CN113416654A - Crocus sativus endophytic fungus and application thereof - Google Patents

Abstract

The invention discloses saffron endophytic fungi and application thereof, and belongs to the technical field of yield increase of medicinal plants and quality improvement of medicinal materials. The endophytic fungi is obtained by separating healthy living bodies of Crocus sativus L of Crocus of Iridaceae by adopting a traditional microorganism separation and purification technology, is classified and named as Penicillium CS29(Penicillium sp.) and has a strain preservation number of CGMCC No. 20732. The invention also discloses the application of the endophytic fungi: promoting the growth of the bulb and stigma of saffron, respectively increasing the yield of bulb and stigma by 23.78% and 39.26%, and respectively increasing the content of medicinal active ingredients of safflorin I and safflorin II by 19.42% and 39.34%. The endophytic fungi has obvious effects of increasing the yield of saffron corms and medicinal materials and improving the quality of the medicinal materials, and brings wide application prospect for high-efficiency and high-quality saffron cultivation.

Description

Crocus sativus endophytic fungus and application thereof

Technical Field

The invention relates to the technical field of cultivation of high-quality and high-yield medicinal plants, in particular to saffron endophytic fungi and application thereof in increasing the yield and improving the quality of saffron.

Background

The medicinal material saffron is dry stigma of Crocus sativus L of Crocus of Iridaceae, has sweet taste and neutral nature, and has effects of invigorating heart and liver channel, promoting blood circulation for removing blood stasis, cooling blood for removing toxic substance, resolving stagnation and tranquilization, and can be used for treating amenorrhea and abdominal mass, puerperal blood stasis, toxic heat and macula, melancholy and stuffiness, palpitation and mania. The stigma is used as the medicine, so that the yield of the medicinal material is extremely low, the medicinal material is more famous and precious, and the saffron has better health care and medicinal values, is called as 'gold in plants' and has high economic value.

The major quality control index components in saffron are crocin I and crocin II, and are rich in flavonoids, terpenoids, organic acids and other compounds. In modern pharmacological research and clinical application, the method also receives considerable attention, such as obvious improvement effect on the intermittent rigidity, the tip expansion degree and the like of male genitals; can be used for improving menstrual disorder and depression caused by menstruation. At present, in agriculture, the saffron is cultivated by adopting bulb vegetative propagation mostly, and after cultivation for multiple generations, the bulbs are degraded, so that the yield is reduced, the content of stigma crocin is reduced, and the quality of medicinal materials is reduced. The saffron field management mostly adopts measures such as chemical fertilizer, crop rotation cultivation and the like, or adopts high-yield variety breeding and the like, but has the defects of soil hardening, environmental pollution, waste of land resources, long breeding period and the like.

Endophytic fungi (Endophytic fungi) refer to a class of fungi that live in plant cells or within plant tissues at a certain period of their life history without causing significant disease to the plant tissues. In a broad sense, these include saprophytic bacteria that grow on the surface at some stage in life history, latent pathogenic fungi and mycorrhizal fungi that are not temporarily harmful to the host. The endophytic fungi is a special biological group, has wide distribution and various types, and has extremely rich biological diversity from various aspects such as host plant types, self types, heredity, ecological environment and the like. The relationship between the endophytic fungi and the host plant is very complex, is different from the relationship of parasitism, pathogeny, saprophytic and the like, and is generally considered as a mutualistic symbiotic relationship, on one hand, the host provides sites and nutrients required by the growth of the endophytic fungi, and on the other hand, the endophytic fungi play a very important role in the growth development, secondary metabolism and system evolution process of the host. More and more researches show that the endophytic fungi can promote the growth and secondary metabolism of medicinal plants and are important microbial resources in the technical field of high-quality and high-yield medicinal plant cultivation.

However, the application of improving the yield of the bulb and stigma of saffron and improving the quality of medicinal materials by using the saffron endophytic fungi is not reported at present.

Disclosure of Invention

The invention aims to provide saffron endophytic fungi and application thereof aiming at the defects of the prior art.

In a first aspect of the invention, there is provided a medicinal plant endophytic fungus designated Penicillium sp CS 29. The endophytic fungi is obtained by separating from living bodies of Crocus sativus L. of Crocus of Iridaceae by adopting a traditional microbial separation and purification technology, and is identified as Penicillium fungus CS29(Penicillium sp.) by microbial classification. The strain is preserved, the preservation number of the strain is CGMCC No.20732, the preservation date is 9 months and 24 days in 2020, the preservation unit is China general microbiological culture Collection center (CGMCC), the address is No. 3 of Xilu No.1 of Beijing, Chaoyang, and the postal code is 100101.

The solid culture of the endophytic fungi is characterized in that:

culturing on potato glucose agar (PDA) at 26 deg.C in dark, with rapid growth, gray-green round colony, obvious powdery scatter, white edge, tooth-like shape, invisible radial pattern in the middle, and yellow-white back, as shown in FIG. 1.

An ITS sequence amplification electrophoresis chart of the endophytic fungi is shown in figure 2, and a base sequence is shown in SEQ ID NO. 1;

the sequencing results were aligned on the NCBI website (http:// blast. NCBI. nlm. nih. gov/blast. cgi) with 99.30% homology to Penicillium thomii (MH858993) and 99.29% homology to Penicillium spinulosum (FR670336) of Penicillium spinulosum. Thus, it was identified as a fungus (Penicillium sp.) belonging to the genus Penicillium. Multiple sequence alignments were performed using MEGA7.0, analyzed by the Neighbor-joining method (NJ), and the alignments were repeated 1000 times. A phylogenetic tree of the endophytes of the invention is shown in FIG. 3.

In a second aspect of the present invention, there is provided an application of the above mentioned endophytic fungi in increasing saffron yield and improving saffron quality.

The application specifically comprises that the CS29 fungal spore suspension is applied to effectively improve the yield of stigma croci Sativi corms and stigma, induce the accumulation of saffron secondary metabolic components and improve the content of crocin I and crocin II, the spore suspension is obtained by scraping living hyphae on a solid culture medium PDB culture medium and filtering with sterile gauze, and the formula of the solid culture medium is as follows: 200g of potato, 20g of glucose, 1000mL of distilled water and 15g of agar, and the pH value is natural.

The application is mainly realized by the following technical scheme:

(1) taking the endophytic fungi strain, picking a small amount of hyphae by using an aseptic inoculating needle under an aseptic condition, inoculating into a sterilized PDA culture medium, and carrying out dark culture for 5d under the conditions of 26 ℃, 65% humidity;

(2) under the aseptic condition, a proper amount of fungus cakes with the diameter of 5mm are punched by a sterilization puncher, the ratio of the number of the fungus cakes to the volume of PDB is 1:10 (one/mL), the fungus cakes are cultured in a constant-temperature shaking table in a shaking way at the culture temperature of 28 ℃, the rotating speed of 180rpm and the humidity of 65 percent for 5d, a sterilization gauze is adopted for filtration, a proper amount of fermentation liquor is absorbed for counting by a blood counting plate, PDB is added into a volumetric flask with the volume of 100mL, and the volumetric flask is prepared to have the final spore concentration of 10⁶Obtaining CS29 spore suspension liquid after each mL;

(3) applying stigma croci Sativi corm in spraying mannerUsing, 6mL/m²Spraying the crocus sativus extract for 1 time every 3 days for 90 days, so that the weights of the corms and stigma of the crocus sativus can be obviously increased, the yields of the corms and the stigma are respectively increased by 23.78% and 39.26% (figure 5), and the contents of medicinal active ingredients of crocin I and crocin II are respectively increased by 19.4% and 39.3% (figure 6).

The invention has the advantages that: the endophytic fungus CS29 of the invention can obviously increase the weight of saffron corms and stigma and improve the content of crocin I and crocin II by using the live spore suspension prepared by shake culture of a liquid culture medium. The endophytic fungus CS29 has great popularization and application values in the agricultural field for improving the saffron yield and the quality of medicinal materials.

Drawings

The invention is further explained below with reference to the figures and examples;

FIG. 1: colony morphology of Penicillium fungus CS29(Penicillium sp.) on PDA solid medium;

FIG. 2: an ITS sequence gel electrophoresis plot of CS29(Penicillium sp.) amplified based on ITS5 and ITS4 primers;

FIG. 3: an ITS sequence-based phylogenetic tree of the endophytic fungus CS29(Penicillium sp.) of Penicillium (NJ method);

FIG. 4: live spore suspensions prepared from endophytic fungi of the genus Penicillium CS29(Penicillium sp.);

FIG. 5: suspension of spores of endophytic fungus CS29(Penicillium sp.) of the genus Penicillium increases the weight of stigma and corm of saffron (II)^**P<0.01, CS 29-treated VS negative control group);

FIG. 6: penicillium endophytic fungus CS29(Penicillium sp.) increases the content of crocin I and crocin II in stigma croci (II)^**P<0.01, CS 29-treated VS negative control).

Detailed Description

The endophytic fungi of the invention is a strain separated from healthy living bodies of saffron produced in Shanghai Chongming.

Example 1:

the endophytic fungi is obtained by separation according to the following steps: taking the bulb of fresh saffron of Shanghai Chongming, washing under running water to remove impurities such as silt on the surface, and sucking water by using filter paper. And (3) carrying out three-step surface disinfection treatment after wrapping gauze: 1min of 75% ethanol solution, 3min of 2.5% sodium hypochlorite solution and 30s of 75% ethanol solution, then washing for 3-5 times by using sterile water, and sucking dry by using sterile filter paper. Tissue blocks of approximately 0.5cm by 0.5cm size were excised from the middle of the tissue and inoculated into a PDA sterile isolation dish containing sodium penicillin (50mg/L) in a formulation of 200g potato, 20g glucose, 1000mL distilled water, 15g agar, pH adjusted to 4 tissue blocks per dish. Sealing with sealing film, recording number, and culturing at room temperature for 7-12 d. The growth of colonies is regularly observed, and single-target colonies are screened according to the flora morphology, the color and the like. Carefully picking up hypha growing out from the edge of a tissue block under aseptic conditions, transferring the hypha to a freshly prepared PDA culture dish for secondary culture, numbering and recording, observing and screening again after the culture is finished, selecting a proper bacterial colony, taking the hypha at the edge of the bacterial colony to connect to a new PDA flat plate, continuously and repeatedly screening, picking and culturing to purify the bacterial strain, and finally obtaining the endophytic fungi strain CS29 of the single bacterial colony, as shown in figure 1, culturing on a potato glucose agar (PDA) medium at 26 ℃ in a dark mode, wherein the bacterial colony grows faster, is gray-green and round, is obviously powdery and scattered, has a circle of white edge, is slightly dentate, has a hidden visible radial pattern in the middle and is yellow and white at the back, and the bacterial strain is stored at low temperature (4 ℃).

Taking a freezing storage tube of CS29 strain, picking a little hypha with a sterile inoculating needle under the aseptic condition, putting the hypha into a newly prepared PDA culture medium for activation, carrying out light-shielding culture at 26 ℃ until the hypha grows to a full plate, picking a proper amount of hypha, putting the hypha into a 2mL sterile centrifuge tube, extracting the DNA of the strain according to a DNAiso kit method, and carrying out PCR amplification on ITS genes by using ITS5 and ITS 4. PCR reaction cycle parameter amplification step: initial denaturation at 1.95 ℃ for 3 min; denaturation at 2.94 ℃ for 40 s; annealing at 3.52 ℃ for 50 s; stretching at 4.70 deg.C for 1 min; 5.2-4 repeating the cycle 35 times; 6.72 ℃, 15min, expand. The PCR product detected by agarose gel electrophoresis is sent to Shanghai's chemical company for sequence determination, and the determined base sequence is shown as SEQ ID NO. 1. The sequence determined from the PCR product was used as a target sequence, the GenBank database in NCBI was searched for homologous sequences, a reference sequence most similar to the CS29 sequence was downloaded, and the phylogenetic status of the strain to be identified was determined using the Neighbor-joining (NJ) method for phylogenetic analysis, as shown in FIGS. 2 and 3. The endophytic fungus strain is classified and named as Penicillium CS29(Penicillium sp.) with the preservation number of CGMCC No. 20732.

Example 2:

taking a freezing storage tube of CS29 strain, picking the endophytic fungi strain by using an aseptic inoculating needle under the aseptic condition, inoculating the sterilized PDA culture medium under the aseptic condition, and carrying out dark culture for 5d under the conditions of 26 ℃ and 65% humidity; under the aseptic condition, beating the fungus cake with a sterilization puncher, wherein the diameter of the fungus cake is 5mm, the ratio of the number of the fungus cake to the volume of a PDB culture medium is 1:10 (one/mL), carrying out shake culture in a constant-temperature shaking table at the culture temperature of 28 ℃, the rotation speed of 180rpm and the humidity of 65%, carrying out culture for 5d, filtering by using a sterilization gauze, absorbing a proper amount of fermentation liquor, counting by using a blood counting plate, adding PDB into a volumetric flask with the constant volume of 100mL, and preparing into a volumetric flask with the final spore concentration of 10⁶cell/mL to obtain CS29 spore suspension, shown in figure 4; applying the harvested stigma croci Sativi corms on the shelf in a spraying manner, wherein the dosage is 6mL/m²Spraying the saffron fron at intervals of 3 days for 1 time, continuously planting for 90 days, and harvesting to obtain the saffron seed seedling raising agent capable of raising the weight of corm and stigma of saffron obviously by 23.78% and 39.26%, (^**P<0.01), see fig. 5.

Example 3:

according to the step of the example 2, after the stigma croci is blossomed, stigma croci is collected, dried to constant weight in a 45 ℃ oven, ground into powder and sieved by a 60-mesh sieve for later use. Taking 10mg of the powder, precisely measuring 20mL of 50% ethanol, performing ultrasonic extraction (power 250W and frequency 35kHz) for 30min at room temperature, filtering, and metering the volume of the filtrate to a 25mL volumetric flask by using 50% ethanol to obtain a sample solution. Taking a proper amount, filtering with a 0.45-micron microporous filter membrane to obtain a saffron sample solution to be detected, and determining the content of crocin I and crocin II by a High Performance Liquid Chromatography (HPLC) method, wherein the specific parameters are as follows: agilent 1100 high performance liquid chromatography system, ZORBAX SB-C18 column (250 mm. times.4.6 mm, 5 μm), mobile phase acetonitrile (A) -0.5% formic acid aqueous solution (B), column temperature 35 ℃. Detection wavelength of 440nm, flowThe speed was 1.0 mL/min. The crocin I and crocin II standard powder were dissolved in 50% ethanol to obtain crocin I (0.43, 0.5, 0.6, 0.75, 1.0, 1.5 and 3mg/mL) and crocin II (0.43, 0.5, 0.6, 0.75, 1.0, 1.5 and 3mg/mL) standard solutions, which were subjected to detection on a 0.45 μm microfiltration membrane under the above HPLC conditions to prepare a standard curve of crocin I and crocin II. The results were calculated from the established standard curve and expressed in mg/mL. We found that safflorin I and crocin II contents in stigma croci treated with endophytic fungus CS29 spore suspension were significantly increased (^**P<0.01), increased by 19.42% and 39.34%, respectively, as shown in fig. 6.

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.

Sequence listing

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Claims (8)

1. The saffron endophytic fungus is obtained by separating healthy living Crocus sativus L of Crocus of Iridaceae and is classified and named as Penicillium fungus CS29(Penicillium sp.) with the strain preservation number of CGMCC No. 20732.

2. Use of the saffron endophytic fungus of claim 1 for increasing saffron yield and improving saffron quality.

3. The use as claimed in claim 2, wherein the increase in saffron yield is achieved by increasing the weight of the bulb and stigma of saffron and increasing the content of the active pharmaceutical ingredients crocin i and ii in stigma of saffron.

4. The application according to claim 2, characterized in that it is specifically: spraying spore suspension of crocus sativus endophytic fungi on harvested crocus sativus corms.

5. Use according to claim 4, wherein the spore suspension is prepared from said endophyte in a PDB medium having the formula: 200g of potato, 20g of glucose and 1000mL of distilled water, and the pH value is natural.

6. Use according to claim 5, wherein the spore suspension is prepared according to the following steps:

(1) taking the saffron endophytic fungi strain, picking a proper amount of hypha by using a sterilization inoculating needle under the aseptic condition, inoculating the hypha into a sterilized PDA culture medium, and carrying out dark culture for 5d under the conditions of 26 ℃, 65% of humidity;

(2) under the aseptic condition, punching a proper amount of fungus cakes by using a sterilization puncher, wherein the diameter of the fungus cakes is 5mm, the ratio of the number of the fungus cakes to the volume of a PDB culture medium is 1: 10/mL, carrying out shake culture in a constant-temperature shaking table, wherein the culture temperature is 28 ℃, the rotation speed is 180rpm, and the humidity is 65%, and carrying out culture for 5 d;

(3) after the culture is finished, filtering by adopting sterile gauze in a sterile environment, sucking a proper amount of fermentation liquor to count a blood counting chamber, adding a sterile PDB culture medium to a constant volume of 100mL brown volumetric flask, wherein the final concentration of spores is 10⁶And (5) obtaining the spore suspension.

7. The use according to claim 6, wherein the spore suspension is sprayed at a concentration of 6mL/m²。

8. Use according to claim 7, wherein the spore suspension is sprayed under the conditions: spraying every 3d for 90d 1 time.

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