Application of induced culture of streptomyces bikini in improving resistance to magnaporthe oryzae
By using γ-butyrolactone to induce the culture of Streptomyces bikini HD-087, the problem of its low lipopeptide synthesis efficiency was solved, the inhibitory effect on rice blast fungus was significantly enhanced, and the yield of Streptomyces antibiotics was increased, which has the potential for environmental remediation.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-15
- Publication Date
- 2026-03-27
AI Technical Summary
In the existing technology, Streptomyces bikini HD-087 has a low lipopeptide synthesis efficiency, which limits its inhibitory effect on rice blast fungus and makes it difficult to effectively control the spread of rice blast.
Using γ-butyrolactone (GBL) as an inducer, the resistance of Streptomyces bikini HD-087 to rice blast fungus was enhanced, and its lipopeptide production and antibacterial activity were increased through cross-species induction culture.
Induced culture of Streptomyces bikini HD-087 significantly improved the inhibitory effect on rice blast fungus, enhanced the oil excretion activity of lipopeptides, showed potential for environmental remediation, and increased the yield of Streptomyces antibiotics.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of microbial prevention and treatment of plant diseases, in particular to the field of application of induced culture of Streptomyces bikiniensis in improving resistance to Magnaporthe oryzae. BACKGROUND
[0002] Rice (Oryza sativa L.) is the staple food of more than half of the world's population, especially in Asia, Africa and Latin America, and is a key crop for ensuring world food security. Magnaporthe oryzae (sexual generation) and Pyricularia oryzae (asexual generation) cause rice blast, which is one of the most destructive pathogenic fungi in global rice production. In addition to rice, Magnaporthe oryzae can also infect millet and barley and other major crops, causing significant economic losses to agricultural production. In 2012, it was listed by the International Molecular Plant Pathology as the world's top ten pathogens that harm crops. Therefore, controlling Magnaporthe oryzae infection is of great significance to maintaining the world's food security supply.
[0003] The lipopeptide of Streptomyces bikiniensis HD-087 can significantly inhibit different growth and development stages of Magnaporthe oryzae, specifically in effectively hindering the spore germination process and the extension growth of mycelium. However, the lipopeptide synthesis efficiency of Streptomyces itself is relatively low, and the yield is limited, so the present application selects cross-species induced culture to improve the yield of lipopeptide, and thus improves the ability of Streptomyces bikiniensis HD-087 to resist Magnaporthe oryzae. SUMMARY
[0004] The application provides the application of induced culture of Streptomyces bikiniensis in improving resistance to Magnaporthe oryzae.
[0005] The application provides the application of induced culture of Streptomyces bikiniensis in improving resistance to Magnaporthe oryzae.
[0006] Further, GBL is used as an inducer for inducing culture of Streptomyces bikiniensis HD-087.
[0007] Further, the concentration of GBL is 10-20 mmol / L.
[0008] Further, GBL extracted from Magnaporthe oryzae is used as an inducer for inducing culture of Streptomyces bikiniensis HD-087.
[0009] The application provides the application of induced culture of Streptomyces bikiniensis in improving resistance to Magnaporthe oryzae.
[0010] Further, the induction culture method is carried out according to the following steps:
[0011] I. 1 mL of conidia suspension of Pyricularia oryzae with a concentration of 1*10 5 The conidia suspension of Pyricularia oryzae with a concentration of 1*10
[0012] II. The inducer and the DBY fermentation medium are mixed at a ratio of 1:50 to obtain an induction culture medium, and Streptomyces bikiniensis HD-087 is inoculated into the induction culture medium for culture.
[0013] The application of GBL and Pyricularia oryzae in the induction culture of Streptomyces bikiniensis for improving the lipopeptide oil discharge activity.
[0014] The antibacterial experiment after the induction culture shows that the metabolic products of Streptomyces bikiniensis HD-087 after induction inhibit the formation of spores and melanin of Pyricularia oryzae, and also inhibit the germination of spores of Pyricularia oryzae, and the inhibitory effect on the germination of spores of Pyricularia oryzae is far superior to that of the non-induction group, and shows higher stability in the time dimension. The antibacterial activity of the lipopeptide of Streptomyces bikiniensis HD-087 after induction by the GBL standard product is improved, and the effect of inhibiting Pyricularia oryzae is enhanced. The induction by the inducer can enhance the oil discharge activity of the lipopeptide, and the lipopeptide has the ability to drive oil, and shows certain potential in the degradation or removal of oil, which provides a theoretical basis for its application in the field of environmental remediation.
[0015] In addition, the induction effect of the Pyricularia oryzae inducer is significantly higher than that of the GBL standard product, which is manifested in the significant increase in the lipopeptide yield of Streptomyces bikiniensis HD-087, and it can be seen that there are substances in the Pyricularia oryzae inducer other than GBL that can induce the increase of lipopeptide yield, which play an auxiliary and synergistic role in the induction of GBL. The phenomenon of increasing the yield of lipopeptide of Streptomyces bikiniensis HD-087 induced by heterologous GBL proves that the application can use GBL to increase the yield of streptomycin antibiotics in production. The application has obvious enhancement in the induction of the antibacterial effect of Streptomyces bikiniensis HD-087, and the application can use GBL to increase the yield of streptomycin antibiotics in production. The application can improve the antibacterial effect of Streptomyces bikiniensis HD-087 while repairing the environment. BRIEF DESCRIPTION OF DRAWINGS
[0016] Figure 1 The results of the influence of the inducer on the antibacterial activity of the metabolic products of Streptomyces bikiniensis HD-087;
[0017] Figure 2 To induce the effect of culture on the growth of Magnaporthe oryzae mycelium;
[0018] Figure 3 To induce the effect of culture on the germination of Magnaporthe oryzae conidia;
[0019] Figure 4 To induce the effect of GBL standard on the antibacterial activity of Streptomyces bikiniensis HD-087 lipopeptide;
[0020] Figure 5 To induce the effect of GBL standard on the oil-repelling activity of Streptomyces bikiniensis HD-087 lipopeptide;
[0021] Figure 6 To compare the production of GBL (S-GBL) produced by Streptomyces bikiniensis HD-087 and GBL (M-GBL) produced by Magnaporthe oryzae;
[0022] Figure 7 To compare the effect of exogenous addition of GBLs on the antibacterial activity of Streptomyces bikiniensis HD-087 fermentation broth;
[0023] Figure 8 To compare the effect of GBL induction on the production of Streptomyces bikiniensis HD-087 lipopeptide;
[0024] Figure 9 To determine the effect of GBL on the expression of GBL pathway genes of Streptomyces bikiniensis HD-087 by qPCR. DETAILED DESCRIPTION
[0025] The technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application.
[0026] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.
[0027] Example 1 Induction culture of Streptomyces bikiniensis HD-087 by Magnaporthe oryzae
[0028] The microorganism used in this example: Streptomyces bikiniensis HD-087 (cucumber wilt biocontrol bacteria HD-087), provided by the Key Laboratory of Microbiology of Heilongjiang Province, Heilongjiang University; Magnaporthe oryzae, provided by the Key Laboratory of Microbiology of Heilongjiang Province, Heilongjiang University.
[0029] DBY fermentation medium: glucose 20 g, soybean powder 5 g, yeast powder 4 g, (NH4)2SO4 5 g, NaCl 1 g, K2HPO4 0.05 g, distilled water to 1 L, pH natural, 121 ℃ high pressure steam sterilization for 15 min. Mainly used for fermentation culture of Streptomyces bikiniensis HD-087.
[0030] CM medium: glucose 10 g, yeast extract 1 g, peptone 2 g, acid hydrolysis casein 1 g, NaNO3 6 g, KH2PO4 1.52 g, KCl 0.52 g, MgSO4·7H2O 0.52 g, vitamin solution 1 mL, trace elements 1 mL, distilled water to 1 L, pH adjusted to 6.5, solid medium added with 2% agar powder, liquid medium without agar powder, 121 ℃ high pressure steam sterilization for 15 min. Mainly used for sporulation culture of Magnaporthe oryzae.
[0031] 1. Preparation of inducer
[0032] The cell-free filtrate of Magnaporthe oryzae was used as the inducer to stimulate the secondary metabolism of Streptomyces bikiniensis HD-087. The preparation of the cell-free filtrate of Magnaporthe oryzae is as follows:
[0033] 1.1 Preparation of Magnaporthe oryzae conidial suspension
[0034] A puncher after high temperature sterilization was used to take a 6 mm diameter fungus cake from the Magnaporthe oryzae colony cultured on CM medium at 28 ℃ for 14 d, and then the fungus cake was inoculated on a newly prepared CM solid plate medium, and cultured at 28 ℃ with 12 h light and dark alternation treatment, for 14 d to induce the formation of conidia. After sporulation, 5 mL of sterile distilled water was added to the plate to wash the conidia off the surface of the fungus, and the collected conidial suspension was placed in a 10 mL centrifuge tube and shaken moderately to disperse the conidia and mycelium. The mycelium was removed by filtering with two layers of sterile lens paper, and the conidial suspension was obtained. The number of conidia in the suspension was counted using an optical microscope combined with a hemocytometer, and the concentration of conidia was adjusted to 10 5 individuals / mL. Finally, the prepared conidial suspension was stored in a 4 ℃ refrigerator for later use.
[0035] 1.2 Obtaining the inducer (cell-free filtrate of rice blast fungus)
[0036] Take 1 mL of a concentration of 1×10 5 A suspension of rice blast fungus conidia per mL was inoculated into an Erlenmeyer flask containing 50 mL of PDB medium and cultured on a shaker at 180 r / min and 28 ℃ for 144 h. During the culture period, 2 mL of culture medium samples were collected every 24 h, centrifuged at 10,000 r / min for 10 min, and filtered (filter membrane pore size 0.45 µm) to remove bacteria. The clear filtrate obtained was the inducer. The inducers collected at different times were named Mo-24, Mo-48, Mo-72, Mo-96, Mo-120, and Mo-144, respectively. For example, the inducer collected at 24 h of culture was Mo-24, and so on.
[0037] 2. Induction methods:
[0038] Take 1 mL of each inducer (Mo-24, Mo-48, Mo-72, Mo-96, Mo-120, Mo-144) and add it to the corresponding 50 mL DBY fermentation medium / 250 mL Erlenmeyer flask. Simultaneously, inoculate 2 mL of Streptomyces bikini HD-087 seed culture. This series is the induction group, denoted as Ind group.
[0039] The non-induction group was not given any inducer, but was inoculated with an equal amount of Streptomyces bikini HD-087 seed liquid, and was designated as the Non-ind group.
[0040] 3. Determination of the activity of Streptomyces bikini HD-087 metabolites against rice blast fungus after induction culture
[0041] Antimicrobial activity was determined using the cup-and-dish method. 50 mL of CM culture medium at 50 °C was poured into a sterile 15 cm diameter petri dish, and 3 mL of a 1×10⁻⁶ solution was added. 5 A suspension of *Streptomyces bikini* spores at a concentration of 100 spores / mL was thoroughly mixed to prepare inoculum plates, on which Oxford cups were evenly placed. 200 μL of supernatant from *Streptomyces bikini* HD-087 fermentation broth, obtained after induction with inducers (Mo-24, Mo-48, Mo-72, Mo-96, Mo-120, Mo-144) for 48, 72, 96, 120, and 144 h, was added to each Oxford cup. Uninoculated DBY liquid medium served as a blank control (CK). The inoculated plates were then incubated at 28 ℃ for 72 h. After incubation, the diameter of the inhibition zone was measured using the cross-sectional method to reflect the antibacterial activity of the fermentation broth at different time points. Each treatment was performed in triplicate.
[0042] The effect of the inducer on the antibacterial activity of Streptomyces chinensis HD-087 metabolites is shown in the figure. Figure 1 Figure (A): Inhibition zone effect diagram; (B): Inhibition zone diameter diagram. It can be seen that the metabolites of the blank control (CK) and Non-ind group showed no activity against rice blast fungus at 48 h. However, the metabolites of the Ind group induced by different inducers (Mo-24~Mo-144) all showed significant activity against rice blast fungus at 48 h, and the diameter of the inhibition zone was greater than 20 mm, indicating that induced culture can advance the production of antibiotics from *Streptomyces bikini* HD-087 on a time scale. Among the inducers, Mo-96 induced the best antibacterial effect of the metabolites produced by *Streptomyces bikini* HD-087 at all time points; therefore, Mo-96 was selected as the representative of the Ind group for subsequent experiments.
[0043] From the fermentation cycle perspective, the antibacterial activity of the metabolites in both the Non-ind and Ind(Mo-96) groups reached its peak at 96 h of fermentation. The inhibition zone diameter of the Ind(Mo-96) group was 51.48 ± 2.06 mm, which was 2.96 mm larger than that of the Non-ind group, showing a significant difference at the p < 0.05 level. Inducers obtained from different culture time points of *Streptomyces bikini* HD-087 stimulated early antibiotic expression in *Streptomyces bikini* HD-087 at 48 h, but their effects showed significant individual differences. Mo-72 also showed a significant inducing effect at 120 h of fermentation of *Streptomyces bikini* HD-087. However, other experimental groups did not show an inducing effect after 72 h of fermentation of *Streptomyces bikini* HD-087; instead, they inhibited the antibacterial effect of *Streptomyces bikini* HD-087. This indicates that the chemical composition of the inducers obtained from different culture times of *Streptomyces bikini* varies significantly.
[0044] 4. Effects of Streptomyces bikini HD-087 metabolites on the growth rate of rice blast fungus after induction culture
[0045] Take 20 mL of pre-melted CM solid medium and cool it to approximately 50 °C. Add 1 mL of supernatant from the fermentation broth of *Streptomyces bikini* HD-087, obtained by culturing under Non-ind and Ind conditions for 96 h, respectively. For the blank control, add an equal volume of sterile distilled water, gently shake, pour into sterile Petri dishes with a diameter of 9 cm, and allow to solidify. Using a high-temperature sterilized punch, extract 6 mm diameter mycelial cakes from plates cultured for 14 days of *Streptomyces bikini*. Inoculate these mycelial cakes into the center of pre-prepared solidified CM solid medium plates and incubate them upside down at 28 °C for 10 days. Measure the colony diameter of the blank control group and the treatment group using calipers according to the cross-sectional method and take photographs for subsequent visualization analysis and comparison of the antibacterial effect.
[0046] Figure 2 The effects of induced culture of each experimental group on the growth of Magnaporthe oryzae mycelium are shown in Figure (A): colony map; (B): colony diameter. The metabolites of Streptomyces bikiniensis HD-087 of the Ind group (Mo-96) and the Non-ind group can both inhibit the expansion of Magnaporthe oryzae colony, but the inhibitory effect of the Ind group is stronger. When the colony diameter of Magnaporthe oryzae of the CK group reaches 70.27 ± 1.23 mm, the colony diameter of the Non-ind group is 34.68 ± 0.74 mm, and the inhibition rate of mycelial growth rate is 50.65%, while the colony diameter of the Ind group is 25.97 ± 0.56 mm, and the inhibition rate of mycelial growth rate is 63.04%, which is 12.39% higher than that of the Non-ind group. This shows that the inhibitory activity of the metabolites of the induced culture group on Magnaporthe oryzae mycelium is higher than that of the non-induced culture group. And from Figure 2 it can also be seen that the Magnaporthe oryzae colony of the Ind group is white, which shows that the metabolites of the Ind group inhibit the formation of Magnaporthe oryzae spores and melanin.
[0047] 5. Effects of metabolites of Streptomyces bikiniensis HD-087 after induced culture on Magnaporthe oryzae spore germination
[0048] An appropriate amount of Magnaporthe oryzae conidial suspension was taken, and 1 / 4 volume of the culture supernatant of the Non-ind and Ind groups (Mo-96) after 96 h of fermentation was added, with an equal amount of sterile water as a blank control. After mixing uniformly with a pipette gun, 40 μL of each sample was taken from each group and dropped on the surface of a hydrophobic glass slide. These glass slides were placed in a constant temperature and humidity incubator at 28 °C, and microscopic observation was performed at 3 h and 3.5 h after treatment. 200 spores were randomly selected for microscopic observation for each treatment sample, and when the length of the germ tube was more than half the radius of the spore, it was determined that the spore had entered the germination state. According to this, the spore germination rate of each group was calculated, and the corresponding germination inhibition rate was further calculated. The spore germination rate and spore germination inhibition rate were calculated according to formulas (1-1) and (1-2).
[0049]
[0050] Figure 3 The effects of induced culture on Magnaporthe oryzae conidial germination are shown in Figure 3 and Table 1; in combination with Figure 3 and the results of Table 1, it can be seen that the metabolites of the Non-ind and Ind groups can both inhibit the germination of Magnaporthe oryzae conidia (P < 0.05) Figure 3The inhibition rate of spore germination of the Non-ind group reached 71.01% at 3 h, and the inhibition rate of the Ind group reached 86.54%, which was 15.53% higher than that of the Non-ind group. At 3.5 h, the inhibition rate of spore germination of the Non-ind group reached 53.13%, and the inhibition rate of the Ind group (Mo-96) was 74.17%, which was 21.04% higher than that of the Non-ind group. This indicates that the inhibitory effect of the metabolites of the Ind group (Mo-96) on spore germination is better than that of the Non-ind group, and it shows higher stability in the time dimension.
[0051] Table 1 Effect of induced culture on spore germination of Magnaporthe grisea
[0052]
[0053] Example 2 Induction of Streptomyces bikiniensis HD-087 by GBL standard (1,4-butyrolactone)
[0054] 1. Cultivation of Streptomyces bikiniensis HD-087 seed liquid and fermentation liquid
[0055] One ring of activated Streptomyces bikiniensis HD-087 was picked from the Gao's No. 1 slope medium and inoculated into 50 mL of Gao's No. 1 liquid medium. The culture was incubated in a constant temperature shaking incubator at 28°C and 180 r / min for 36 h to obtain the seed liquid of Streptomyces bikiniensis HD-087. The Gao's No. 1 medium: soluble starch 20 g, KNO3 1 g, NaCl 0.5 g, K2HPO4 0.5 g, MgSO4 0.5 g, FeSO4 0.01 g, distilled water to 1 L, pH 7.2-7.4, solid medium with 2% agar powder, liquid medium without agar powder, 121°C high pressure steam sterilization for 15 min.
[0056] Two mL of Streptomyces bikiniensis HD-087 seed liquid was inoculated into a conical flask containing 60 mL of DBY liquid medium, and the culture was incubated under the same parameters (28°C, 180 r / min) as the seed liquid to obtain the fermentation liquid of Streptomyces bikiniensis HD-087. The DBY fermentation medium: glucose 20 g, soybean powder 5 g, yeast powder 4 g, (NH4)2SO4 5 g, NaCl 1 g, K2HPO4 0.05 g, distilled water to 1 L, pH natural, 121°C high pressure steam sterilization for 15 min.
[0057] 2. Induction of Streptomyces bikiniensis HD-087 by GBL standard (1,4-butyrolactone) (GBL group)
[0058] 1 mL of 1,4-butyrolactone GBL standard (GBL) with a concentration of 1-100 mmol / L (mM) was added to 50 mL of DBY medium / 250 mL flask. Meanwhile, 2 mL of Streptomyces bikiniensis HD-087 seed liquid was inoculated for culture to obtain Streptomyces bikiniensis HD-087 induced fermentation broth (GBL group).
[0059] 2.1 Effect of GBL on antibacterial activity of HD-087 lipopeptide
[0060] 2.1.1 After Streptomyces bikiniensis HD-087 was induced to culture for different times (48, 96, 144 h) with different concentrations (1, 10, 20, 100 mM) of GBL standard, the fermentation supernatant was obtained. The control group was the fermentation supernatant of Non-ind, and the test method was the same as that described in Example 1 "determination of the anti-rice blast fungus activity of HD-087 metabolites after induction culture".
[0061] After the Streptomyces bikiniensis HD-087 fermentation broth of each group induced by different GBL was obtained after 96 h of culture, it was centrifuged at 10 000 r / min for 20 min, the precipitate was removed, and the supernatant was collected. It was concentrated under reduced pressure at 50°C, the pH was adjusted to 2.0 with HCl, a large amount of flocculent precipitate was precipitated, and it was placed in a 4°C refrigerator overnight. It was centrifuged at 10 000 r / min for 20 min, the precipitate was collected, 1 / 4 volume of methanol was added to the total volume of the fermentation supernatant, and it was fully stirred with a magnetic stirrer for more than 2 h (4°C overnight), then it was centrifuged at 10 000 r / min for 20 min, the precipitate was discarded, and the supernatant (methanol extract) was collected, and the pH was adjusted to 7.0 with NaOH. The methanol was removed by rotary evaporation at 50°C, and the lipopeptide of each group was obtained.
[0062] 2.1.2 Figure 4 To study the effect of GBL standard on the antibacterial activity of Streptomyces bikiniensis HD-087 lipopeptide, Figure 4 (A): antibacterial circle effect diagram; (B): antibacterial circle diameter diagram. From Figure 4 It can be seen that the antibacterial activity of Streptomyces bikiniensis HD-087 lipopeptide changed after induction by GBL. The lipopeptide antibacterial circle diameters of GBL-1 (GBL standard experimental group with a concentration of 1 mM) and GBL-10 (GBL standard experimental group with a concentration of 10 mM) were 26.59 ± 1.02 mm and 25.32 ± 1.14 mm, respectively. Compared with the Non-ind group and the Ind group (Mo-96), the lipopeptide antibacterial activity was significantly up-regulated, indicating that GBL affected the antibacterial activity of lipopeptide.
[0063] 2.2.3 Effect of GBL on the oil displacement activity of Streptomyces ginsengii HD-087 lipopeptides
[0064] A sterilized flat plate with an inner diameter of 90 mm was placed on the water surface, 1 / 2 volume of distilled water was added, and a drop of Sudan III dye was added at the center of the water surface. After the dye spread over the flat plate, 100 μL of each group of lipopeptides with a concentration of 50 μg / mL was added at the center of the dye, with methanol as the blank control. The diameter of the oil displacement ring was measured, and the test was repeated three times.
[0065] Figure 5 To investigate the effect of GBL standard on the oil displacement activity of Streptomyces ginsengii HD-087 lipopeptides, Figure 5 (A): Oil displacement activity diagram; (B): Oil displacement activity column chart, as shown in Figure 5 No oil displacement ring was observed in the methanol control group, while oil displacement rings were observed in the other test groups. Among them, the oil displacement activity of lipopeptides induced by Ind group (Mo-96) and GBL-10 was the best, with oil displacement diameters of 51.68 ± 1.17 mm and 50.76 ± 1.70 mm, respectively. Compared with the Non-ind group, the oil displacement activity increased by 27.60% and 21.34%, respectively. In addition, the oil displacement activity of all treatment groups was significantly better than that of the Non-ind group except for the GBL-1 treatment group. This shows that the induction and GBL treatment can enhance the oil displacement activity of Streptomyces ginsengii HD-087 lipopeptides. The above phenomenon shows that the lipopeptides of all treatment groups have oil displacement ability and exhibit certain potential in oil degradation or removal, which can be applied in the field of environmental remediation, etc.
[0066] 3. Extraction of GBL from Magnaporthe oryzae as an inducer to induce Streptomyces ginsengii HD-087
[0067] 3.1. Extraction of M-GBL
[0068] 1 mL of M-GBL with a concentration of 1 × 10 5Myceliophthora thermophila spore suspension of 108 spores / mL was inoculated into a 250 mL flask containing 50 mL PDB medium, and cultured at 180 r / min and 28 °C for 144 h. During the culture, 2 mL of culture solution was collected every 24 h, centrifuged at 10 000 r / min for 10 min, filtered (filter membrane pore size: 0.45 µm) to remove bacteria, and then 1 mL of ethyl acetate was added to the obtained clear filtrate, and mixed thoroughly. The mixture was allowed to stand for 24 h to separate the organic phase from the aqueous phase. Then, the upper organic phase was collected and concentrated under reduced pressure in a rotary evaporator at 40 °C until the solvent was completely evaporated. Then, 1 mL of ethyl acetate was added to redissolve the evaporated components, and the supernatant was obtained by centrifugation at 12 000 r / min for 10 min, to obtain the cell-free filtrate of M. thermophila as an inducer, named M-GBL, which was used to induce the culture of S. bikiniensis HD-087.
[0069] 3.2 Induction method
[0070] 1 mL of the inducer M-GBL was added to the corresponding 250 mL flask containing 50 mL DBY medium. At the same time, 2 mL of S. bikiniensis HD-087 seed solution was inoculated for culture to obtain M-GBL-induced S. bikiniensis HD-087 fermentation broth (M-GBL group).
[0071] 4. GBL extracted from S. bikiniensis HD-087 was used as an inducer to induce the culture of S. bikiniensis HD-087.
[0072] 4.1 Extraction of S-GBL
[0073] The supernatant of S. bikiniensis HD-087 fermentation broth was added with an equal amount of ethyl acetate at a volume ratio of 1:1, and mixed thoroughly. The mixture was allowed to stand for 24 h to separate the organic phase from the aqueous phase. Then, the upper organic phase was collected and concentrated under reduced pressure in a rotary evaporator at 40 °C until the solvent was completely evaporated. Then, 1 mL of ethyl acetate was added to redissolve the evaporated components, and the supernatant was obtained by centrifugation at 12 000 r / min for 10 min, to obtain the fermentation broth filtrate of S. bikiniensis HD-087, which was named S-GBL and used as an inducer to induce the culture of S. bikiniensis HD-087.
[0074] 4.2 Induction method
[0075] 1 mL of inducer S-GBL was added to the corresponding 50 mL DBY medium / 250 mL flask. Meanwhile, 2 mL of B. bikiniensis HD-087 seed liquid was inoculated for culture to obtain S-GBL induced B. bikiniensis HD-087 fermentation broth (S-GBL group).
[0076] 5. Comparison of the production of GBL produced by B. bikiniensis HD-087 (S-GBL) and GBL produced by M. grisea (M-GBL)
[0077] The production of GBL produced by B. bikiniensis HD-087 (S-GBL) and GBL produced by M. grisea (M-GBL) was compared, from Figure 6 It can be seen that the production of M-GBL is 31.67 ± 2.67 mg / L, and the production of S-GBL is 21.95 ± 1.15 mg / L, and the production of M-GBL is 44.28% higher than that of S-GBL, showing a significant difference (P<0.01). Figure 6 Among them, ** represents a significant difference at the level of p<0.01.
[0078] 6. Effect of GBL on the secondary metabolism and growth and development of B. bikiniensis HD-087
[0079] 6.1 Effect of GBLs on the antibacterial activity of B. bikiniensis HD-087
[0080] Different concentrations (1, 10, 20, 100 mM) of GBLs (GBL, M-GBL, S-GBL) and Ind group (Mo-96) were used as inducers, and B. bikiniensis HD-087 was induced for different times (48, 96, 144 h), and the fermentation broth supernatant was collected, and the antibacterial activity was detected. The fermentation broth supernatant of Non-ind was used as the control group. The detection method is the same as the method described in Example 1 "determination of the anti-M. grisea activity of the metabolic products of HD-087 after induction culture".
[0081] Figure 7 The effect of exogenous addition of GBLs on the antibacterial activity of B. bikiniensis HD-087 fermentation broth, Figure 7 Among them, (A): GBL (1,4-butyrolactone); (B): M-GBL; (C): S-GBL; (D): best antibacterial effect figure. From Figure 7 It can be seen that within the appropriate concentration range, three kinds of GBLs inducers (GBL, M-GBL, S-GBL) can induce B. bikiniensis HD-087 to produce antibiotics in the early stage of fermentation (48 h), and reach the peak at 96 h of fermentation (M-GBL: 1.25 ± 0.05 mg / L, S-GBL: 0.85 ± 0.05 mg / L, GBL: 0.75 ± 0.05 mg / L). Figure 7). Among them, GBL (1,4-butyrolactone) showed the best induction effect, and the bacteriostatic effect of the metabolites of Streptomyces bikiniensis HD-087 induced by GBL was significantly higher than that of the Non-ind group at 96 h, and there was no significant difference between the Ind group and the Ind group (Mo-96) Figure 7 D), which shows that GBL plays a key role in the induction of Mo-96.
[0082] In the GBL (1,4-butyrolactone) treatment group ( Figure 7 A), less than 20 mM concentration of GBL showed positive induction effect on HD-087 antibiotic production, and the induction effect of 10 mM was the best, and the inhibition zone diameter reached the maximum value of 51.30 ± 0.86 mm at 96 h of fermentation, while the inhibition zone diameter of the Ind group (Mo-96) under the same conditions was 52.31 ± 0.88 mm, which shows that the induction effect of 10 mM GBL (1,4-butyrolactone) is almost equal to that of the inducer Mo-96, so it can be seen that exogenous GBL can stimulate the production of streptomycin. However, 100 mM GBL showed strong inhibition effect on HD-087 antibiotic production.
[0083] In the M-GBL ( Figure 7 B) and S-GBL ( Figure 7 C) treatment group, less than 20 mM of each GBL showed certain induction effect, and 20 mM concentration was the best. But the induction effect was significantly lower than that of the Ind group, and significantly higher than that of the Non-ind group ( Figure 7 D). From Figure 8 D, when the Ind group (Mo-96) can increase the bacteriostatic effect of HD-087 metabolites by 12.43%, 10 mM GBL (1,4-butyrolactone) can increase by 9.90%. 20 mM M-GBL and S-GBL can increase by 6.09% and 5.48% respectively. The phenomenon of heterologous GBL inducing the enhancement of bacteriostatic effect of Streptomyces bikiniensis HD-087 can be used in production to improve the yield of streptomycin antibiotic by GBLs.
[0084] 6.2 GBL on lipopeptide of Streptomyces bikiniensis HD-087
[0085] 6.2.1 Effect of GBL on lipopeptide yield of Streptomyces bikiniensis HD-087
[0086] (1) Effect of GBL on lipopeptide yield of Streptomyces bikiniensis HD-087
[0087] The fermentation broth of *Streptomyces bikini* HD-087, obtained after induction culture with various inducers (GBL, M-GBL, S-GBL) for 96 h, was centrifuged at 10,000 r / min for 20 min, the precipitate was removed, and the supernatant was collected. After concentration by rotary evaporation under reduced pressure at 50 °C, the pH was adjusted to 2.0 with HCl, resulting in the precipitation of a large amount of flocculent material. The broth was then placed in a refrigerator at 4 °C overnight. After centrifugation at 10,000 r / min for 20 min, the precipitate was collected, and 1 / 4 volume of methanol (the total volume of the fermentation supernatant) was added. The mixture was stirred thoroughly with a magnetic stirrer for at least 2 h to ensure complete dissolution (and then allowed to stand overnight at 4 °C). After centrifugation at 10,000 r / min for 20 min, the precipitate was discarded, and the supernatant (methanol extract) was collected. The pH was adjusted to 7.0 with NaOH. The methanol was removed by rotary evaporation at 50 °C to obtain the lipopeptides.
[0088] Figure 8 To investigate the effect of GBLs on the lipopeptide production of Streptomyces glomeruli HD-087, Figure 8 (A): GBL (1,4-butyrolactone); (B): M-GBL; (C): S-GBL; (D): Optimal lipopeptide yield diagram. Figure 8 It can be seen that, within the appropriate concentration range, all three GBL inducers (GBL(1,4-butyrolactone), M-GBL, and S-GBL) can induce Streptomyces bikini HD-087 to increase its lipopeptide production, which is significantly higher than that of the Non-ind group, but the induction effect is not as good as that of the Ind group (Mo-96).
[0089] In the GBL (1,4-butyrolactone) treatment group ( Figure 8 In group A, GBL (1,4-butyrolactone) at concentrations ≤20 mM showed a positive induction effect on lipopeptide production in *Streptomyces bismuth subtilis*, with 10 mM showing the best induction effect, achieving a lipopeptide yield of 449.74 ± 13.09 mg / L, significantly lower than that in group Ind (Mo-96) (lipopeptide yield of 498.67 ± 10.04 mg / L). However, GBL (1,4-butyrolactone) at a concentration of 100 mM showed a strong inhibitory effect on lipopeptide synthesis in *Streptomyces bismuth subtilis*. This indicates that an appropriate concentration (≤20 mM) of GBL (1,4-butyrolactone) has an inducing effect on lipopeptide synthesis.
[0090] In M-GBL ( Figure 8 B) and S-GBL ( Figure 8C) In the treatment group, the induction effect of 20 mM M-GBL and S-GBL was the best, and M-GBL and S-GBL with a concentration lower than 20 mM also had a certain induction effect. However, 100 mM M-GBL and S-GBL not only had no induction effect, but also had an inhibitory effect. The induction effect of 20 mM M-GBL and S-GBL was less than that of the Ind group (Mo-96).
[0091] From Figure 9 As can be seen from D, the Ind group can increase the lipopeptide yield of Streptomyces bikiniensis HD-087 by 37.03%, 10 mM GBL (1,4-butyrolactone) can increase it by 27.44%, and 20 mM M-GBL and S-GBL can increase it by 14.30% and 11.84%, respectively.
[0092] The above results not only illustrate the important role of GBL in inducing lipopeptide synthesis, but also illustrate that there are substances other than GBL in the Ind group that can induce an increase in lipopeptide yield, which play an auxiliary role in the induction of GBL. The phenomenon that heterologous GBL induces an increase in the lipopeptide yield of Streptomyces bikiniensis HD-087 proves that the present application can be used in production to increase the yield of streptomycin lipopeptide antibiotics by using GBL.
[0093] 6.3 Effect of GBL on the GBL cascade pathway of Streptomyces bikiniensis HD-087
[0094] Figure 9 The effect of GBL (1,4-butyrolactone) on the expression of genes in the GBL pathway of Streptomyces bikiniensis HD-087 was determined by qPCR. Figure 9 It is shown that GBL significantly affects the expression of key genes in the GBL cascade pathway of Streptomyces bikiniensis HD-087, with low concentration (≤20 mM) promoting and high concentration (100 mM) inhibiting. However, the optimal induction effect of exogenous GBL is less than that of the Ind group (Mo-96). At 48 h of fermentation of Streptomyces bikiniensis HD-087, the Ind group (Mo-96) and the low-concentration GBLs treatment group both promote the up-regulation of the expression of the antibiotic synthesis global regulator adpA and the cluster site regulator atrA gene, which means that the exogenous GBL relieves the repression of the repressor protein ArpA on adpA, thereby activating the expression of atrA, leading to the up-regulation of antibiotic gene transcription and improving the early expression of antibiotics of Streptomyces bikiniensis HD-087.
[0095] The expression of the key genes in the GBL cascade pathway was most significantly different in the induction groups compared with the Non-ind group at 96 h of fermentation of S. bikiniensis HD-087. In particular, the expression of adpA and atrA in the GBL-10 group reached a peak, which was 7.72 times and 6.04 times that of the Non-ind group, respectively. The expression of adpA and atrA in the Ind group (Mo-96) was 4.57 times and 4.98 times that of the Non-ind group, respectively, which was lower than that in the GBL-10 group but significantly higher than that in the Non-ind group. From the above, it can be seen that the expression of the key genes in the GBL cascade pathway was significantly up-regulated in the GBL-1 and GBL-20 groups, which was the main reason for the increase in antibiotic production. The expression of the four key genes in the GBL cascade pathway was down-regulated in the GBL-100 group, which inhibited the production of antibiotics. At this time, the GBL synthase gene afsA and the receptor adpA did not change significantly, and it was speculated that this was the period of vigorous metabolism, and the addition of exogenous GBL did not have a greater impact. It can also be seen that the GBL cascade pathway adpA and atrA in the GBL-1 and GBL-20 groups were significantly up-regulated, which may be the main reason for the increase in antibiotic production. The expression of the four key genes in the GBL cascade pathway in the GBL-100 group was down-regulated, which inhibited the production of antibiotics. At this time, the GBL synthase gene afsA and the receptor adpA did not change significantly, and it was speculated that this was the period of vigorous metabolism, and the addition of exogenous GBL did not have a greater impact.
[0096] The expression trend of adpA and atrA at 144 h was basically the same as that at 96 h. The expression was significantly up-regulated in the Ind group and the GBL-10 group, and the expression in the Ind group was 3.41 and 2.98 times that of the Non-ind group, respectively. The expression in the GBL-10 group was 4.73 and 5.03 times that of the Non-ind group, respectively, which was still higher than that in the Ind group. At the same time, there was a downward trend with the increase of the concentration of the GBL treatment group, and the expression of the four key genes was down-regulated in the GBL-100 mM group. It is worth noting that when the concentration of GBL was ≥10 mM, the expression of GBL synthase afsA was down-regulated, indicating that the exogenous addition of GBL triggered the negative feedback regulation of afsA, which inhibited the transcription of afsA.
[0097] In summary, low concentration (≤20 mM) GBL can activate the expression of the gene cluster in the GBL cascade pathway that regulates antibiotic synthesis, and high concentration (100 mM) GBL inhibits it.
Claims
1. Application of GBL induced culture of Streptomyces bikiniensis to improve its resistance to Magnaporthe oryzae.
2. The use of GBL-induced Streptomyces bikiniensis culture for increasing the ability of the bacteria to resist Pyricularia oryzae according to claim 1, characterized in that, GBL is used as inducer to induce culture of Streptomyces bikiniensis HD-087.
3. The use of GBL-induced Streptomyces bikiniensis culture for increasing the ability of the bacteria to resist Pyricularia oryzae according to claim 2, characterized in that, The concentration of GBL is 10-20 mmol / L.
4. The use of GBL-induced Streptomyces bikiniensis culture for enhancing its ability to resist Pyricularia oryzae according to claim 1, characterized in that, GBL extracted from Magnaporthe oryzae is used as inducer to induce culture of Streptomyces bikiniensis HD-087.
5. Application of Magnaporthe oryzae induced culture of Streptomyces bikiniensis to improve its resistance to Magnaporthe oryzae.
6. The use of the G. oryzicola induced culture of S. bikiniensis according to claim 5 for increasing the resistance against G. oryzicola, characterized in that The induction culture method is carried out according to the following steps: I. Take 1 mL of 1×10 5 The conidia suspension of Magnaporthe grisea with a concentration of 1×10 The conidia suspension of Magnaporthe grisea with a concentration of 1×10 II. The inducer is mixed with DBY fermentation medium at a ratio of 1:50 to obtain an induction culture medium, and then Streptomyces bikiniensis HD-087 is inoculated into the induction culture medium for culture.
7. Application of GBL and Magnaporthe oryzae in inducing culture of Streptomyces bikiniensis to improve lipopeptide oil-repellent activity.