A strain of Streptomyces lactis and its application in resisting bacterial wilt

Abstract

The present invention discloses a strain of Streptomyces lactacystinicus and its application in resistance to bacterial wilt. The strain is named Streptomyces lactacystinicus ZY‑20, with a deposit number of CCTCC NO: M2022561, and was deposited in the China Type Culture Collection at Wuhan University, Wuhan, China on May 6, 2022. The strain of the present invention has a significant antagonistic effect on Ralstonia solanacearum, providing another way to prevent and treat bacterial wilt. In addition, the present invention discloses the application of piericide B1 and piericide A in resistance to bacterial wilt. The inventors of the present invention have discovered for the first time that piericide B1 and piericide A have a strong inhibitory effect on Ralstonia solanacearum, providing a new option for the preparation of a new type of biological control agent for bacterial wilt.

Description

Technical Field

[0001] The invention relates to the field of agricultural biotechnology, and in particular to a lactic acid streptomyces strain and application thereof in resisting bacterial wilt. Background Art

[0002] Bacterial bacterial wilt of plants is a devastating soil-borne bacterial disease of the vascular bundle caused by Ralstonia solanacearum. Ralstonia solanacearum is a notorious soil-borne bacterium that can cause catastrophic bacterial wilt in hundreds of plants, including important crops such as tomatoes, potatoes, bananas, eggplants and peppers. It is a devastating soil-borne disease with widespread systemic infection in subtropical, tropical and temperate regions, commonly known as "plant blast". Ralstonia solanacearum is considered to be a complex composed of many genetically different strains, commonly referred to as the Ralstonia species complex. Ralstonia solanacearum can produce a variety of virulence factors during infection, causing typical wilting symptoms in host plants. At present, the prevention and control of bacterial bacterial wilt is a major problem recognized by the world. It is widely distributed all over the world and is seriously occurring in southern provinces and cities in my country, especially in Zhejiang. In recent years, not only Solanaceae and melons have been affected, but the large-scale occurrence of mulberry bacterial wilt has also caused serious losses to silkworm production.

[0003] At present, the main methods for preventing and controlling bacterial wilt include strengthening field management, cultivating disease-resistant varieties, using chemical agents and applying biocontrol agents. However, practice has shown that field management is difficult to effectively control the occurrence of bacterial wilt, the breeding of resistant materials takes a long time, and chemical control pollutes the environment and destroys the ecological balance. In addition, the long-term use of chemical agents can easily make pathogens resistant. Therefore, the use of biocontrol strains and their active products to prevent and control ginger bacterial wilt has attracted more and more attention. A large number of studies have shown that some bacteria and actinomycetes are ideal antagonists of bacterial wilt.

[0004] At present, there are no research reports on the use of Streptomyces lactis as a biocontrol agent for bacterial wilt. Therefore, it is necessary to find a more effective compound for preventing and controlling bacterial wilt. Summary of the invention

[0005] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a strain of Streptomyces lactis.

[0006] Another object of the present invention is to provide a method for producing piericidin using the above-mentioned Streptomyces lactis.

[0007] Another object of the present invention is to provide an application of the above-mentioned Streptomyces lactis and piericidin in resisting bacterial wilt.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A strain of Streptomyces lactacystinicus, named Streptomyces lactacystinicus ZY-20, with a deposit number of CCTCC NO:M 2022561, was deposited in the China Type Culture Collection at Wuhan University, Wuhan, China on May 6, 2022.

[0010] The 16S rRNA sequence of the lactic acid Streptomyces is shown in SEQ ID NO: 1.

[0011] The colony morphology of the lactic acid Streptomyces is as follows: the strain cells are rod-shaped, and when cultured on an LB agar plate, the colonies are round, with neat edges, a smooth surface, and opaque colonies.

[0012] The physiological and biochemical characteristics of the lactic acid Streptomyces are as follows: gelatin liquefaction, milk coagulation, milk peptone, fructose utilization, sucrose utilization, glucose utilization, starch hydrolysis, and nitrate reduction are positive, and melanin production, arabinose utilization, raffinose utilization, cellulose hydrolysis, xylose utilization, melibiose utilization, and mannitol utilization are negative.

[0013] The application of Streptomyces lactis in resisting bacterial wilt is preferably to apply the Streptomyces lactis to the soil for cultivating crops or to spray it on the surface of crops.

[0014] A bacterial wilt control agent contains the above-mentioned Streptomyces lactis.

[0015] A method for producing piericidin using the above-mentioned Streptomyces lactis, the specific steps are as follows:

[0016] The lactic acid streptomyces is fermented, separated, centrifuged and extracted to obtain piericidin.

[0017] Application of piericide in resistance to bacterial wilt.

[0018] The piericides are piericide B1 and piericide A, wherein the structural formula of piericide B1 is shown in Formula I, and the structural formula of piericide A is shown in Formula II:

[0019]

[0020]

[0021] The application of the piericide in the preparation of a bacterial wilt prevention and treatment agent.

[0022] A bacterial wilt control agent contains the above-mentioned piericide.

[0023] The bacterial wilt prevention and control agent refers to an agent that can inhibit the proliferation, growth, pathogenicity and β-galactosidase activity of Ralstonia solanacearum.

[0024] The bacterial wilt disease is caused by Ralstonia solanacearum.

[0025] A method for treating bacterial wilt caused by Ralstonia solanacearum is to apply a drug containing Streptomyces lactis or piericidin to the soil where crops are cultivated or to spray it on the surface of crops.

[0026] The crops are crops that are easily infected by Ralstonia solanacearum; preferably Solanaceae crops; more preferably tomatoes.

[0027] The crops are crops that have been infected by Ralstonia solanacearum, or crops that have not been infected by Ralstonia solanacearum.

[0028] The method for producing piericidin using Streptomyces lactis comprises the following specific steps:

[0029] (1) fermenting Streptomyces lactis ZY-20, and performing solid-liquid separation on the obtained fermentation liquid; adding ethyl acetate to the obtained supernatant for extraction, collecting the upper organic phase, dissolving it in methanol after rotary evaporation, and removing insoluble matter with a filter membrane with a pore size of 0.22 μm to obtain an organic solution I containing an active ingredient;

[0030] (2) The column was loaded with C18 reverse phase silica gel, and the organic solution I containing the active ingredient was loaded, and the active ingredient was adsorbed by the C18 reverse phase silica gel; then, 10% to 100% chromatographic methanol was used for gradient elution, and each 500 mL of the eluate was collected in a clean conical flask as a fraction; the 80% methanol fraction was collected, dissolved in methanol after rotary evaporation, and insoluble matter was removed by filtering through a 0.22 μm filter membrane;

[0031] (3) The 80% methanol fraction after removing the insoluble matter in step (2) was subjected to semi-preparative high performance liquid chromatography; wherein the solvent A phase was chromatographically pure water; the solvent B phase was chromatographically pure acetonitrile; the elution procedure was as follows: the 80% methanol: 20% water fraction with antibacterial activity was subjected to semi-preparative high performance liquid chromatography (reversed phase semi-preparative column XSelect HSS T3 Prep 5μm, 250×10.0mmColumn; solvent A phase is chromatographic pure water; solvent B phase is chromatographic pure methanol; elution procedure is: 75% B, 0~1.5min; 75% B to 100% B, 1.5~7.5min; 100% B, 7.5~17.5min; 100% B to 75% B, 17.5~18.5min; 75% B, 18.5~20min; flow rate is 3.0mL / min, collect for 10~12min to obtain compound 1 piericide B1, collect for 13~14min to obtain compound 2 piericide A.

[0032] The lactic acid Streptomyces ZY-20 described in step (1) has a preservation number of CCTCC NO:M 2022561 and was deposited in the China Type Culture Collection at Wuhan University, Wuhan, China on May 6, 2022.

[0033] The fermentation medium used in the fermentation in step (1) is preferably LB medium.

[0034] The composition of the LB medium is as follows: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0-7.2.

[0035] The fermentation conditions in step (1) are preferably at 26-28° C. and 200-250 rpm; more preferably at 28° C. and 220 rpm.

[0036] The fermentation degree in step (1) is preferably to reach the logarithmic growth phase or stable phase of Streptomyces lactis; more preferably OD 600 =3.

[0037] The solid-liquid separation method in step (1) is preferably centrifugation.

[0038] The centrifugal conditions are preferably 8000-12000×g for 10-20 min; more preferably 10000×g for 15 min.

[0039] The amount of ethyl acetate used in step (1) is preferably equal in volume to the supernatant.

[0040] The column packed with C18 reverse phase silica gel in step (2) preferably has a height of 0.3 m and a diameter of 2 cm.

[0041] The column used in the semi-preparative high performance liquid chromatography described in step (3) is preferably a reverse phase semi-preparative column XSelectHSS T3 Prep 5μm, 250×10.0mm Column.

[0042] Compared with the prior art, the present invention has the following advantages and effects:

[0043] (1) The inventors of the present invention have discovered for the first time that piericidin B1 and piericidin A have a strong inhibitory effect on Ralstonia solanacearum, providing a new option for the preparation of a novel biological control agent for bacterial wilt.

[0044] (2) The preparation method of piericidin B1 and piericidin A provided by the present invention is to use Streptomyces lactacystinicus as the strain, and obtain them through cultivation, separation and purification; the method has simple steps and simple operation, which is conducive to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a photograph showing the antagonistic effect of strain ZY-20 of the present invention on Ralstonia solanacearum.

[0046] Figure 2 This is a biological control experiment effect diagram of antagonistic bacteria of bacterial wilt in the present invention, in which V GMI1000 Ralstonia solanacearum GMI1000, V S.latis It is Streptomyces lactis ZY-20.

[0047] Figure 3 It is a photograph of a single colony of strain ZY-20 in the present invention.

[0048] Figure 4 This is the NMR result of piericide B1.

[0049] Figure 5 This is the mass spectrometry result of piericidin B1.

[0050] Figure 6 This is the NMR result of piericide A.

[0051] Figure 7 This is the mass spectrometry result of piericidin A.

[0052] Figure 8 This is a graph showing the results of the antibacterial activity test of piericide B1 and piericide A against Ralstonia solanacearum. Figure 8 (a) is piericidin B1, Figure 8 (b) is piericidin A.

[0053] Fig. 9 This is a graph showing the results of the detection of piericidin B1 and piericidin A on the expression level of epsA gene of Ralstonia solanacearum.

[0054] Fig.10 This is a graph showing the effect of piericide A in biological control experiments on bacterial wilt. DETAILED DESCRIPTION

[0055] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0056] Example 1 Bacteria are isolated and purified

[0057] 1. Culture medium

[0058] LB agar medium (LB solid medium): peptone 10 g, yeast extract 5 g, NaCl 10 g, agar 15 g, dilute to 1000 mL with H2O, pH 7.0-7.2. Sterilize at 121°C for 20 min.

[0059] 2. Experimental Procedure

[0060] (1) Sample collection: Select a representative location (soil from the roots of healthy plants in a field with bacterial wilt, Longyan City, Fujian Province), shovel away the topsoil, and use a soil collecting tool to collect soil from a layer 5 to 10 cm below the surface. Collect 5 samples from each location, put them in bags, and keep records.

[0061] (2) Preparation of soil suspension: Weigh 10 g of soil and place it in a conical flask containing 90 mL of sterile water and shake it thoroughly to make a soil suspension with a concentration of 1:10. After the soil particles settle, take 1 mL of the supernatant and transfer it to a test tube containing 9 mL of sterile water to make a soil suspension with a concentration of 1:100. Similarly, gradient dilution is performed to make a soil suspension with a concentration of 10:10. -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 Times the soil suspension for later use.

[0062] (3) Bacterial isolation: select 10 -3 , 10 -4 , 10 -5 and 10 -6 0.1 mL of soil suspension of four concentrations was added to each LB agar culture dish. The coating stick was fully sterilized by burning in an alcohol lamp, and then coated on the plate after cooling. The inoculated culture dish was inverted and cultured at a constant temperature of 30°C with three replicates.

[0063] (4) Results: A total of 1288 colonies were isolated.

[0064] Example 2 Plate antagonism test of isolated bacteria against Ralstonia solanacearum

[0065] 1. Culture medium

[0066] TTC solid medium:

[0067] (1) 10 g of peptone, 1 g of casein acid hydrolysate, 5 g of glucose, and 15 g of agar were diluted with H2O to a volume of 1000 mL, pH 6.8-7.2; sterilized at 121°C for 20 minutes to obtain solution I;

[0068] (2) TTC (2,3,5-triphenyltetrazolium chloride) was prepared into a 1% by mass solution with distilled water, and sterilized by filtration with a 0.22 μm filter membrane to obtain solution II;

[0069] (3) When solution I has cooled to about 45°C, add 0.5 mL of solution II to every 100 mL of solution I and pour the plate.

[0070] CPG liquid culture medium: The preparation method refers to TTC solid culture medium, without adding agar and TTC.

[0071] 2. Experimental Procedure

[0072] 2.1 Plate antagonism test

[0073] 2.1.1 Bacterial Activation

[0074] Pseudomonas solanacear and bacteria were activated: Pseudomonas solanacear GMI1000 (ATCC) was transferred to a TTC solid culture medium plate and cultured at 28°C for 2 days; 1288 colonies isolated in Example 1 were transferred to an LB solid culture medium plate and cultured at 30°C for 1 day for later use.

[0075] 2.1.2 Preparation of Ralstonia solanacearum carrying pME2-pepsA-lacZ

[0076] The pME2-lacZ plasmid was used to construct the reporter plasmid pME2-pepsA-lacZ of the exopolysaccharide EPS synthase gene epsA of Ralstonia solanacearum (the construction method of the plasmid and strain is referred to the literature Ralstonia solanacearum promotes pathogenicity by utilizing l-glutamic acid from host plants, doi.org / 10.1111 / mpp.12963). The constructed pME2-pepsA-lacZ plasmid was introduced into Ralstonia solanacearum GMI1000 by electroporation to obtain Ralstonia solanacearum containing the epsA gene and carrying pME2-pepsA-lacZ. A single colony of Ralstonia solanacearum carrying pME2-pepsA-lacZ was picked and inoculated into 10 mL of CPG liquid culture medium, and cultured at 28°C and 220 rpm / min for one day to obtain a bacterial solution. Then 3 mL of the Ralstonia solanacearum bacterial solution carrying pME2-pepsA-lacZ was added to 30 mL of melted TTC solid culture medium (cooled to about 45°C), and then 150 μL of x-gal was added to pour it into a plate, and after solidification, a Ralstonia solanacearum plate was obtained.

[0077] 2.1.3 Preliminary screening of plate antagonism test

[0078] Use a sterilized toothpick to pick up 288 activated colonies and spot-inoculate them on Ralstonia solanacearum plates. Inoculate 36 different colonies on each plate. Repeat 3 times and culture at 28°C. Observe the experimental results at 24h, 36h and 48h, mainly to observe the presence or absence of inhibition zone.

[0079] 3. Results

[0080] Through the initial screening test, 10 bacterial strains with antagonistic effects were obtained, and strain ZY-20 was one of them.

[0081] Depend on Figure 1 It can be seen that the inhibition zone is obvious, indicating that strain ZY-20 has a significant antagonistic effect on Ralstonia solanacearum.

[0082] Example 3 Biological control effect test of strain ZY-20

[0083] In order to test the biocontrol effect of strain ZY-20, the fermentation liquid of strain ZY-20 was used to test the biocontrol effect of bacterial wilt on potted plants.

[0084] Experimental steps:

[0085] 1.1 Preparing tomato seedlings

[0086] Purchase tomato seedlings grown in greenhouses through commercial channels.

[0087] 1.2 Seedling transplanting

[0088] Fill a flower pot (size 170mm×160mm) with substrate soil, slightly compact it, then move one tomato seedling into each pot, press the roots slightly with two fingers after watering, and cultivate in a greenhouse. Cultivate in the flower pot for about 4 weeks.

[0089] 1.3 Activation of Ralstonia solanacearum and antagonistic bacteria and preparation of fermentation broth

[0090] Ralstonia solanacearum GMI1000 and antagonistic bacteria ZY-20 were activated separately (for specific steps, refer to step 2.1.1 in Example 2), and then single colonies were picked and inoculated into CPG liquid culture medium and LB liquid culture medium for shaking flask culture at 28°C, 200rpm, and Ralstonia solanacearum GMI1000 was cultured overnight (to OD600 of about 0.5) to obtain Ralstonia solanacearum fermentation broth, and antagonistic bacteria ZY-20 was cultured for about two days (to OD600 of about 2.5).

[0091] 1.4 Inoculation of Ralstonia solanacearum and antagonistic bacteria

[0092] Two control groups were set up in this experiment, namely CK1 and CK2. CK1: no bacteria were added, only 20 μL CPG liquid culture medium was added; CK2: 5 μL Ralstonia solanacearum fermentation liquid was added, and no antagonistic bacteria fermentation liquid was added.

[0093] The experimental group was set up for strain ZY-20 treatment: the fermentation broth of Ralstonia solanacearum and the culture broth of strain ZY-20 were mixed according to the volume ratio of 1:0.5 and 1:2, respectively, to prepare two experimental groups with different ratios, and the total addition amount of each group was 7.5μL and 15μL, respectively.

[0094] The specific experimental method is: take the prepared experimental group and control group according to the added amount to directly infect 4-week-old tomato seedlings, use a capillary to inject the sample into the stem above the petiole of the first true leaf, and cultivate the plants under standard conditions. Each group of experiments has 10 pots.

[0095] 1.5 Observation and Recording

[0096] The number of diseased plants and the severity of the disease were recorded every other day.

[0097] 2. Results

[0098] like Figure 2 As shown, after the inoculation experiment, the control group CK1 inoculated with only CPG liquid culture medium did not develop the disease, with a mortality rate of 0, the control group CK2 inoculated with only Ralstonia solanacearum GMI1000 fermentation liquid all developed the disease, with a mortality rate of 100%, and the treatment groups inoculated with Ralstonia solanacearum GMI1000 fermentation liquid and strain ZY-20 fermentation liquid at a ratio of 1:0.5 and 1:2 had a mortality rate of 37.5%. It can be seen that the treatment with strain ZY-20 fermentation liquid can effectively control the occurrence of bacterial wilt in tomato plants.

[0099] Example 4 Identification of isolated bacteria

[0100] After testing by the Guangdong Microbiological Analysis and Testing Center, the testing basis is the "Common Bacteria System Identification Manual" (Edited by Dong Xiuzhu, Science Press), the "Berger Bacteria Identification Manual" 9th edition and the "Molecular Cloning Experiment Guide" 2nd edition (Translated by Huang Peitang, Science Press, 2002). The identification results are: The 16S rDNA sequence of strain ZY-20 is 99.78% homologous to Streptomyces lactacystinicus (Streptomyces lactacystinicus), and its morphological characteristics and physiological and biochemical characteristics are most similar to Streptomyces lactacystinicus (Streptomyces lactacystinicus). The specific test results are as follows:

[0101] 1. The morphological, cultural, physiological and biochemical characteristics of the deposited strain

[0102] Streptomyces lactacystinicus ZY-20: Belongs to Streptomyces lactacystinicus; the mature spore chains of the sample Streptomyces are linear, each chain contains more than 20 spores, and the spores are cylindrical, 1.1-1.3×0.6-0.7μm. (such as Figure 3 shown).

[0103] 2. Physical and chemical experiments of strain ZY-20

[0104] The experimental results are shown in Table 1:

[0105] Table 1

[0106] Test Project result Test Project result Test Project result Test Project result Gelatin liquefaction + Fructose Utilization + Cellulose hydrolysis － Nitrate reduction + Milk curdling + Sucrose Utilization + Xylose Utilization － Melibiose Utilization － Milk peptonization + Arabinose Utilization － Glucose utilization + Mannitol Utilization － Melanin production － Raffinose Utilization － Starch hydrolysis +

[0107] Note: “+” indicates positive, “-” indicates negative.

[0108] 3.16S rRNA sequence alignment

[0109] The results of 16S rRNA gene sequence determination of strain ZY-20 are shown in SEQ ID NO: 1. The 16S rRNA gene sequence was subjected to BLAST comparison analysis on the NCBI website, and the similarity with Streptomyces lactacystinicus was 99.78%. Its morphological characteristics and physiological and biochemical properties were most similar to those of Streptomyces lactacystinicus. Therefore, based on the results of 16S rRNA gene sequence determination and morphological characteristics and physiological and biochemical characteristics, the strain can be classified as Streptomyces lactacystinicus.

[0110] The strain ZY-20 was named Streptomyces lactacystinicus ZY-20, with the deposit number of CCTCC NO:M 2022561. It was deposited in the China Type Culture Collection, Wuhan University, Wuhan, China on May 6, 2022.

[0111] Example 5: Extraction, separation and structural identification of Streptomyces lactis metabolites

[0112] 1. Bacterial strains and culture medium

[0113] Streptomyces lactacystinicus ZY-20, with the deposit number of CCTCC NO:M2022561, was deposited in the China Type Culture Collection, Wuhan University, Wuhan, China on May 6, 2022.

[0114] Ralstonia solanacearum GMI1000 was purchased from ATCC. LB solid medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 15 g agar, and H2O to 1000 ml, pH 7.0-7.2. Sterilize at 121°C for 20 minutes.

[0115] LB liquid medium: The only difference from LB solid medium is that it does not contain agar.

[0116] Preparation of TTC solid medium: 10g of tryptone, 1g of acid hydrolyzed casein, 5g of glucose, 15g of agar, and H2O to 1000ml, pH 6.8-7.2; sterilize at 121℃ for 20 minutes to obtain basic medium. TTC (2,3,5-triphenyltetrazolium chloride) is made into a 0.1% (w / v) solution with distilled water and sterilized by filtering with a bacterial filter. Before pouring the basic medium onto a plate (cooled to about 45℃), add 0.5mL of 0.1% (w / v) TTC solution per 100mL of basic medium.

[0117] CPG liquid medium: does not contain agar.

[0118] 2. Experimental steps

[0119] 2.1 Culture medium preparation

[0120] Prepare the culture medium as in step 1.

[0121] 2.2 Activation of Streptomyces lactis ZY-20

[0122] Streptomyces lactis ZY-20 stored at -80°C was streaked on an LB plate for activation and cultured in a 28°C incubator overnight.

[0123] 2.3 Fermentation of Streptomyces lactis ZY-20

[0124] Take the lactic acid Streptomyces ZY-20 strain activated by LB solid medium, pick a single colony and inoculate it into 10mL LB liquid medium, shake and culture at 28℃ and 220r / min for 12h to obtain seed liquid, inoculate 1000mL LB liquid medium at a 1% (v / v) inoculum volume, shake and culture at 28℃ and 220r / min until OD 600 When the concentration reached 3.0, the cells were centrifuged at 10,000 × g for 15 min to remove the bacteria and collect the supernatant.

[0125] 2.4 Extraction of compounds

[0126] Add an equal volume of ethyl acetate to the supernatant, mix well and pour into a separatory funnel to let stand for stratification, discharge the lower aqueous phase, pour out the upper organic phase, evaporate the ethyl acetate on a rotary evaporator, add 1 mL of methanol to dissolve, pass through a 0.22 μm organic filter membrane to remove insoluble matter, obtain a crude extract, and store at 4°C.

[0127] 2.5 Preliminary separation of compounds

[0128] Weigh C18 reverse phase silica gel into a clean conical flask, add chromatographic methanol to soak for 24 hours and then install it in a column with a height of 0.3m and a diameter of 2cm. Rinse it with chromatographic methanol and then load the sample (i.e. the crude extract obtained in 2.4), and then use 10% to 100% chromatographic methanol for gradient elution. Every 500mL of eluate is collected in a clean conical flask as a fraction. After evaporating the methanol on a rotary evaporator, dissolve it in 0.5mL of chromatographic methanol, pass it through a 0.22μm organic filter membrane to remove insoluble matter, and then detect the antibacterial activity of each part respectively.

[0129] 2.6 Antibacterial activity detection

[0130] The antibacterial activity was detected by agar diffusion method. Ralstonia solanacearum GMI1000 stored at -80°C was streaked on a TTC plate for activation, cultured at 28°C for 3 days, and a single colony was picked and inoculated in 10mL CPG liquid medium. The culture was shaken at 28°C and 220r / min for one day. The Ralstonia solanacearum bacterial solution was then added to the melted TTC solid medium (cooled to about 45°C) and poured into a plate. After solidification, a sterile puncher (8mm in diameter) was used to punch a hole in the center of the bacterial plate, one hole per plate, and the bacterial block was removed and discarded. 10μL of the test solution was added to each well with a pipette, and after drying, it was inverted and cultured at 28°C for 2 days, repeated 3 times, and the inhibition zone was observed. It was found that the 80% methanol fraction was active.

[0131] 2.7 Secondary separation of compounds

[0132] The 80% methanol fraction with antibacterial activity was subjected to semi-preparative high performance liquid chromatography (reverse phase semi-preparative column XSelectHSS T3 Prep 5μm, 250×10.0mm Column; solvent A phase was chromatographic pure water; solvent B phase was chromatographic pure methanol; elution program was: 75% B, 0-1.5min; 75% B to 100% B, 1.5-7.5min; 100% B, 7.5-17.5min; 100% B to 75% B, 17.5-18.5min; 75% B, 18.5-20min; flow rate was 3.0mL min -1 , collect for 10 to 12 minutes to obtain compound 1, and collect for 13 to 14 minutes to obtain compound 2.

[0133] 2.8 ESI-MS and NMR detection of compounds

[0134] ESI-MS detection: The compound was dissolved in an appropriate amount of chromatographic methanol and filtered, and analyzed by ESI-MS (ultra-performance liquid quadrupole tandem time-of-flight mass spectrometry).

[0135] NMR detection: The compound's solvent is evaporated, dissolved in deuterated methanol, and then transferred to a clean NMR tube for NMR measurement. Measurement types include: 1H NMR, 13C NMR, Dept135.

[0136] 2.9 Results

[0137] Compound 1 was detected by ESI-MS and NMR (see Figures 4-5 ), compound 1 was determined to be Piericidin B1 N-oxide, with a relative molecular mass of 446 and a structure as shown in Formula I. Compound 2 was detected by ESI-MS and NMR (see Figures 6-7 ), compound 2 was determined to be Piericidin A, with a relative molecular mass of 415 and a structure as shown in Formula II.

[0138]

[0139] Example 6: Determination of the antibacterial activity of compounds against Ralstonia solanacearum

[0140] 1. Culture medium

[0141] TTC culture medium: same as in Example 2.

[0142] CPG liquid culture medium: same as Example 2.

[0143] 2. Experimental steps

[0144] 2.1 Culture medium preparation

[0145] Same as Example 2.

[0146] 2.2 Bacteria Activation

[0147] Same as Example 2.

[0148] 2.3x-gal activity detection

[0149] A single colony of Ralstonia solanacearum with pME2-pepsA-lacZ prepared in Example 2 was inoculated in 10 mL of CPG liquid culture medium, and cultured at 28°C and 220 r / min for one day. Then 2 mL of Ralstonia solanacearum with pME2-pepsA-lacZ (preparation process reference Example 2) was added to 20 mL of melted TTC solid culture medium (cooled to about 45°C), and then 100 μL of x-gal was added to form a plate. After solidification, a sterile hole puncher (diameter 8 mm) was used to punch a hole in the center of the bacterial plate, 1 hole per plate, and the bacterial block was removed and discarded. 10 μL of the compound solution prepared in Example 5 was added to each well with a pipette, and after drying, it was inverted and cultured at 28°C for 2 days, repeated 3 times, and the inhibition zone was observed.

[0150] 2.4 Detection of the effect of compounds on β-galactosidase activity of Ralstonia solanacearum

[0151] (1) After R. solanacearum carrying pME2-pepsA-lacZ was activated on a TTC plate, a single colony was picked and inoculated into 10 mL CPG liquid medium at 28°C and 220 rpm overnight;

[0152] (2) Dilute the cultured bacterial solution with fresh CPG liquid medium to OD600 = 0.1, take a sterile 15 mL centrifuge tube, and add 3 mL of the diluted bacterial solution to each tube;

[0153] (3) Antibacterial substances (i.e., the compounds prepared in Example 5) were added, and the concentrations of piericide B1 were 0.5, 0.25, and 0.125 μg / mL, respectively; the concentrations of piericide A were 0.2, 0.1, and 0.05 μg / mL, respectively. Methanol was added as a control, and the cells were cultured at 28°C and 200 rpm until OD 600＝ 2.0.

[0154] (4) By measuring the expression of the lacZ gene in the reporter strain, the activity of the inserted promoter in the reporter strain can be compared and analyzed. In the β-galactosidase activity assay, ONPG (2-nitrophenyl-β-D-pyranogalactoside) is used as a substrate, and the activity of β-galactosidase can be relatively quantitatively analyzed by the color change in the culture medium.

[0155] The experimental steps for determining the activity of β-galactosidase are as follows:

[0156] 1) The reporter strain was streaked on a TTC plate (containing tetracycline and X-gal) for activation, and a single colony of Ralstonia solanacearum carrying pME2-pepsA-lacZ was picked and inoculated into 10 mL of CPG liquid medium (containing tetracycline), and cultured overnight at 28°C and 200 rpm with shaking;

[0157] 2) Determine the OD600 value of each strain, dilute with fresh culture medium to OD600 = 0.01, and culture at 28°C and 200 rpm with shaking;

[0158] 3) Take samples every 4 hours, take out 200 μL of bacterial solution and transfer it to a new centrifuge tube, and record the OD600 value of each test strain;

[0159] 4) Centrifuge at 12000 rpm for 1 min to collect the cells, and add 200 μL of Z-buffer / β-mercaptoethanol to resuspend the cells;

[0160] 5) Add 20 μL of 0.1% SDS, shake for 5 min, add 20 μL of chloroform, shake for 5 min;

[0161] 6) Quickly add 200 μL of ONPG and incubate at 37°C until the solution turns yellow (reaction time should not exceed 30 min). Record the incubation time.

[0162] 7) Immediately add 600 μL of 1 M Na2CO3 to terminate the reaction;

[0163] 8) Centrifuge at 12000 rpm for 1 min, take the supernatant and measure the OD420 and OD550 values.

[0164] 9) Calculation of β-galactosidase activity

[0165] 3. Results

[0166] The experimental results show that piericide B1 and piericide A have strong antibacterial effects on Ralstonia solanacearum. Figure 8 ; It has an inhibitory effect on the β-galactosidase activity of Ralstonia solanacearum, see Fig. 9 .

[0167] Example 7: Biological control effect test of Streptomyces lactis metabolites

[0168] In order to test the biocontrol effect of Streptomyces lactis metabolites, the compound piericidin A was used to conduct a biocontrol test on bacterial wilt in potted plants.

[0169] Experimental steps:

[0170] 1.1 Preparing tomato seedlings

[0171] Purchase tomato seedlings grown in greenhouses through commercial channels.

[0172] 1.2 Seedling transplanting

[0173] Fill the flowerpot (size 170mm*160mm) with substrate soil, slightly compact it, then move one tomato seedling into each pot, press the roots slightly with two fingers after watering, and cultivate in the greenhouse. Cultivate in the flowerpot for about 4 weeks.

[0174] 1.3 Activation of Ralstonia solanacearum and preparation of fermentation broth

[0175] The Ralstonia solanacearum GMI1000 was activated, and then single colonies were picked and inoculated into CPG liquid medium, and the Ralstonia solanacearum GMI1000 was cultured overnight at 28°C and 200 rpm (to OD 600 About 1.0), to obtain Ralstonia solanacearum fermentation broth.

[0176] 1.4 Inoculation of Ralstonia solanacearum and Streptomyces lactis metabolites

[0177] Two control groups were set up in this experiment, namely CK1 and CK2. CK1: No bacteria were added, only 5 μL CPG liquid medium was added; CK2: 2 μL methanol was added to 100 μL Ralstonia solanacearum fermentation liquid and mixed well, and 5 μL of CK1 / CK2 was respectively drawn with a capillary and injected into the stem above the petiole of the first true leaf to directly infect 4-week-old tomato seedlings.

[0178] The experimental group compound was treated with piericidin A: the piericidin A solution prepared in step 2.7 of Example 5 was added to the fermentation broth of Ralstonia solanacearum to prepare reagents with piericidin A concentrations of 50 ng / mL and 25 ng / mL. Two treatments were performed, with 10 pots in each treatment. 5 μL of the reagent was aspirated with a capillary and injected into the stem above the petiole of the first true leaf to directly infect 4-week-old tomato seedlings, and the tomato seedlings were cultivated under standard conditions.

[0179] 1.5 Records

[0180] The number of diseased plants and the severity of the disease were recorded every other day.

[0181] 2. Results

[0182] like Fig.10 As shown, after artificial inoculation, the control group CK1 inoculated with CPG liquid culture medium only did not develop the disease, with a mortality rate of 0, the control group CK2 inoculated with Ralstonia solanacearum GMI1000 fermentation broth with methanol added all developed the disease, with a mortality rate of 100%, and the mortality rates of the groups inoculated with Ralstonia solanacearum GMI1000 fermentation broth and compound piericide A 50ng / mL, 25ng / mL treatment groups were 62.5% and 50%, respectively. It can be seen that the compound piericide A treatment can effectively control the occurrence of bacterial wilt in tomato plants.

[0183] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A strain of Streptomyces lactis, characterized in that: Streptomyces lactis ( Streptomyces lactacidis ) ZY-20, with the deposit number of CCTCC NO:M2022561, was deposited on May 6, 2022 in the China Center for Type Culture Collection, Wuhan University, Wuhan, China.

2. Use of the lactic acid Streptomyces according to claim 1 in resisting bacterial wilt; The bacterial wilt disease is caused by Ralstonia solanacearum.

3. A bacterial wilt control agent, characterized in that: Contains the lactic acid Streptomyces according to claim 1.

4. A method for producing piericidin using the Streptomyces lactis according to claim 1, characterized in that The specific steps are as follows: The lactic acid streptomyces is fermented, separated, centrifuged and extracted to obtain piericide; The piericides are piericide B1 and piericide A; The structural formula of the piericidin B1 is shown in Formula I, and the structural formula of the piericidin A is shown in Formula II: Formula I; Formula II.

5. Use of the piericidin according to claim 4 in resisting bacterial wilt; The bacterial wilt disease is caused by Ralstonia solanacearum.

6. The use according to claim 5, characterized in that: The application is to prepare a drug for preventing and controlling bacterial wilt; The bacterial wilt disease is caused by Ralstonia solanacearum.

7. A method for preventing and treating bacterial wilt caused by Ralstonia solanacearum, characterized in that: The method comprises applying the agent containing the lactic acid streptomyces described in claim 1 or the piericide described in claim 4 to the soil for cultivating crops or spraying it on the surface of the crops.

8. The method according to claim 7, characterized in that: The crops are crops that are easily infected by Ralstonia solanacearum.

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