using low-temperature vacuum freeze drying to obtain a lipopeptide mixture powder.
4. Use of the lipopeptide mixture according to any one of claims 1 to 3 in preventing and controlling wet rot of Pseudostellaria heterophylla during storage.
5. The use of the lipopeptide mixture according to claim 4 in the prevention and treatment of wet rot of Pseudostellaria heterophylla during storage, characterized in that: The lipopeptide mixture powder is added into fine loess at a rate of 0.05-0.5% and mixed thoroughly. The loess containing antimicrobial lipopeptides is mixed with Pseudostellaria heterophylla seeds at a mass ratio of 1:(4-6) and stored at 2-8°C.