Polyketone metabolite of trichoderma QTYC44 and application

Abstract

The invention belongs to the technical field of microbial engineering, and particularly relates to a polyketone compound extracted from a Trichoderma sp.QTYC44 liquid fermentation product as well as a preparation method and application of the polyketone compound. Specifically, the invention discloses a polyketone compound, the chemical structural formula of the polyketone compound is # imgabs0, and the polyketone compound can be used for inhibiting the growth of rice bacterial streak pathogen, staphylococcus aureus, tetracoccus and kiwi fruit canker pathogen.

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 sp. QTYC44, a preparation method thereof and an application of its antibacterial activity. Background Art

[0002] Plant pathogens can cause serious diseases in crops, ranging from mild symptoms to large areas of crops being destroyed, thus reducing yields. Taking bacterial diseases in agricultural production as an example, it has now developed into the second largest disease after fungi, and it occurs in almost every crop, usually causing a 20%-30% reduction in crop yields. According to incomplete statistics, the current area of ​​bacterial diseases in my country is 120 million mu. In particular, bacterial diseases represented by citrus canker, rice streak disease, and rice bacterial leaf blight are becoming more and more serious, causing huge yield losses of 40%-60% in agricultural production.

[0003] Reliance on chemical pesticides has caused high pesticide residues in crops, which has become a global problem. Compared with chemical pesticides, biological pesticides are relatively safe, among which microbial pesticides are highly selective, not prone to resistance, and green and pollution-free. They are very safe for crops, which not only meets people's demand for green food, but also provides a guarantee for the sustainable development of agriculture.

[0004] The active metabolites produced by fungi have special and diverse chemical structures and can be used as a new resource for finding new antimicrobial active substances. Therefore, studying fungi and their secondary metabolites is of great significance for the development and discovery of new biofungicides. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a Trichoderma polyketide compound and a preparation method and use thereof - as an antibacterial agent.

[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 sp. 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 CCTCCM2014267.

[0009] The present invention also provides a method for preparing the above-mentioned Trichoderma polyketide compound, comprising the following steps:

[0010] 1) Inoculate Trichoderma into PDA plate medium and culture in a constant temperature incubator at 28±0.5℃ for 3 days;

[0011] 2) Then, the fresh mycelium was inoculated into 100 mL of high-pressure sterilized liquid PDA medium, and cultured on a shaker at a temperature of 28±0.5°C and a speed of 180 rpm for 3 days to obtain seed liquid;

[0012] 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 1;

[0013] 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;

[0014] 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;

[0015] 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 to obtain a polyketone compound.

[0016] The present invention also provides the use of the polyketide compound in an antibacterial agent, specifically: for inhibiting the growth of pathogenic bacteria, wherein the bacteria are rice bacterial leaf streak bacteria, Staphylococcus aureus, Tetragenococcus and kiwifruit canker bacteria.

[0017] The present invention has the following beneficial effects:

[0018] 1. The compounds of the present invention have inhibitory effects on a variety of pathogenic bacteria and can be used as antibacterial agents or their precursors.

[0019] 2. The compounds of the present invention can be produced by liquid fermentation using microorganisms, which has a simple process, a short cycle, low cost and a guaranteed source.

[0020] 3. The present invention utilizes biological methods to synthesize compounds without causing pollution to the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0022] Figure 1 is the growth of QTYC-44 colonies on solid PDA medium;

[0023] Figure 2This is a graph showing the inhibitory effect of the compound Bisvertinolone on five pathogenic bacteria (a, b, c, d, and e are Escherichia coli, Staphylococcus aureus, kiwifruit canker bacteria, Tetragenococcus, and rice bacterial leaf streak bacteria, respectively)

[0024] Figure 3 This is the inhibitory effect of the positive control gentamicin sulfate on five pathogenic bacteria (a, b, c, d, and e are Escherichia coli, Staphylococcus aureus, kiwifruit canker bacteria, Tetragenococcus, and rice bacterial leaf streak bacteria, respectively) DETAILED DESCRIPTION

[0025] The present invention will be further explained below in conjunction with specific implementation cases.

[0026] Example 1, liquid fermentation of QTYC44 Trichoderma

[0027] The Trichoderma strain Trichoderma sp. QTYC44 (deposit number M2014267) was obtained from the China Type Culture Collection Administration Center. The Trichoderma strain was inoculated into a PDA plate culture medium and cultured in a constant temperature incubator at 28±0.5°C for 3 days; then, fresh mycelium was inoculated into 100 mL of high-pressure sterilized liquid PDA culture medium, and cultured on a shaker at a temperature of 28±0.5°C and a rotation speed of 180 rpm for 3 days to obtain a seed solution; the seed solution was transferred to a fermentation medium at an inoculation rate of 10%-15%, and cultured on a shaker at a temperature of 28±0.5°C and a rotation speed of 180 rpm for 7 days.

[0028] Example 2: Extraction and separation of polyketide compounds from Trichoderma QTYC44

[0029] 32L of fermentation broth prepared in Example 1 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 4g 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 ratios of 100:1, 100:2, and 100:4 were used in sequence (the amount of each eluent was about 10 times the column volume). Finally, 1.2 g of the 100:0 elution portion was obtained. After drying, it was separated by gel column chromatography using petroleum ether as the eluent. The resulting precipitate was recrystallized in methanol to obtain 45 mg of a polyketide compound. Its structural formula is:

[0030]

[0031] Example 3: Structural Identification of Polyketide Compounds from Trichoderma QTYC44

[0032] 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.

[0033] The properties and spectral data are as follows:

[0034] Orange-yellow paste, soluble in dichloromethane. 13 CNMR(150MHz,cdcl3)δ199.96,196.59,191.37,185.89,170.23,164.22,148.37,144.11,139.66,137.54,131.36,131.15 ,122.12,120.21,110.93,107.37,104.19,100.02,79.99,79.44,60.00,54.62,25.78,23.03,19.19,18.80,18.78,7.06. 1 HNMR(600MHz,cdcl3)δ7.55(dd,J=15.2,9.2Hz,1H),7.39(d,J=15.0Hz,1H),7.30(d d,J=14.9,10.8Hz,1H),6.40(d,J=14.8Hz,1H),6.32(d,J=8.2Hz,2H),6.27(d,J=11 .8Hz,1H),6.10(dd,J=14.8,7.2Hz,1H),3.78(s,1H),2.02(s,1H),1.88(d,J=4.9Hz,3H),1.85(d,J=6.8Hz,4H),1.49–1.43(m,10H),1.40(s,3H),1.23(t,J=7.0Hz,1H). The compound was identified as Bisvertinolone.

[0035] Example 4: Inhibitory effect of QTYC44 Trichoderma polyketide compounds on pathogenic bacteria

[0036] The antibacterial activity of QTYC44 Trichoderma polyketide compounds was tested by the filter paper diffusion method. The compound was dissolved in acetone and prepared to a concentration of 30 μg / mL. Pure acetone and gentamicin sulfate were used as blank and positive controls. The test bacteria were inoculated into LB liquid culture medium and then cultured in a constant temperature shaker at 37°C for 24 hours for activation. Take 1 mL of sterile water in a 1.5 mL centrifuge tube, use an inoculation loop to collect a small amount of bacteria in sterile water and mix evenly to make a bacterial suspension, and adjust the concentration of the bacterial suspension to about 10 by comparing with the turbidity tube. 6cFU / mL. Use a 6mm hole puncher to punch the filter paper into several filter paper discs for sterilization. Use a pipette to take 100μL of bacterial suspension and add it to 100mL50-60℃ LB liquid culture medium, mix evenly and pour on a plate. After the culture medium solidifies, take 5μL of crude extract and drop it on a 6mm filter paper disc. Use tweezers to carefully stick the filter paper disc on the culture medium, and culture at 37℃ for 24-36h. Repeat three times for each group, and use the cross method to determine the diameter of the inhibition zone.

[0037] The growth inhibition effect of polyketide compounds 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, polyketide compounds have a better inhibitory effect on rice bacterial leaf spot pathogens, with an inhibition zone diameter of 14.3 mm; they have a moderate inhibitory effect on Staphylococcus aureus, Tetragenococcus, and kiwifruit canker bacteria, with inhibition zone diameters of 9.0 mm, 15.0 mm, and 8.5 mm, respectively.

[0038] Table 1. Inhibitory effect of polyketide compounds of Trichoderma sp. QTYC44 on the growth of pathogenic bacteria (mm)

[0039]

[0040] Note: The results are expressed as the standard of inhibition zone diameter ± 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. Polyketide compounds of Trichoderma sp. QTYC44, characterized by The structural formula is:

2. The method for preparing a Trichoderma compound as claimed in claim 1, characterized in that The steps include: 1) The Trichoderma sp. QTYC44 of 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 QTYC44 Trichoderma sp. was inoculated in a PDA plate medium and cultured in a constant temperature incubator at 28±0.5°C for 3 days; 2) Then, the fresh mycelium was inoculated into 100 mL of high-pressure sterilized liquid PDA medium, and cultured on a shaker at a temperature of 28±0.5°C and a speed of 180 rpm for 3 days to obtain seed liquid; 3) Transfer the seed solution to the fermentation medium at an inoculum rate of 10% to 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 to obtain a polyketone compound.

3. The polyketide compound of QTYC44 Trichoderma according to claim 1 has antagonistic effects on rice bacterial leaf streak pathogen, Staphylococcus aureus, Tetragenococcus and kiwifruit canker pathogen during preparation, and can be used to develop fungicides.

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