Application of thiogamboge in prevention and treatment of plant phytophthora root rot

Abstract

The invention discloses application of thiogamboge in prevention and treatment of phytophthora phytophthora. The invention finds that the thiogamboge can effectively inhibit the growth of pathogenic bacteria of the plant phytophthora root rot and has the application of preventing and treating the plant phytophthora root rot. Researches show that the sulfur gamboge can inhibit the growth of phytophthora nicotianae hyphae and cause damage to organelles of phytophthora nicotianae pathogenic bacteria, so that the occurrence of phytophthora nicotianae is effectively prevented and treated, and the prevention effect is 85.81%.

Description

Technical Field

[0001] The invention relates to the technical field of plant disease prevention and control, and in particular to application of thiolutein in preventing and controlling plant phytophthora. Background Art

[0002] Phytophthora has a wide range of hosts, mainly attacking tens of thousands of plants such as forests, fruit trees, vegetables, flowers, cotton, hemp, oilseeds and Chinese herbal medicines, posing a serious threat to agricultural, food and forestry production. Because Phytophthora is highly destructive and harmful to host plants, it often causes serious losses to agricultural production. Therefore, plant diseases caused by Phytophthora are usually called "blight". For example, late blight of potato caused the Great Famine in Ireland, nearly one million people starved to death, and more than 2 million people immigrated to other places. Traditional prevention and control methods rely heavily on chemical fungicides, but this method often causes pathogen resistance and is harmful to the environment and human health. At present, there is a lack of green pesticide control agents that are effective against Phytophthora. Therefore, there is an urgent need to explore "green, efficient, low-toxic, healthy and sustainable" control agents.

[0003] Thiolutin (THL) has a molecular formula of C8H8N2O2S2 and a relative molecular weight of 228.29. It is a dithiopyrrolidine compound, mainly derived from Gram-positive bacteria, and is a yellow crystalline substance with antibacterial activity. It has been reported that thiolutin has antagonistic activity against Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, tumor cells, fragrant pear rot, Shigella, pear black spot, and Acinetobacter baumannii. However, there is no report on the use of thiolutin in the prevention and treatment of plant blight. Summary of the invention

[0004] The present invention aims to provide a preparation method and application of thiogarcinol. The present invention firstly finds that thiogarcinol has significant antibacterial activity against plant phytophthora: tobacco phytophthora (Phytophthora nicotiana), camphor phytophthora (P. cinnamomi), palm phytophthora (P. palmivora), pepper phytophthora (P. capsici), cowpea phytophthora (P. vignae), melon phytophthora (P. melonis), taro phytophthora (P. colocasiae), soybean phytophthora (P. sojae), etc., thus providing a new control idea for controlling diseases caused by plant phytophthora.

[0005] In order to achieve the above-mentioned object of the invention, the present invention proposes the following technical solutions:

[0006] Application of thiogarcinol in preventing and controlling plant phytophthora diseases. The phytophthora diseases are plant diseases caused by one or any combination of two or more of tobacco phytophthora (Phytophthoranicotiana), camphor phytophthora (P.cinnamomi), palm phytophthora (P.palmivora), pepper phytophthora (P.capsici), cowpea phytophthora (P.vignae), melon phytophthora (P.melonis), taro phytophthora (P.colocasiae), and soybean phytophthora (P.sojae).

[0007] Preferably, the preparation method of thiolutein comprises the following steps:

[0008] Streptomyces sp.ASG80 was inoculated into ISP2 liquid culture medium, and cultured in a constant temperature shaking box at 28°C and 180rpm for 3 days; inoculated into 120L improved soybean powder infusion culture medium at a 1% (volume percentage) inoculation amount, and cultured at 28°C and 180rpm for 8 days to obtain a fermentation liquid;

[0009] The fermentation liquid is filtered to obtain a bacterial liquid, and the bacterial liquid is extracted to obtain a fermentation extract;

[0010] The fermentation extract is separated to obtain thiolutein.

[0011] Preferably, the method for extracting the bacterial liquid is: extracting the bacterial liquid with ethyl acetate, and concentrating the ethyl acetate phase to dryness to obtain a crude fermentation extract;

[0012] Preferably, the separation method is: dissolving the fermentation crude extract with 3 volumes of methanol, filtering through a Buchner funnel, the dissolved liquid is component A1, and the solid in the Buchner funnel is component A2;

[0013] Component A2 was separated by gel column chromatography and eluted with methanol solvent to obtain three components, which were named component A2-1, component A2-2, and component A2-3 in the order of their elution.

[0014] The A2-2 component was subjected to high performance liquid chromatography to obtain thiocarbazine;

[0015] The mobile phase used for the HPLC purification is 75% methanol and 25% water (volume percentage);

[0016] In one embodiment of the present invention, the application of thiogarcinia in preventing and treating sisal zebra stripe disease (Phytophthoranicotiana) is disclosed, and the application method is a potted plant experiment. The 1000-fold diluted thiogarcinia is used to prevent and treat sisal zebra stripe disease, and the prevention and treatment effect of this embodiment reaches 85.81%.

[0017] The present invention first discovered the antibacterial activity of thiogarcin against plant phytophthora, and applied thiogarcin to the prevention and treatment of plant phytophthora, with good prevention and treatment effect and broad prospects; further, the present invention also provides a method for preparing thiogarcin. The results of the embodiment showed that thiogarcin could destroy the cell wall and cell membrane of tobacco phytophthora hyphae, deform the hyphae, damage the organelles, increase the reactive oxygen species (ROS) in the hyphae, inhibit the growth of tobacco phytophthora, and inhibit the EC of tobacco phytophthora. 50 The value was 0.8266 μg / mL, which effectively prevented and controlled the occurrence of sisal zebra stripe disease caused by tobacco phytophthora, with a prevention efficiency of up to 85.81%. The above results show that thiocanthosides have significant activity in inhibiting plant phytophthora. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the molecular structure of Thioconitine in Example 1;

[0019] Figure 2 Thiogarcinol in Example 1 1 H NMR spectrum;

[0020] Figure 3 Thiogarcinol in Example 1 13 C NMR spectrum;

[0021] Figure 4 The antibacterial activity test diagram of different concentrations of Thiocarpon against Phytophthora nicotianae in Example 3;

[0022] Figure 5 The hyphal morphology and hyphal ultrastructure of Phytophthora nicotianae in Example 4 at different concentrations of Thiocarpon in V8 medium;

[0023] Figure 6 Effect of Thiocarpin on the integrity of the hyphal cell wall of Phytophthora nicotianae in Example 4;

[0024] Figure 7 The effect of thiogarcinol on the integrity of the hyphal cell membrane of Phytophthora nicotianae in Example 4;

[0025] Figure 8 Effect of Thiocarpon on Reactive Oxygen Species (ROS) in Phytophthora nicotianae hyphae in Example 4;

[0026] Fig. 9 Actual picture of the potted plant test of thiogarcinol in preventing sisal zebra stripe disease in Example 4. DETAILED DESCRIPTION

[0027] Example 1. Preparation of Thiogarcinol

[0028] The Streptomyces strain ASG80 (the deposit number of the General Microbiological Center of the China National Microbiological Culture Collection Administration is CGMCC No. 28698, recorded in the patent document CN118048273A) was subjected to 120 L fermentation treatment, and the fermentation crude product of Streptomyces ASG80 was obtained by ethyl acetate extraction, and then the fermentation crude product of Streptomyces ASG80 was separated and purified to obtain the Streptomyces secondary metabolite thioglucosidone. The specific process is as follows:

[0029] The strain ASG80 stored in a -80℃ refrigerator was transferred to a culture dish covered with ISP2 solid culture medium and cultured at 28℃ for 5 days. In a sterile operating table, a sterile toothpick was used to inoculate the strain ASG80 into a 250mL conical flask containing 100mL of ISP2 liquid culture medium, and cultured at 28℃ and 180r / min for 3 days to obtain the primary seed solution.

[0030] Preparation of fermentation broth: The first-level seed liquid was inoculated at a rate of 1% (volume percentage) into a 1L conical flask containing 400mL of improved soybean powder infusion medium fermentation medium, and cultured at 28°C and 180rpm for 8 days to obtain fermentation broth, a total of 120L of fermentation broth was obtained, and 23.58g of crude fermentation extract of strain ASG80 was obtained by extraction with ethyl acetate.

[0031] After obtaining the fermentation extract, the present invention separates thiocarboxylic acid from the fermentation extract. In the present invention, the separation method is preferably: dissolving the crude fermentation extract with 3 times methanol. Filter through a Buchner funnel, the dissolved liquid is component A1, and the solid in the Buchner funnel is component A2. Component A2 is separated by gel column chromatography and eluted with 100% methanol solvent to obtain 3 components, which are recorded as A2-1 to A2-3 in the order of component elution. Component A2-2 is subjected to high performance liquid chromatography with a mobile phase of 75% methanol and 25% water to obtain thiocarboxylic acid, with the structural formula being Figure 1 As shown. The structure of thiolutin was identified by NMR. 1 H NMR spectrum is shown in Figure 2 , 13 C NMR spectrum is shown in Figure 3 , which is consistent with the thiolutein reported in the literature. The NMR data for identifying the compound are shown in Table 1. The yield of thiolutein is 41.7 mg / L.

[0032] Table 1 NMR data of thiolutin (DMSO-d6, 1 H:600MHz, 13 C:151MHz)

[0033]

[0034] SP2 solid medium configuration: yeast extract 4.0g; malt extract 10.0g; glucose 4.0g; trace salt solution 1.0mL; agar powder 20.0g; distilled water 1000mL; pH 7.2; sterilized at 121℃ for 20min. Among them, trace salt solution: FeSO4·7H2O 0.1g; MnCl2·4H2O 0.1g; ZnSO4·7H2O 0.1g; distilled water 1000mL.

[0035] ISP2 liquid culture medium configuration: yeast extract 4.0g; malt extract 10.0g; glucose 4.0g; trace salt solution 1.0mL; distilled water 1000mL; pH 7.2; sterilization at 121℃ for 20min. Among them, trace salt solution: FeSO4·7H2O 0.1g; MnCl2·4H2O 0.1g; ZnSO4·7H2O 0.1g; distilled water 1000mL.

[0036] The configuration of the improved soybean powder extract medium is as follows: sucrose 10.0 g; peptone 2.0 g; soluble starch 5.0 g; yeast extract powder 2.0 g; NaCl 2.0 g; K2HPO4 0.5 g; MgSO4·7H2O 0.5 g; CaCO3 1.0 g; soybean extract powder 20.0 g; distilled water 1000 mL; pH 7.2; sterilization at 121°C for 20 min.

[0037] Configuration of V8 solid culture medium: 100 mL of V8 juice; 0.2 g of CaCO3; 20 g of agar powder; 900 mL of distilled water; pH 7.0; sterilization at 121°C for 20 min.

[0038] Example 2: Determination of the antibacterial spectrum of thiocarpin against Phytophthora

[0039] Take the thiocarbazine prepared in Example 1 and dissolve it in dimethyl sulfoxide (DMSO) to form a sample to be tested at a concentration of 10 mg / mL. Metalaxyl (effective content 98%) and azoxystrobin (effective content 97%) are used as positive controls for the pathogenic fungus Phytophthora nicotiana. Phytophthora cinnamomi, Phytophthora palmivora, Phytophthora capsici, Phytophthora vignae, Phytophthora melonis, Phytophthora colocasiae, and Phytophthora sojae are used as indicator bacteria for antimicrobial spectrum determination. Thiocarbazine, metalaxyl and azoxystrobin are added to a V8 solid culture medium at a temperature of about 50°C-60°C, and the final concentrations are set to 1 μg / mL and 3 μg / mL, respectively. In the clean bench, the plant disease fungus that had been cultured in advance was prepared into a bacterial cake using a hole puncher with a diameter of 5 mm, and the bacterial cake was inoculated into the center of a V8 medium plate and cultured in a constant temperature incubator at 28°C. When the colony growth almost reached the edge of the plate, the colony diameter was measured and the mycelial growth inhibition rate was calculated, and the inhibition rate (%) = [(control group pathogen growth diameter - pathogen block diameter) - (treatment group pathogen growth diameter - pathogen block diameter)] / (control group pathogen growth diameter - pathogen block diameter) × 100%. The inhibition rate of each fungus is shown in Table 2 below.

[0040] Table 2. Determination of the antibacterial spectrum of thiocarbazine

[0041]

[0042] It can be seen from the data in Table 2 that the antibacterial spectrum of thiogarcin is relatively wide. At a concentration of 1 μg / mL, the antibacterial rate of thiogarcin against tobacco phytophthora and camphor phytophthora reached more than 50%. When the concentration was 3 μg / mL, the antibacterial rate of thiogarcin against the tested phytophthora pathogens reached more than 50%, and the antibacterial rate against tobacco phytophthora, camphor phytophthora, cowpea phytophthora, soybean phytophthora, and taro phytophthora reached more than 90%. Compared with metalaxyl and myclobutanil, thiogarcin has a better inhibitory effect on phytophthora pathogens overall. Therefore, thiogarcin has important potential in the field of green and environmentally friendly microbial pesticides for plant diseases caused by phytophthora.

[0043] Example 3: Determination of the antibacterial activity of thiocanthosides against Phytophthora nicotianae

[0044] The antibacterial activity of thiogarcinol against Phytophthora nicotianae was measured by the mycelial growth rate method, and the EC value of thiogarcinol for inhibiting the mycelial growth of Phytophthora nicotianae was calculated by the mycelial growth inhibition test. 50The thiocarbazine prepared in Example 2 was added to a V8 solid culture medium at a temperature of about 50°C-60°C, and the final concentration of thiocarbazine was set at 0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL and 2 μg / mL, a total of 5 gradient concentrations. Further, the mycelium growth inhibition test ( Figure 4 ), calculate the EC value of thiocanthosides to inhibit the growth of tobacco phytophthora mycelium 50 Through calculation, the regression equation of thiocanthosides toxicity to tobacco phytophthora is: y = 1.1912x + 5.0985 (r = 0.9953), EC 50 It is 0.8266μg / mL.

[0045] Example 4: Effects of Thiogarcinol on Physiological Indexes of Phytophthora nicotianae

[0046] 1. Effect of Thiocarpon on Mycelial Morphology

[0047] 1. Effect of Thiogarcinol on the Microstructure of Phytophthora nicotianae

[0048] Prepare V8 solid culture medium containing 1μg / mL and 2μg / mL thiolutein, respectively, and use 0.5% DMSO as a control, and spread sterilized cellophane on the V8 culture medium. Inoculate the tobacco blight cake into the center of the V8 culture medium plate covered with cellophane, and culture it in a constant temperature incubator at 28℃ for 3 days. Use scissors to cut the cellophane containing hyphae about 5mm×5mm where the colony grows on the edge of the cellophane, place it on a slide, and observe the hyphae morphology at the edge of the tobacco blight colony under a microscope. Figure 5 -A It can be seen that with the increase of thiolutein, the hyphae branches after treatment increased significantly and appeared in clusters.

[0049] 2. Effects of Thiogarcinol on the Ultrastructure of Phytophthora nicotianae

[0050] V8 solid culture medium containing 1μg / mL and 2μg / mL of thiogarcinia was prepared, and 0.5% DMSO was used as a control. The tobacco phytophthora cake was inoculated into the center of the V8 culture medium plate and cultured in a constant temperature incubator at 28℃ for 3d. Then, about 8mm×5mm×3mm fungus blocks were cut from the edge of the colony, and 3 fungus blocks were cut for each treatment. They were immediately put into pre-cooled 4% glutaraldehyde fixative and kept at 4℃ overnight for scanning electron microscopy and transmission electron microscopy sample preparation. By observing the scanning electron microscopy and transmission electron microscopy images of tobacco phytophthora, it can be seen that thiogarcinia significantly affects the ultrastructure of tobacco phytophthora hyphae.

[0051] Scanning electron microscope observation showed that ( Figure 5-B), the mycelium of the control group was normal in morphology, uniform in thickness, smooth and round, and grew well, while the mycelium treated with thiolutein was deformed, broken, and had small spikes. And with the increase of treatment concentration, the mycelium deformation became more serious. Observation under transmission electron microscope showed ( Figure 5 -C), the mycelial cytoplasm of the control group was uniform, the organelles were intact, and the mitochondrial morphology was normal. However, after the treatment with thiocarbazine, the cytoplasm was aggregated rather than evenly distributed, the organelles were damaged, the cell membrane and cell wall were damaged, the mitochondria ruptured, and the vacuoles of different sizes were fused. As the concentration of thiocarbazine treatment increased, the cell components were severely damaged and vacuolization intensified.

[0052] 3. Effect of Thiocarpon on the Integrity of Mycelial Cell Wall

[0053] Preparation of hyphae samples: 1.5 g of tobacco phytophthora hyphae were suspended in PBS buffer (pH = 7.2-7.4), and thiocarbazine was added to make the final concentrations of 1 μg / mL and 2 μg / mL, respectively, with 0.5% DMSO as a control, and cultured at 28°C and 180 rpm for 12 hours, and washed 3 times with PBS buffer to obtain hyphae samples treated with thiocarbazine. Mycelial samples in subsequent experiments were prepared according to this method.

[0054] The hyphae sample treated for 12 hours was picked up with tweezers and placed in 500 μL of 10% tannic acid aqueous solution, incubated in dark for 60 minutes, washed three times with PBS buffer, treated with 5% crystal violet aqueous solution for 5 minutes, washed three times with PBS buffer, treated with 5% Congo red aqueous solution for 2 minutes, rinsed with distilled water, placed on a slide, and photographed with a microscope. The results are shown in the figure. Figure 6 shown.

[0055] according to Figure 6 It can be seen that the cell wall structure of the mycelium in the control group is clearly visible, and very little cytoplasm is dark purple. As the concentration of thiocarbapenem treatment increases, the cell wall staining in the mycelium of the treatment group becomes seriously blurred, and the complete cell wall cannot be seen. Almost all mycelium is stained dark purple. This indicates that the thiocarbapenem treatment has caused damage to the cell wall of the mycelium of tobacco phytophthora.

[0056] 4. Effects on the integrity of mycelial cell membrane

[0057] The hyphae sample treated for 12 h was picked up with tweezers and placed in 500 μL of PI staining solution (40 μg / mL), incubated for 15 min in the dark, washed three times with PBS buffer, placed on a slide, and covered with a coverslip. The results were obtained under a laser scanning confocal microscope. Figure 7 shown.

[0058] according to Figure 7It can be seen that after 12 hours of mycelium treatment, almost no fluorescence was observed in the control group, while strong fluorescence could be detected in mycelium treated with thiogarcinol. The higher the concentration of thiogarcinol treatment, the stronger the fluorescence signal. This indicates that after thiogarcinol treatment of Phytophthora mycelium, mycelial cell viability decreased and cell membrane integrity was destroyed.

[0059] 5. Determination of reactive oxygen species (ROS) levels

[0060] The hyphae sample treated for 12 h was picked up with tweezers and placed in 500 μL H2DCF-DA (10 μM / L) staining solution, incubated for 30 min in the dark, washed three times with PBS buffer, placed on a slide, and covered with a coverslip. The results were obtained under a laser scanning confocal microscope. Figure 8 shown.

[0061] The main source of ROS is the mitochondrial respiratory chain. Excessive ROS can cause oxidative damage to important biological macromolecules in cells (such as proteins, nucleic acids, lipids, etc.), thereby affecting normal biochemical functions. Figure 8 The mycelium treated with thiocarbazone showed obvious green fluorescence under fluorescence, and the fluorescence intensity increased with the increase of concentration, while only a small amount of fluorescence was observed in the control group. The green fluorescence signal reflects the level of reactive oxygen species, and the stronger the fluorescence signal, the higher the level of reactive oxygen species. This shows that thiocarbazone can induce an increase in the level of ROS in the mycelium, and the degree of ROS increase is positively correlated with the concentration of thiocarbazone.

[0062] 2. Thiogarcinol in potted plants to prevent sisal zebra disease (Phytophthora nicotiana)

[0063] Millet was boiled with distilled water, filtered through gauze, air-dried to about 40% water content, and sterilized by high pressure (121°C, 20 min) in a conical flask. Phytophthora nicotianae was cultured on V8 medium for 4 days, and 5 cakes of Phytophthora nicotianae (5 mm in diameter) were inoculated on sterilized millet medium and cultured at 28°C for 14 days.

[0064] The test soil was purchased from Huizhou BiDa Landscape Materials Co., Ltd. Organic nutrient soil was mixed with tobacco phytophthora to obtain diseased soil, and 2 kg of soil was added to each pot (4 g of infected millet / 1 kg of soil). One-year-old sisal seedlings with consistent growth were selected and planted in 4 treatments (CK: not inoculated with tobacco phytophthora, sterile water; Pn: inoculated with tobacco phytophthora, sterile water; PnMe: inoculated with tobacco phytophthora, 1000 times of metalaxyl-M; PnTh: inoculated with tobacco phytophthora, 1000 times of thiocarbazin).

[0065] Each pot had one sisal plant, and each treatment had 10 plants, which were repeated three times. According to different treatments, 40 mL of treatment solution was added to each pot of sisal for root irrigation. The incidence of zebra stripe disease in sisal plants in each treatment was investigated 30 days after transplanting, and the disease index and disease prevention effect were calculated. The results of the potted experiment are shown in Table 3 and Fig. 9 shown.

[0066] Incidence rate (%) = (number of diseased plants / total number of plants) × 100%;

[0067] Disease prevention effect (%) = [(Pn disease index - PnMe or PnTh disease index) / Pn disease index] × 100%;

[0068] (N i : the number of leaves in the i-th disease level; i: disease level; M: the total number of leaves investigated)

[0069] Table 3. Thiocarpon sulphate control potted plant test efficacy data

[0070] Pot treatment Incidence (%) Disease index Disease prevention effect (%) CK 0 / / Pn 100 53.02 / PnMe 33.33 10.57 80.06 PcqI 26.67 7.53 85.81

[0071] The pot experiment confirmed that thiogarcinol has a significant control effect on sisal zebra stripe disease. As shown in Table 3, 30 days after treatment, the disease severity of thiogarcinol treatment was significantly reduced compared with the control group, the disease index was 7.53, and the control effect reached 85.81%. Compared with metalaxyl, thiogarcinol has a lower disease index and incidence rate in the control of sisal zebra stripe disease than metalaxyl. This shows that thiogarcinol is a biological pesticide with great potential for the control of plant phytophytophthora.

[0072] The above-described embodiments only illustrate several implementation methods of the present invention, and the descriptions thereof are relatively specific, but are only illustrative and not restrictive of the present invention. For those skilled in the art, many modifications, variations and improvements may be made without departing from the spirit and scope defined by the appended claims, and all of these belong to the protection scope of the present invention.

Claims

1. A method for extracting thiolutein, comprising the following steps: 1) inoculating Streptomyces ASG80 into ISP2 liquid culture medium to obtain seed liquid, and inoculating the obtained seed liquid into improved soybean powder infusion medium fermentation medium to ferment and obtain fermentation liquid; 2) extracting the fermentation liquid obtained in step 1) with ethyl acetate to obtain a fermentation extract; 3) dissolving the fermentation extract with 3 volumes of methanol and filtering through a Buchner funnel; the dissolved liquid is component A1, and the solid in the Buchner funnel is component A2; 4) separating component A2 by gel column chromatography and eluting with methanol solvent to obtain three components, which are respectively recorded as component A2-1, component A2-2, and component A2-3 in the order of elution of the components; 5) subjecting the A2-2 component to high performance liquid chromatography to obtain thiolutein; The Streptomyces ASG80 has been deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with the deposit number being CGMCC No.28698.

2. Use of thiolutein or the method according to claim 1 in preventing and controlling plant blight.

3. The use according to claim 2, characterized in that: The plant pathogenic phytophthora is at least one of tobacco phytophthora (Phytophthora nicotiana), camphor phytophthora (P. cinnamomi), palm phytophthora (P. palmivora), pepper phytophthora (P. capsici), cowpea phytophthora (P. vignae), melon phytophthora (P. melonis), taro phytophthora (P. colocasiae), and soybean phytophthora (P. sojae).

4. The use according to claim 2, characterized in that: It is applied to plants through root irrigation to achieve the purpose of preventing and controlling plant blight.

5. Application of thiolutein in changing the hyphae morphology of tobacco Phytophthora.

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