Screening and cultivating method of mutagenic bolete strain

Abstract

The invention discloses a method for screening and cultivating a bolete mutant strain, which belongs to the field of microorganisms, and mainly comprises the following steps: obtaining the bolete mutant strain by using a carbon ion beam irradiation technology, naming the bolete mutant strain as ng2, and preserving the bolete mutant strain with the preservation number of CCTCC (China Center for Type Culture Collection) M 2025077; inoculating the ng2 strain into a hypha rapid germination and propagation solid culture medium PDA or PDA + peptone + beef extract + yeast extract powder, wherein hypha begins to germinate after 4 days; the method comprises the following steps: inoculating a solid strain into a hypha rapid propagation liquid culture medium including cane sugar, corn flour, soybean flour, wheat bran, monopotassium phosphate and yeast extract powder, and generating a large amount of fungus balls within 7 days; inoculating a liquid strain into a fruiting culture raw material self-made fermentation material, corn straw, turfy soil and rice, wherein hyphae overgrow in 30 days; the ng2 strain is high in activity, high in growth speed and suitable for being used as an excellent bolete strain in the Heilongjiang region; the screening and cultivating method of the bolete mutant strain is mainly used for solving the problems that the propagation efficiency of the bolete strain is low, germplasm resources in northern areas such as Heilongjiang are deficient and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and more particularly, to a method for screening and cultivating mutagenized strains of Boletus edulis. Background Art

[0002] Boletus edulis is a general term for fungi such as Boletaceae and Strobilomyces spp. It is named after its thick and fleshy texture, which closely resembles that of a bovine liver. There are mainly white, yellow, black and other varieties. Except for a few varieties that are poisonous or bitter and inedible, most varieties are edible. Data shows that every 100 g of dried Boletus edulis contains 20.2 g of protein, 64.2 g of carbohydrates, 338 kcal of calories, 4.0 g of ash, 23 mg of Ca, 500 mg of P, 50 mg of Fe, and 3.68 mg of riboflavin. In addition to its edible value, Boletus edulis also has unique medicinal value. Research has proven that this fungus has the effects of clearing heat and relieving restlessness, nourishing blood and regulating the middle energizer, dispelling wind and cold, relaxing tendons and activating blood circulation, tonifying deficiency and refreshing the mind, etc. It is one of the raw materials for the Chinese patent medicine "Shujin Pills" and is also a good gynecological medicine, which can treat women's leucorrhea and infertility. In addition, the water extract of Boletus edulis has an inhibition rate of 100% against the sarcoma S-180 of mice and an inhibition rate of 90% against Ehrlich ascites carcinoma. It also has the effects of anti-influenza virus and preventing and treating colds.

[0003] The edible and medicinal values of Boletus edulis endow it with an indispensable position in edible and medicinal fungi. However, the quantity of wild resources in places such as Yunnan cannot meet the market's annual demand for consumers. So far, only Phlebopus portentosus can be artificially domesticated among Boletus edulis species. Only a very few enterprises such as Hongzhen in Jinghong can produce it on an industrial scale. There are many research institutions in the northern regions, but they have not overcome the bottleneck problem of efficient propagation of liquid strains. It can be seen that the artificial domestication of Boletus edulis is extremely difficult.

[0004] Through a large number of screenings, separations, purifications and mutageneses, the present invention finally obtained the excellent mutagenized strain ng2 of Phlebopus portentosus. The ng2 mycelium has strong vitality, fast growth rate and short propagation cycle. The present invention uses the solid-liquid mixed alternating amplification method to solve the problems of long propagation time of solid strains and difficult proliferation of liquid strains, shortening the propagation cycle of the mutagenized strain from 30 days to 17 days compared with before mutagenesis. The successful domestication of Phlebopus portentosus in northern regions such as Heilongjiang meets the market's demand for high-end edible and medicinal fungi, fills the blank of successful artificial domestication of wild Boletus edulis in northern regions such as Heilongjiang, and adds valuable germplasm resources of rare edible and medicinal fungi to northern regions such as Heilongjiang Province. Summary of the Invention

[0005] The technical problems to be solved by the present invention are as follows: In the prior art, there is a lack of Boletus varieties suitable for cultivation in northern regions such as Heilongjiang. There are often problems such as low success rate of tissue separation induction, slow propagation of mycelium on solid medium, non-proliferation of liquid spawn, and inappropriate cultivation raw material formula. The present invention provides a mutagenized Boletus strain, which has strong mycelial growth potential, fast growth rate, and short propagation cycle, and is suitable as an excellent Boletus strain in northern regions such as Heilongjiang.

[0006] Specifically, the present invention is achieved through the following technical solutions: In the first aspect, the present invention provides the screening of a mutagenized Boletus ng2 strain.

[0007] As an optional method, during the cultivation of the above mutagenized strain, the mutagenesis method is carbon ion beam irradiation at 80 MeV / u.

[0008] As an optional method, during the cultivation of the above mutagenized strain, the preservation number of Boletus ng2 (Boletus sp. ng2) is CCTCC M 2025077, and it was preserved at the China Center for Type Culture Collection on January 10, 2025, with the preservation address being Wuhan University, China.

[0009] In the second aspect, the present invention provides a cultivation method for the ng2 strain.

[0010] As an optional method, during the cultivation of the above ng2 strain, the solid medium formula for rapid mycelial germination and propagation is potato dextrose agar medium (PDA), PDA + peptone, PDA + beef extract, PDA + yeast powder, PDA + peptone + beef extract, PDA + peptone + yeast powder, PDA + beef extract + yeast powder, PDA + peptone + beef extract + yeast powder, and the temperature is 28°C.

[0011] Preferably, in the above formula, the solid medium formula for rapid mycelial germination is PDA or PDA + peptone + beef extract + yeast powder, and the temperature is 28°C.

[0012] As an optional method, during the cultivation of the above ng2 strain, the liquid medium formula for rapid mycelial proliferation is sucrose + potato, sucrose + corn flour, sucrose + corn flour + soybean powder, sucrose + corn flour + soybean powder + wheat bran, sucrose + corn flour + soybean powder + wheat bran + potassium dihydrogen phosphate, sucrose + corn flour + soybean powder + wheat bran + potassium dihydrogen phosphate + yeast extract powder, and the temperature is 28°C.

[0013] Preferably, in the above formula, the liquid medium formula for rapid mycelial propagation is sucrose + corn flour + soybean powder + wheat bran + potassium dihydrogen phosphate + yeast extract powder, and the temperature is 28°C.

[0014] As an alternative, the mushroom-growing culture medium is composed of crushed wood chips + corn straw, crushed wood chips + peat soil + rice, crushed wood chips + corn straw + peat soil, crushed wood chips + corn straw + peat soil + rice, self-made fermented material + corn straw, self-made fermented material + peat soil + rice, self-made fermented material + corn straw + peat soil, and self-made fermented material + corn straw + peat soil + rice.

[0015] Preferably, in the above formula, the formula of the mushroom-growing culture medium is self-made fermented material + corn straw + peat soil + rice.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention obtains the boletus strain ng2 through the carbon ion beam irradiation technology. This mutagenesis technology is applied for the first time in the mutagenesis of edible and medicinal fungi and has achieved excellent results.

[0017] The ng2 strain of the present invention has strong growth potential and fast color transformation on the selected solid culture medium.

[0018] The ng2 strain of the present invention has fast proliferation and uniform mycelial pellets in the selected liquid culture medium.

[0019] The present invention uses alternating cultivation in solid and liquid culture media to solve the problems of long solid spawn propagation time and difficult proliferation of liquid spawn, shortening the propagation cycle of the mutagenized strain from 30 days to 17 days compared with that before mutagenesis.

[0020] The present invention simplifies the formula of the mushroom-growing culture medium, making it easier for the mycelium of boletus to propagate in rice and black soil. Description of the Drawings

[0021] The drawings described herein are used to provide a further understanding of the present application and form a part of the present application. The schematic embodiments and descriptions thereof are used to explain the present application and do not constitute an improper limitation to the present application. In the drawings: Figure 1 shows the antagonism test between the mutagenized strain ng2 and the control implemented in the present invention; Figure 2 shows the ITS detection result of the mutagenized strain ng2 implemented in the present invention; Figure 3 shows the liquid spawn of the mutagenized strain ng2 implemented in the present invention. Detailed Embodiments

[0022] To enable those skilled in the art to better understand the solution of the present invention, the exemplary embodiments or examples of the present invention will be described below in conjunction with the accompanying drawings. Obviously, the described embodiments or examples are only a part of the embodiments or examples of the present invention, rather than all of them. All other embodiments or examples obtained by those of ordinary skill in the art based on the embodiments or examples of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0023] To make the above objects, features and advantages of the present invention more obvious and understandable, the following will describe the specific embodiments of the present invention in detail with reference to the drawings. Embodiment

[0024] The collected bolete resources were subjected to mother culture isolation and purification, and experiments such as mycelium induction and propagation were completed in the laboratory of the Harbin Academy of Agricultural Sciences.

[0025] 1.1 Mutagenesis treatment of the starting strain (1) Tissue isolation of the collected bolete resources was carried out in a laminar flow hood. Take a tissue of 0.5 cm × 0.5 cm × 0.5 cm at the junction of the cap and the stipe, inoculate it into the PDA medium, and incubate it in the dark at 28 °C. After 50 - 60 days of incubation, a small amount of induced mycelium together with the tissue block and a little solid medium were transferred into the potato sucrose liquid medium, and cultured for proliferation at 28 °C and 150 rpm. The mycelial balls were collected, centrifuged at 12000 rpm to remove the supernatant, and the mycelial balls were added again and centrifuged at 12000 rpm. This process was repeated 3 - 4 times, and after suspension, it was diluted into a bacterial suspension with a concentration of 10 -5 as the starting strain and sent to Harbin Institute of Technology for mutagenesis treatment.

[0026] (2) The carbon ion beam 80Mev / u 12 C 6+ was used for irradiation in this experiment.

[0027] 1.2 Screening of mutagenized strains The mutagenized strains were numbered ng1, ng2, ng3......ng6 respectively, and spread on the PDA medium with a spreading rod and incubated in the dark at 28 °C. The control was the strain before mutagenesis, and it was cultured under the same conditions. After 30 days, it was found that the ng2 strain germinated earliest and started to turn color after 50 days. Taking the number of germinated mycelial balls as a parameter, the 6 mutagenized strains and the control strain were statistically analyzed, and the results are shown in Table 1.

[0028] Table 1 Statistical results of the number of germinated mycelial balls

[0029] 1.3 Antagonism test The mutagenized ng2 strain and the control strain before mutagenesis were inoculated in the same petri dish with a spacer left in the middle, and incubated in the dark at 28°C for 50 days to observe the antagonistic effect. The test showed that the growth of the mutagenized ng2 strain was significantly stronger than that of the control, and an obvious antagonistic line was formed with the control product. The results are as Figure 1 shown.

[0030] 1.4 Sequencing and preservation The ng2 strain was sent to Hangzhou Lianchuan Company for ITS sequencing. The results are as Figure 2 shown. After confirmation, the strain was preserved. The preservation number is CCTCC M 2025077, and it was preserved in the China Center for Type Culture Collection on January 10, 2025. The preservation address is Wuhan University, China.

[0031] Example 2 2.1 Optimization of the rapid germination and propagation medium for the strain Using the strain before mutagenesis and the ng2 mother strain as test materials, and PDA as the basic medium for strain germination, by adding different nutrients or designing different culture conditions in the basic medium, the effects of various factors on the germination of the Boletus strain were explored. Each combination had 3 replicates, and the average number of days for mycelium germination was used as the detection index. (1) Carbon source: The potato and glucose in the PDA medium were used as the carbon source for strain germination, and the optimal addition amount was adjusted to 45 g / L. (2) Nitrogen source: Peptone, beef extract, and yeast powder were added as nitrogen sources, and the optimal addition amount was adjusted to 2.5 g / L. The best medium for rapid strain germination obtained through the test was: PDA or PDA + peptone + beef extract + yeast powder, as shown in Table 2.

[0032] Table 2 Effects of different medium formulations on mycelium germination

[0033] 2.2 Optimization of the liquid medium for rapid mycelium proliferation The ng2 strain was propagated on PDA medium and used as the material for the liquid medium after propagation. Using potato and sucrose as controls, different excipients were added to the control medium to explore the effects of different combinations on the propagation of the ng2 strain. Each combination was repeated 3 times, and the average number of mycelial pellets formed in 7 days and 15 days was used as the detection index. (1) Carbon source: Potato and sucrose were used as the carbon sources for mycelial growth, and the optimal addition amount was adjusted to 1%. (2) Nitrogen source: Corn flour, soybean powder, wheat bran, yeast extract powder, and potassium dihydrogen phosphate were added as nitrogen sources, and the optimal addition amount was adjusted to 1%. The best liquid medium for rapid mycelial growth was obtained through experiments: sucrose + corn flour + soybean powder + wheat bran + potassium dihydrogen phosphate + yeast extract powder. Among them, corn flour, soybean powder, and wheat bran were made into a self-made basic culture medium, and the results are shown in Table 3. The key technology for the artificial cultivation of Boletus edulis lies in the quality of the liquid spawn and the nutrient ratio of the liquid medium. The liquid formula used in the present invention is rich in nutrients, making the mycelial pellets uniform in size and strong in vitality, and can be used for fruiting and liquid fermentation tank propagation in 7 - 10 days. For details, see Figure 3 。

[0034] Table 3 Effects of different medium formulations on mycelial proliferation

[0035] 2.3 Optimization of the raw material formula for fruiting Prepare the raw materials for fruiting, formulate them according to a carbon-nitrogen ratio of 40:1, and sterilize them after bottling. At the same time, prepare the cultured liquid spawn and inject 20 mL per bag into the raw materials for fruiting. Each combination was repeated 3 times, and the average time for the mycelium to fill the culture bottle was used as the detection index to screen the optimal raw material formula for fruiting. The raw materials were: crushed wood chips, self-made fermented material, corn straw, peat soil, and rice, etc. The best raw material formula for fruiting was obtained through experiments: self-made fermented material + peat soil + rice + corn straw (a certain amount of lime and gypsum can be added), and the results are shown in Table 4.

[0036] Table 4 Effects of different culture raw materials on the growth state of mycelium

Claims

1. A mutagenized boletus ng2 strain, characterized in that: The strain has strong vitality and fast growth rate, and is suitable as an excellent strain of boletus in Heilongjiang area.

2. The ng2 strain according to claim 1, characterized in that: The mutagenesis method was carbon ion beam irradiation, 80MeV / u.

3. The ng2 strain according to claim 1 and claim 2, characterized in that: The solid culture medium for rapid mycelium germination and propagation is PDA or PDA+peptone+beef extract+yeast extract powder.

4. The ng2 strain according to claim 1 and claim 2, characterized in that: The mycelium rapid proliferation liquid culture medium is sucrose+corn flour+soybean flour+wheat bran+potassium dihydrogen phosphate+yeast extract powder.

5. The ng2 strain according to claim 1 and claim 2, characterized in that: The mushroom culture medium is homemade fermentation material + corn stalks + peat soil + rice.

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