Method for improving quality of radix peucedani based on radix peucedani endophyte-restrictive penicillium

Abstract

The invention relates to the technical field of peucedanum praeruptorum planting, and discloses a method for improving the quality of peucedanum praeruptorum based on peucedanum praeruptorum endophyte-restrictive penicillium, which comprises the following steps: step 1, separating a sample plant with high abundance from the root of peucedanum praeruptorum, separating restrictive penicillium from the sample plant, performing metabonomics analysis on the restrictive penicillium, and culturing; and 2, preparing a bacterium solution of restrictive penicillium, and inoculating the bacterium solution back to the root of peucedanum praeruptorum by using a root irrigation method. In the step 1, endophyte-restrictive penicillium is obtained from the root of peucedanum praeruptorum. According to the method disclosed by the invention, various coumarin compounds can be generated by tieback of the bacterial liquid of the restrictive penicillium, and bolting and coumarin accumulation of peucedanum praeruptorum can be influenced after tieback. According to the method, restrictive penicillium has an inhibiting effect on bolting of peucedanum praeruptorum dunn and has a promoting effect on coumarin accumulation, the quality of peucedanum praeruptorum dunn can be improved by applying bacterial liquid, and good economic benefits are achieved.

Description

Technical Field

[0001] The invention relates to the technical field of peucedanum planting, and in particular to a method for improving the quality of peucedanum based on peucedanum endophyte-Penicillium restrictum. Background Art

[0002] Peucedanum peucedanum is used as medicine with its root, and its pharmacologically active ingredients are coumarin compounds. In recent years, the medicinal quality of Peucedanum peucedanum has been declining year by year, and the content of Peucedanum peucedanum index components on some medicinal material markets is lower than the requirements specified in the pharmacopoeia. And as the area of ​​Peucedanum peucedanum cultivation gradually expands, how to improve the coumarin content in Peucedanum peucedanum has become one of the key issues to be solved. Peucedanum peucedanum planted in the field generally bolts in 1-2 years, and its insufficient growth years lead to insufficient accumulation of medicinal ingredients. At present, a kind of endophytic fungus has not been found, which can be used as a bacterial fertilizer to inhibit the bolting of Peucedanum peucedanum, so as to promote the accumulation of its medicinal ingredients. For this reason, the present invention has been studied, in order to find a kind of endophytic fungus that can inhibit the bolting of Peucedanum peucedanum, and promote the accumulation of coumarin. Summary of the invention

[0003] In order to solve the technical problems raised in the background technology, the present invention provides a method for improving the quality of Peucedanum peucedanum based on the endophyte Peucedanum restrictum.

[0004] The present invention is implemented by the following technical scheme: a method for improving the quality of Peucedanum peucedanum based on the endophyte Peucedanum restrictum, comprising the following steps:

[0005] Step 1, separating a sample with high abundance from the root of Peucedanum peucedanum, isolating restrictive Penicillium from the sample, performing metabolomics analysis on restrictive Penicillium, and culturing;

[0006] Step 2: prepare a bacterial solution of Penicillium restrictum and inoculate the bacterial solution back into the roots of Peucedanum peucedanum using the root irrigation method.

[0007] As a further improvement of the above scheme, in step 1, the endophyte Penicillium restrictum obtained from the roots of Peucedanum peucedanum is subjected to ITS sequence amplification, sequencing and comparison with the control strain of the China Center for Type Culture Collection, and culture is performed if the similarity meets the requirements.

[0008] As a further improvement of the above scheme, the operation in step 2 is as follows:

[0009] First, the strain was cultured in PDA medium at 28°C without light for 7 days. After it filled the culture dish, the endophytic fungal blocks were picked out and cultured in a conical flask containing 300 ml of PDB medium at 28°C and 140 rpm in a shaking flask. The fermentation liquid and mycelium were then separated to obtain the mycelium, and then sterile water was added to prepare the bacterial solution.

[0010] As a further improvement of the above scheme, in step 2, the culture is carried out in a conical flask for 5-7 days.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The invention utilizes the inhibitory effect of Penicillium restrictum on the bolting of Peucedanum peucedanum and promotes the accumulation of coumarin, and finally improves the quality of Peucedanum peucedanum by applying bacterial liquid, thus having good economic benefits and being convenient for popularization and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the morphological observation of the mycelium after fermentation in the embodiment of the present invention;

[0014] Figure 2 : It is a treatment group arrangement diagram proposed in the embodiment of the present invention; wherein: single H: single bacteria high concentration area; single M: ​​single bacteria medium concentration area; single L: single bacteria low concentration area; mixed H: mixed bacteria high concentration area; mixed M: mixed bacteria medium concentration area; mixed L: mixed bacteria low concentration area; CK: sterile water control;

[0015] Figure 3 This is a statistical bar graph of bolting rates of Peucedanum pekinensis in different treatment groups in the embodiment of the present invention;

[0016] Figure 4 The coumarin fluorescence colorimetric diagram of the root sections of Peucedanum peucedanum in different treatment groups in the embodiment of the present invention at a mixed wavelength of 450-490nm and 460-500nm; wherein: Figure 4 (A) Part is the section of Peucedanum peucedanum without bolting in the single bacteria low concentration treatment group; Figure 4 (B) Part is the section of the non-bolting white flower peucedanum in the control group; Figure 4 (C) Part is the section of Peucedanum peucedanum after bolting in the single bacteria low concentration group; Figure 4 Part (D) is a section of Peucedanum peucedanum after bolting in the control group. DETAILED DESCRIPTION

[0017] The present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0018] Embodiment 1:

[0019] Referring to 1-4, the endophytic bacteria of Peucedanum peucedanum in this embodiment were separated from the root of Peucedanum peucedanum in the early stage, and the ITS sequence was amplified, sequenced and compared with the control strain of China Center for Type Culture Collection (CCTCC), and it had a high sequence similarity with Penicillium restrictum, and the accession number was AF033459.1, with a similarity of 99.82%. The isolated Penicillium restrictum was cultured with PDA medium (potato dextrose agar medium) and PDB medium (potato dextrose medium), respectively, and the formula of PDB medium was: 200g potato, 20g glucose, 1L sterilized deionized water. PDA medium was added with 15-20g agar in PDB medium.

[0020] First, the strain was placed in PDA medium at 28°C and cultured without light for 7 days. After it grew all over the culture dish, the endophytic fungal block was picked and cultured in a conical flask containing 300ml PDB medium at 28°C and 140rpm. A total of 40 bottles were fermented. Samples were taken every two days, 6 bottles were sampled each time, and the longest fermentation culture was 12 days. One bottle of fermentation liquid was observed for morphology (including mycelium size, number, fresh weight and dry weight, etc.), and the sample was photographed and retained for later observation of morphological changes. The other 5 bottles of fermentation liquid were filtered with 4 layers of gauze, the filtered fermentation liquid was centrifuged, the precipitate was discarded, the supernatant was taken, and it was quickly placed in liquid nitrogen for quenching for 30s, and then placed in a -80°C refrigerator for freezing. The filtered mycelium was collected, rinsed with ultrapure water in a Buchner funnel, and then filtered for 20min. After being completely drained, it was weighed, which was the fresh weight of the mycelium. The mycelium after filtration was collected and placed in a vacuum tube freeze dryer for freeze drying for 3 days, and then freeze-dried into powder and weighed dry.

[0021] Reference Figure 1 ,Results analysis: During the growth process, the hyphae gradually entangled with the shaking of the shaker, forming smaller hyphae balls. As time went by, the mycelium became more and more entangled, the shape of the mycelium became larger, the cell contents increased, the color gradually deepened and browned, and the formed hyphae balls continued to expand over time (such as Figure 1 ). The biomass of the mycelium was measured continuously from the second day to the 12th day of the shake flask culture. The results showed that the dry weight and fresh weight of the mycelium gradually increased with time. On the 12th day of culture, the fresh weight and dry weight reached the maximum value, which was about 7.3g / dw and 2.26g / dw, respectively.

[0022] The white peucedanum used in this experiment was planted in the Botanical Garden of Wanxi University. The local temperature is suitable, the light is sufficient, the cultivated land is flat, the soil fertility is uniform, and the drainage and irrigation are convenient, which is suitable for the growth of white peucedanum and can be used for later experiments. Fresh peucedanum samples were picked on October 28. Peucedanum variety: white peucedanum produced in Zhongxi, Ningguo

[0023] In this experiment, the land was prepared in the botanical garden of Wanxi University on February 20, 2023, and 21 experimental fields with a length of 3 meters and a width of 2 meters were designed. The seeds of Peucedanum were disinfected and pretreated before sowing. There are a total of 21 plots, with 100 Peucedanum plants in each plot, adjusted to the appropriate spacing. The experimental fields require daily management. Go to the field every week to observe the growth of Peucedanum. In spring and summer, weeds grow vigorously, and weed regularly every 3-4 days to prevent affecting the growth of Peucedanum. Around the end of March to the beginning of April, Peucedanum began to sprout one after another. Observe the growth of Peucedanum. When it grows to about 5 cm, replant it in time as needed

[64] . After it survives, it can be inoculated with fungi.

[0024] Treatment of restrictive Penicillium: After activation, transfer to PDB medium, place on a shaker at 28°C, 140rpm for 5-7 days, separate the fermentation liquid and mycelium to obtain mycelium. Add sterile water and resuspend to prepare three concentration gradient bacterial suspensions, dilute the high concentration group 20 times, the medium concentration group 40 times, the low concentration mycelium 60 times, and irrigate the control with the same amount of sterile water. The mixed bacteria were operated in the same manner as above. The 18 strains included Trichoderma harzianum, Helotialessp., Diaporthe unshiuensis, Didymella segeticola, Acrocalymmavagum, Acrocalymma vagum, Phialophora mustea, Phialophoramustea, Penicillium restrictum, Fusarium sp., Setophoma terrestris, Pleosporales sp., Clonostachys rossmaniae, Alternaria sp., Setophoma sp., Alternaria alternata, and Periconia macrospinosa. Previous studies have shown that all of these 18 strains can produce coumarins

[64] . These 18 fungi were mixed in a 1:1 ratio, and the bolting rate of each test plot was counted every 15 days until no new early bolts of Peucedanum were observed.

[0025] There are 7 treatments in this experiment:

[0026] Every half a month, three concentrations of Penicillium restrictum and mixed bacteria suspension were inoculated back to the roots of Peucedanum praeruptorum using root irrigation method. The control group was operated in the same way with sterile water, and the bolting rate was counted at the same time. After 8 treatments, samples were taken from Peucedanum praeruptorum;

[0027] Each treatment set up 7 plots, and the plot layout was as follows Figure 2 Shown

[0028] Note: Single H: single bacteria high concentration plot; Single M: ​​single bacteria medium concentration plot; Single L: single bacteria low concentration plot; Mixed H: mixed bacteria high concentration plot; Mixed M: mixed bacteria medium concentration plot; Mixed L: mixed bacteria low concentration plot; CK: sterile water control.

[0029] Coumarin fluorescence colorimetry

[0030] Root sections of Peucedanum peucedanum from the single-bacterium low-concentration group and the control group before and after bolting were taken for coumarin fluorescence colorimetry. The roots of the fresh samples were washed and dried, and the middle part was cut into tissues with a thickness of about 0.5 cm. After embedding with OTC embedding medium, the tissues were placed in a cryostat and pre-frozen at -20°C

[63] . After complete freezing, the sections were sliced ​​with a cryostat to a thickness of 20-30 μm and the fluorescence colorimetry was immediately observed under a fluorescence microscope at a mixed wavelength of 450-490 nm and 460-500 nm.

[0031] Effects of different concentrations of bacterial suspension on the content of peucedanum coumarin

[0032] (1) Preparation of reference solution

[0033] Take appropriate amount of peucedanum praeruptorin A, peucedanum praeruptorin B, xanthoxylum toxin, bergamot lactone, isoscopolamine, coumarin II, peucedanum praeruptorin E and scopoletin reference substances, add methanol to prepare peucedanum praeruptorin A at 23.2 μg / mL, isoscopolamine at 26.4 μg / mL, peucedanum praeruptorin B at 56.4 μg / mL, peucedanum praeruptorin E at 69.54 μg / mL, xanthoxylum toxin at 29.2 μg / mL, bergamot lactone at 65.2 μg / mL, scopoletin at 27.2 μg / mL, coumarin II at 38 μg / mL and isoscopolamine at 63.6 μg / mL respectively.

[0034] (2) Preparation of test solution

[0035] After the bolting rate of Peucedanum peucedanum was stabilized, samples of Peucedanum peucedanum before and after bolting were collected from each test plot on October 28, 2023, when the coumarin components reached the maximum accumulation value. Wash the soil, remove the roots, separate the roots, stems, leaves and flowers of the unbolted Peucedanum peucedanum and the bolted Peucedanum peucedanum, and then extract the effective ingredients: mark the tissue samples and dry them naturally in the shade, crush them through an 80-mesh sieve, weigh 0.2g in a stoppered conical bottle, accurately add 25mL of methanol, weigh the weight, extract with ultrasound (power 250W, frequency 33kHz) for 30min, cool, weigh again, add methanol to make up for the weight loss, shake well and filter, and take the filtrate as the test sample.

[0036] Liquid chromatography conditions

[0037] The chromatographic column model is AglientZORBAX RRHD Eclipse Plus 95AC18 (specific parameters: 100mm×2.1mm, 1.8μm); the mobile phase is water (A)-methanol (B), gradient elution: 0-2min: 5%; 2-5min: 5%~40%; 5-7min: 40%~45%; 7-7.5min: 45%~45.5%; 7.5-8min: 45.5%~46%; 8-9min: 46%~46.5%; 9-10min :46.5%~50.2%;10-10.5min:50.2%~50.3%;10.5-11min:50.3%~50.4%;11-17min:50.4%~55%;17-22min:55%~80%;22-25min:80%~100%;25-27min:100%;27-29min:100%~5%;29-30:5%. Flow rate is 0.25mL / min; column temperature is 30℃; detection wavelength is 321nm; injection volume is 2μL.

[0038] Results and Analysis

[0039] Effects of Differential Bacteria Inoculation on Bolting Rate of Peucedanum chinense

[0040] After the white flower peucedanum was treated with single bacteria and mixed bacteria for 8 times, the bolting rate was analyzed until it stabilized and no longer changed

[64] . The statistical results are shown in Table 2-2. The bolting rate was analyzed by single factor variance analysis

[64] using SPSS software. It was found that the bolting rate of the plots treated with single bacteria low concentration suspension was lower than that of the control plot and the plots treated with other concentrations; the medium and high concentration suspensions had no significant inhibitory effect on the bolting of peucedanum; the bolting rates of the mixed bacteria high, medium and low concentration groups were all higher than those of the control group;

[0041] Effects of Differential Bacteria Inoculation on the Accumulation of Peucedanum Coumarin

[0042] Coumarin fluorescence colorimetric experiments were performed on the root sections of Peucedanum peucedanum before and after bolting at 450–490 nm and 460–500 nm. Since visual comparison of the fluorescence intensity of the sections is highly subjective, the image color intensity parameters were unified, with the background value set to 0, the gamma value to 1.3, and the brightness to 150. Figure 2-3 It can be seen that the phloem and secretory ducts of the root section of Peucedanum leucoderma emit yellow-green fluorescence before bolting, and the fluorescence of the secretory duct is not obvious after bolting, and the xylem area increases. Comparing the single-bacteria low-concentration treatment group with the control group before bolting, it was found that the fluorescence intensity of the single-bacteria low-concentration treatment group was slightly higher than that of the control group, and the fluorescence of the secretory ducts of the treatment group and the control group after bolting was not obvious, and the xylem area was significantly larger than before bolting.

[0043] In this scheme, after the metabolomics analysis of the restricted Penicillium, it was diluted 20, 40, and 60 times with sterile water and inoculated into the Peucedanum peucedanum seedlings by the root irrigation method. The bolting rate was regularly counted during the growth of Peucedanum peucedanum. After inoculation 8 times, the white flower Peucedanum peucedanum plants were sampled before and after bolting in the low-concentration treatment group and the control group for root section coumarin fluorescence colorimetry and coumarin content determination, as well as transcriptome and metabolomics analysis. By analyzing the content differences, coumarin fluorescence colorimetry, differential metabolites and differential genes before and after bolting, the phenylalanine metabolic pathway related to coumarin synthesis was enriched:

[0044] Low concentration of restrictive Penicillium suspension can inhibit the bolting of Peucedanum chinense to a certain extent and promote the accumulation of coumarin compounds.

[0045] The present invention treats Peucedanum peucedanum with bacterial suspensions of high, medium and low concentrations of single bacteria and mixed bacteria, and finds that the single bacteria low concentration treatment group has an inhibitory effect on the bolting of Peucedanum peucedanum through bolting rate statistics. Through coumarin fluorescence colorimetric observation of root slices, it is found that the coumarin of Peucedanum peucedanum before bolting is mainly distributed in the secretory duct, while the fluorescence of the secretory duct weakens after bolting, and the coumarin fluorescence of the treatment group before bolting is slightly more obvious than that of the control group, indicating that the accumulation of coumarin before bolting is promoted, and there is no obvious difference after bolting.

[0046] The results showed that the inoculation of fungi had a certain effect on the bolting and coumarin components of Peucedanum peucedanum.

[0047] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A method for improving the quality of Peucedanum peucedanum based on the endophyte Peucedanum restrictum, characterized in that: The steps include: Step 1, separating a sample with high abundance from the root of Peucedanum peucedanum, isolating restrictive Penicillium from the sample, performing metabolomics analysis on restrictive Penicillium, and culturing; Step 2: prepare a bacterial solution of Penicillium restrictum and inoculate the bacterial solution back into the roots of Peucedanum peucedanum using the root irrigation method.

2. The method for improving the quality of Peucedanum peucedanum based on the endophyte Peucedanum restrictum as claimed in claim 1, characterized in that: In the step 1, the endophyte Penicillium restrictum obtained from the root of Peucedanum peucedanum is subjected to ITS sequence amplification, sequencing and comparison with the control strain of China Center for Type Culture Collection, and culture is performed if the similarity meets the requirement.

3. The method for improving the quality of Peucedanum peucedanum based on the endophyte Peucedanum restrictum as claimed in claim 1, characterized in that: The operations in step 2 are as follows: First, the strain was cultured in PDA medium at 28°C without light for 7 days. After it filled the culture dish, the endophytic fungal blocks were picked out and cultured in a conical flask containing 300 ml of PDB medium at 28°C and 140 rpm in a shaking flask. The fermentation liquid and mycelium were then separated to obtain the mycelium, and then sterile water was added to prepare the bacterial solution.

4. The method for improving the quality of Peucedanum peucedanum based on the endophyte Peucedanum restrictum as claimed in claim 3, characterized in that: In the step 2, the culture is carried out in a conical flask for 5-7 days.

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