Metabolites of dragon fly intestinal fungus trichoderma QTYC44 and application

Abstract

The invention belongs to the technical field of microbiological engineering, and particularly relates to a compound extracted from trichoderma QTYC44 obtained from the intestinal tract of a dragon fly larva and a liquid fermentation product of the trichoderma QTYC44 as well as a preparation method and antibacterial activity application of the compound. Specifically, the invention discloses a polyketone compound. The compound can be used for inhibiting growth of rice bacterial xanthomonas oryzae, rice bacterial leaf streak, staphylococcus aureus, tetracoccus and kiwi fruit pathogenic bacteria, and can be applied to antibacterial agents.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a compound extracted from liquid fermentation product of Trichoderma QTYC44 obtained from the intestine of yellow dragonfly larvae, a preparation method thereof and antibacterial activity application thereof. Background Art

[0002] Plant pathogenic bacteria have become the second largest plant pathogen after fungi, causing significant crop yield reduction and economic losses every year. They are extremely harmful and difficult to control. For example, citrus canker, rice bacterial leaf blight and rice bacterial leaf streak are plant quarantine diseases in many countries, and their prevention, control or eradication are very difficult and thorny.

[0003] Long-term and continuous use of chemical agents has led to a series of problems such as resistance of plant pathogenic bacteria, excessive pesticide residues, and serious environmental pollution, which seriously affects the safety of agricultural products, ecological environment safety, and sustainable control of pests. Compared with chemical pesticides, biological pesticides are relatively safe, especially microbial pesticides, which are not easy to develop resistance and can take into account both disease prevention and control and green prevention and control.

[0004] Trichoderma fungi are a recognized class of biocontrol fungi with rich antimicrobial secondary metabolites. Therefore, it is of great significance to explore and discover agricultural antibiotics with application value from Trichoderma. Summary of the invention

[0005] The purpose of the present invention is to provide a Trichoderma polyketide compound obtained from the intestine of the yellow dragonfly and its preparation method and use - as an antibacterial agent to solve technical problems such as plant bacterial diseases.

[0006] In order to solve the above technical problems, the present invention provides a polyketide compound of Trichoderma chrysogenum, the structural formula of which is:

[0007]

[0008] The Trichoderma QTYC44 described in the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on June 17, 2014, and the strain deposit number is CCTCC M 2014267.

[0009] The present invention achieves the above object through the following technical solutions:

[0010] 1) Starve and disinfect the larvae of the tested yellow dragonfly, then dissect them and completely remove their intestines;

[0011] 2) Place the removed intestine in a mortar with 0.5 mL of sterile water and a few grains of quartz sand, and grind thoroughly to obtain dragonfly intestine grinding liquid;

[0012] 3) Dilute the dragonfly intestinal grinding solution into 10 -1 , 10 -2 , 10 -3 times, and 100 μL of intestinal grinding solution of each concentration was respectively applied evenly on PDA, MEA, and ISP2 plates containing 1% ampicillin and streptomycin, and cultured at 28°C for 14 days;

[0013] 4) During the culture period, observe the growth of the colonies on the plate every day. When colonies appear on the plate, pick the hyphae from the edge of the colony and transfer them to a new PDA medium for culture for 3-4 days until a single strain is obtained;

[0014] 5) Obtain Trichoderma QTYC44 through steps 1-4 and preserve it;

[0015] 6) Inoculate Trichoderma into PDA plate medium and culture in a constant temperature incubator at 28±0.5℃ for 3 days;

[0016] 7) Then take 3 pieces of mushroom cake and inoculate them into 100 mL of autoclaved liquid PDA medium at a temperature of 28 ± 0.5 °C and a rotation speed of 180

[0017] Cultivate on a shaker at rpm for 3 days to obtain seed solution;

[0018] 8) Transfer the seed solution to the fermentation medium at an inoculum rate of 10%-15%, and ferment and culture for 7 days under the shaking conditions in step 2;

[0019] 9) Filter the fermentation liquid obtained in step 8, extract the filtrate with ethyl acetate, and vacuum concentrate and dry to obtain a crude extract;

[0020] 10) Using the crude extract of step 9, a silica gel column was prepared with 10 times the mass of silica gel, and eluted with pure dichloromethane at the beginning, and then eluted with dichloromethane and methanol in a ratio of 100:1, 100:2, and 100:4 in sequence to obtain different elution fractions;

[0021] 11) drying the 100:0 eluted portion of step 10, separating it by gel column chromatography, using petroleum ether as eluent, and recrystallizing the obtained precipitate in petroleum ether to obtain the compound.

[0022] The present invention also provides the use of the above compound in an antibacterial agent, specifically: for inhibiting the growth of pathogenic bacteria, the bacteria are rice bacterial leaf blight fungus, rice bacterial leaf streak fungus, Staphylococcus aureus, Tetragenus coccus and kiwifruit pathogenic bacteria

[0023] The present invention has the following beneficial effects:

[0024] 1. The compounds of the present invention can inhibit a variety of pathogens and can be used as antibacterial agents or their precursors.

[0025] 2. The strain and its metabolites of the present invention can be preserved, and the sources are guaranteed.

[0026] 2. The compounds of the present invention can be produced by liquid fermentation using microorganisms, which has a simple process, short cycle and low cost.

[0027] 3. The present invention utilizes biological methods to synthesize compounds without causing pollution to the environment. DETAILED DESCRIPTION

[0028] The present invention will be further explained below in conjunction with specific implementation cases.

[0029] Example 1: Obtaining active strains from the intestines of yellow dragonfly larvae

[0030] Sterilize the starved dragonflies in a clean bench: soak in 75% alcohol for 3 minutes, wash with sterile water 3 times, and dry with sterile filter paper. Then dissect them, carefully cut open the dragonfly's abdomen with tweezers and a dissecting needle, and remove its intestines completely. Put the removed intestines in a mortar with 0.5 mL of sterile water, add a few quartz sands to help grind them thoroughly, and obtain the dragonfly intestine grinding liquid.

[0031] The dragonfly intestinal grinding solution was diluted to 10 -1 , 10 -2 , 10 -3 Times, respectively, draw 100 μL of intestinal grinding liquid of each concentration onto PDA, MEA, and ISP2 plates containing 1% ampicillin and streptomycin and spread evenly, let it stand until the grinding liquid is absorbed by the culture medium, repeat three times for each group, turn the plate upside down, and culture it at 28°C for 14 days. During the culture period, observe the growth of colonies on the plate every day. When colonies appear on the plate, pick the hyphae from the edge of the colony and transfer it to a new PDA culture medium for 3-4 days until a single strain is obtained. Finally, the isolated and purified single strain is stored on the PDA slope and in a 25% glycerol tube for subsequent research.

[0032] Example 2, liquid fermentation of QTYC44 Trichoderma

[0033] The QTYC44 Trichoderma strain (deposit number M2014267) was obtained from the China Type Culture Collection. The Trichoderma strain was inoculated on a PDA plate culture medium and cultured in a constant temperature incubator at 28±0.5°C for 3 days. For vial culture, three cakes were picked and inoculated into a 250mL conical flask (containing 150mL of liquid culture medium), and cultured at 28°C and 180r / min for 3 days as a seed solution. The seed solution was inoculated into a 1000mL conical flask (containing 400mL of liquid culture medium) at a volume ratio of 1:20.

[0034] Example 3: Extraction and separation of polyketide compounds from Trichoderma QTYC44

[0035] 32L of fermentation broth prepared in Example 2 was filtered through 4 layers of gauze to obtain a filtrate, and the filtrate was extracted several times with a medium-polarity organic solvent ethyl acetate in a 1:1 ratio until no extract was obtained in the organic phase, and all extracts were combined and concentrated under reduced pressure using a rotary evaporator to obtain 3g of a crude extract of the corresponding strain; the crude extract was mixed with an equal mass of 100-200 mesh silica gel, the sample was dried in an oven at 37°C, and further ground into a powder for standby use; a silica gel column was made using silica gel 10 times the mass of the crude extract, and eluted from low to high polarity using dichloromethane and methanol in different proportions as eluents. At the beginning, pure dichloromethane was used for elution, and then dichloromethane and methanol were eluted in a ratio of 100:1, 100:2, and 100:4 (the amount of each eluent was about 10 times the column volume). Finally, 0.3 g of the 100:0 elution portion was obtained. After drying, it was separated by gel column chromatography with petroleum ether as the eluent. The obtained precipitate was recrystallized in methanol to obtain 5 mg of polyketone compound. Its structural formula is:

[0036]

[0037] Example 4: Structural Identification of QTYC44 Trichoderma Polyketide Compounds

[0038] The structures of the polyketide compounds from Trichoderma QTYC44 were determined based on analysis of their properties, mass spectrometry, and nuclear magnetic resonance spectroscopy data.

[0039] The properties and spectral data are as follows:

[0040] Yellow powder. 1 H NMR (600MHz, CDCl3), δ: 7.32 (t, J = 12.8Hz, 1H), 6.38 (d, J = 14.5Hz, 1H), 6.31 (t, J = 13.6 Hz,1H),6.14(dd,J=14.5,6.3Hz,1H),5.50(d,J=6.0Hz,1H),5.46(s,1H),4.40(s,1H),4 .09(s,1H),3.78(s,1H),3.06(m,1H),2.90(m,1H),2.35(m,1H),2.25(m,1H),1.88(d,J =6.9Hz,3H),1.64(d,J=6.3Hz,3H),1.51(s,3H),1.47(s,3H),1.45(s,3H),1.37(s,3H). 13C NMR (150MHz,CDCl3),δ:202.9,197.4,196.2,191.3,170.6,163.6,140.0,137.8,131.2,129.3,126.5,120.1,110.7,108.4,104.2,99.7,79.8,78.6,58.8,54.3,38.9,28.1,25.8,23.2,18.9,18.5,18.0,7.1. The compound was identified as dihydrobisvertinolone.

[0041] Example 5: Inhibitory effect of QTYC44 Trichoderma polyketide compounds on pathogenic bacteria

[0042] The antibacterial activity of QTYC44 Trichoderma polyketide compounds was tested by the filter paper diffusion method. The test bacteria were inoculated in LB liquid medium and activated and cultured at 37°C for 24 hours. 1 mL of sterile water was taken in a 1.5 mL centrifuge tube, and a small amount of bacteria was collected with an inoculation loop and mixed evenly in sterile water to make a bacterial suspension. The concentration of the bacterial suspension was adjusted to about 10% by comparing with the turbidity tube. 6 cFU / mL. The compound, negative control (solvent acetone) and positive control (gentamicin) were prepared into a 6 mg / mL solution and sterilized by filtering with a 0.22 μm filter membrane. Then 5 μL of the solution was dripped onto a 5 mm sterile filter paper. After the solvent evaporated, the filter paper containing the drug was attached to the bacteria-containing plate. The filter paper was 2 cm away from the edge of the plate. Three filter papers were attached to each plate as parallel. The plate was inverted and cultured at 37 ° C for 24 hours. The diameter of the inhibition zone was measured using the "cross method".

[0043] The growth inhibition effect of the compound on pathogenic bacteria is shown in Table 1 below. From the data in the table, it can be seen that when the test concentration is 30 μg / filter paper, compared with the positive control gentamicin sulfate, the compound has a better inhibitory effect on rice bacterial leaf blight fungus, with an inhibition zone diameter of 13.8 mm; it has a moderate inhibitory effect on rice bacterial leaf streak fungus, Staphylococcus aureus, Tetracoccus, and kiwifruit pathogens, with inhibition zone diameters of 9.3 mm, 9.0 mm, 15.3 mm, and 12.7 mm, respectively.

[0044] Table 1. Inhibitory effect of QTYC44 Trichoderma polyketide compounds on the growth of pathogenic bacteria (mm)

[0045]

[0046] Note: The results are expressed as the standard of the diameter of the inhibition zone ± 3 parallel measurements; gentamicin sulfate is the positive control for pathogenic bacteria; NI means no activity; the test concentration is 30 μg / filter paper.

Claims

1. A metabolite and application of the intestinal fungus Trichoderma QTYC44 of yellow dragonfly larvae, characterized in that Provided are a trichoderma polyketide compound obtained from the intestine of a yellow dragonfly, a preparation method thereof and a use thereof - as an antibacterial agent.

2. The method for preparing symbiotic bacteria from the intestine of a yellow dragonfly as claimed in claim 1, characterized by the following steps: 1) Starve and disinfect the larvae of the tested yellow dragonfly, then dissect them and completely remove their intestines; 2) Place the removed intestine in a mortar with 0.5 mL of sterile water and a few grains of quartz sand, and grind thoroughly to obtain dragonfly intestine grinding liquid; 3) Dilute the dragonfly intestinal grinding solution into 10 -1 , 10 -2 , 10 -3 times, and 100 μL of intestinal grinding solution of each concentration was respectively applied evenly on PDA, MEA, and ISP2 plates containing 1% ampicillin and streptomycin, and cultured at 28°C for 14 days; 4) During the culture period, observe the growth of colonies on the plate every day. When colonies appear on the plate, pick up mycelium from the edge of the colony and transfer it to a new PDA medium for culture for 3-4 days until a single strain is obtained.

3. The method for preparing the Trichoderma compound according to claim 1, It is characterized by the following steps: 1) The Trichoderma sp. QTYC44 described in the present invention was deposited in China Center for Type Culture Collection (CCTCC) on June 17, 2014, and the strain deposit number is CCTCC M 2014267. The Trichoderma sp. QTYC44 was inoculated in a PDA plate medium and cultured in a constant temperature incubator at 28±0.5°C for 3 days; 2) Then take 3 pieces of mushroom cake and inoculate them into 100mL of autoclaved liquid PDA medium at a temperature of 28±0.5℃ and a speed of 180 Cultivate on a shaker at rpm for 3 days to obtain seed solution; 3) Transfer the seed solution to the fermentation medium at an inoculum rate of 10%-15%, and ferment and culture for 7 days under the shaking conditions in step 2; 4) Filter the fermentation liquid obtained in step 3, extract the filtrate with ethyl acetate, and vacuum concentrate and dry to obtain a crude extract; 5) Using the crude extract of step 4, a silica gel column was prepared with 10 times the mass of silica gel, and eluted with pure dichloromethane at the beginning, and then eluted with dichloromethane and methanol ratios of 100:1, 100:2, and 100:4 in sequence to obtain different elution fractions; 6) drying the 100:0 eluted portion of step 5, separating it by gel column chromatography, using petroleum ether as eluent, and recrystallizing the obtained precipitate in petroleum ether; The compound is obtained.

4. The polyketide compound of the intestinal fungus Trichoderma QTYC44 of the yellow dragonfly larvae according to claim 1 has antagonistic effects on rice bacterial leaf blight, rice bacterial leaf streak, Staphylococcus aureus, Tetragenococcus and kiwifruit pathogens, and can be used to develop fungicides.

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