CN111205988A

CN111205988A - New strain of grifola frondosa

Info

Publication number: CN111205988A
Application number: CN202010044227.1A
Authority: CN
Inventors: 谢红艳; 宫志远; 黄春燕; 万鲁长; 韩建东; 李瑾; 姚强; 杨鹏; 任海霞; 王艳
Original assignee: Institute of Agricultural Resources and Environment of Shandong Academy of Agricultural Sciences
Current assignee: Institute of Agricultural Resources and Environment of Shandong Academy of Agricultural Sciences
Priority date: 2020-01-15
Filing date: 2020-01-15
Publication date: 2020-05-29
Anticipated expiration: 2040-01-15
Also published as: CN111205988B

Abstract

The invention relates to a new strain of grifola frondosa, which has a preservation number of: CGMCC No.18143, and compared with parent strain, the new strain of Grifola frondosa has the advantages of high single yield, high biological efficiency, high polysaccharide content in fruiting body, and high CO concentration resistance₂Poor ventilation in the fruiting room, CO₂Under high concentration environmental conditions, the new strain 86 of Grifola frondosa (T5Q9) is more differentiated than the primordia of the parent strains Tigri No.1 and Qing Hui 151 and the average single yield of the new strain 86 of Grifola frondosa (T5Q9) is 2 times that of the parent strains Tigri No.1 and Qing Hui 151 respectively.

Description

New strain of grifola frondosa

The technical field is as follows:

the invention relates to a novel strain of grifola frondosa, belonging to the technical field of edible fungi.

Background art:

grifola frondosa (Dicks.) Gray is a rare edible and medicinal dual-purpose bacterium, and has unique taste and flavor and high-efficiency medical health care effect.

Cancer is one of the major diseases threatening the health of residents in China at present, and is the leading cause of death in urban areas. The incidence of cancer in China is on the rising trend year by year, and the search for effective cancer prevention measures is urgent. The twenty-first century is a period of rapid development of the major health industry, and people pay more and more attention to prevention of diseases through daily diet.

Grifola frondosa (Dicks.) Gray, also known as chestnut mushroom, maitake mushroom (Japan), and the like, is a rare edible and medicinal dual-purpose bacterium, has crisp and tender meat quality and flavor like shredded chicken, and is rich in various bioactive substances such as proteins, vitamins, minerals, polysaccharides, sterols, alkaloids, triterpenes and the like. The grifolan is the strongest antitumor activity of all fungal bioactive substances, the inhibition rate of the grifolan on S180 solid tumor of a mouse is 98%, and the inhibition rate of the grifolan on the solid tumor of the mouse is 7/10%. Grifola frondosa polysaccharide has been developed into pharmaceuticals and nutraceuticals for cancer treatment for over 20 years.

The ash tree flower is known as 'fungus king and anticancer wonderful flower' by Japanese, the first artificial cultivation of ash tree flower is one male of Japanese 'yiteng', Japan starts commercial cultivation in the 70 th century, factory and annual production is realized, the level is in the lead level in the world, North America also reports, Zhejiang and Hebei are the main countries, areas such as Fujian, Shandong, Heilongjiang, Sichuan, Beijing, Yunnan and the like have large-scale cultivation, mainly the traditional cultivation modes of 'small arched shed wild imitation cultivation method' and 'fungus stick type cultivation and soil covering secondary mushroom production', and the factory production is not large-scale.

At present, the domestic grifola frondosa cultivation strain is mainly from wild domestication or Japanese introduction, has the defects of poor adaptability, weak anti-mixed bacterium capability, low conversion rate and the like, cannot be directly used for industrial production, and has the more main problem that the current strain has CO₂Poor tolerance. The Grifola frondosa belongs to the field of CO gas₂Sensitive variety, high concentration of CO₂Can lead to the undifferentiation of primordia or the formation of malformed mushrooms, resulting in reduced or even no production. As the humidity requirement in the cultivation process of the grifola frondosa is high, CO is mainly reduced in a ventilation mode during the factory production₂Concentration, too frequent ventilation can result in a sharp drop in humidity, humidity and CO₂The coordination of concentration puts high requirements on the optimization combination of mushroom house facilities and equipment and environmental parameters, so that the industrialized production of grifola frondosa is not large-scale, and the breeding of grifola frondosa capable of tolerating high-concentration CO is urgently needed₂The new product is a new product specially used for factory production. For a long time, due to the severe pressure faced by the development of the edible fungus industry, the breeding target is a multiple injection and heavy yield factor, and the functional index is neglected. The grifola frondosa polysaccharide has efficient medical care effect, breeds high-polysaccharide factory special varieties, improves the functional effect of the varieties, and has great significance for meeting national nutritional and health requirements, accelerating the promotion of edible mushroom supply side structure optimization and variety replacement improvement. The polysaccharide content of the fruit body of the Grifola frondosa determined by adopting an anthrone sulfate method in the Sun Peperon et al (2001) is 3.5 percent, the polysaccharide content of the fruit body of the Grifola frondosa cultured by the sawdust extracted by adopting a water extraction and alcohol precipitation method in the Cao Xiuming et al (2019) is 4.2867 percent, and the polysaccharide content of the fruit body of the Grifola frondosa is lower.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a strain with high single yield, high biological efficiency, high polysaccharide content of fruiting bodies and high-concentration CO resistance₂New strain of Grifola frondosa 86(T5Q 9).

A Grifola frondosa gronodosa (Dicks.) Gray new strain 86(T5Q9) that has been deposited at the chinese common microbial cultures collection management center on 7/11 th 2019, address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, with the deposit number: CGMCC No. 18143.

The morphological characteristics of the novel grifola frondosa strain 86(T5Q9) are as follows:

the fruiting body is large in flower shape, multiple in branches, overlapped into a tile shape, compact in mushroom shape, 14.6-21 cm in length, 11.1-18 cm in width and 6.7-11.2 cm in height; the pileus is spoon-shaped to be fan-shaped, the surface is gray black, the pileus has radial stripes, the edge is thin and wavy, the pileus is meat, the length of the pileus is 26.49-34.42 mm, and the width of the pileus is 18.99-25.25 mm; the mushroom flesh is white and 1.2-1.93 mm thick; white fungus tubes with the length of 0.5-0.96 mm and polygonal tube openings are fully distributed on the back of the fungus cap; the stipe is white, thick, short, full and not in a right cylinder shape, the length is 4.39-7.13 mm, and the width is 3.14-6.35 mm; the spores are oval to elliptical, colorless, smooth, 4.39-5.21 x 5.27-6.31 μm.

The cultivation method of the novel grifola frondosa strain 86(T5Q9) comprises the following specific steps:

1) preparation of liquid spawn

Inoculating activated maitake mushroom 86(T5Q9) into fungus liquid culture medium at 25 deg.C for 140-160 r.min^-1Culturing for 14-16 days to obtain liquid strains;

2) cultivation of mushroom

Preparing a culture medium bag, sterilizing, inoculating liquid strains prepared in the step 1) when the temperature of the culture medium bag is reduced to below 25 ℃, wherein the inoculation amount is (8-11) mL/kg, sealing the opening, placing the culture medium bag in a culture room for 20-25 ℃ constant-temperature dark culture, the relative humidity is 60-70%, after culturing for 40-50 days, transferring the culture medium bag to a fruiting room for fruiting management after hyphae grow over the fungus bag and become thick and white due to physiological maturity, cutting the fungus bag, and culturing the fungus bag in the fruiting room at 18-20 ℃, the humidity is 90-95%, the daytime illumination intensity is 200-500 lx, no illumination is provided at night, and CO is not used for illumination, and the temperature is 90-95%₂The concentration is 1000mL/m³Culturing to harvest;

the culture medium in the step 2) comprises the following components in parts by weight: 30-45 parts of cottonseed hulls, 35-45 parts of chestnut sawdust, 10-20 parts of bran, 1-2 parts of brown sugar and 1-2 parts of gypsum, and water is supplemented until the water content reaches 60-65% and the pH is natural.

Preferably, according to the invention, the activation medium in step 1) is a PDA enriched medium consisting of: 200g/L of potato, 10g/L of bran, 2g/L of yeast powder, 1g/L of monopotassium phosphate, 1g/L of anhydrous magnesium sulfate, 3g/L of peptone, 20g/L of glucose, 20g/L of agar and the balance of water.

Further preferably, the liquid medium in step 1) is PDA enriched medium without agar.

According to the invention, the activation culture temperature in the step 1) is preferably 25-28 ℃, and the culture time is 14-16 d.

Preferably, according to the invention, the composition of the medium in step 2) is, in parts by weight: 42 parts of cottonseed hulls, 40 parts of chestnut sawdust, 16 parts of bran, 1 part of brown sugar and 1 part of gypsum, and supplementing water until the water content reaches 65 percent and the pH is natural.

According to the invention, the culture medium bags in the step 2) are prepared by filling 1.2kg of the culture medium bags with each bag, wherein the filling height is 19cm and the bag opening is sleeved, and the sterilization condition is 121 ℃ and 2 hours.

Further preferably, the amount of inoculation of the medium bag in step 2) is 8.5 mL/kg.

According to the invention, the preferable mode of cutting in the step 2) is to select the dense part of the hypha on the wall of the fungus bag and cut a V-shaped opening for fruiting, wherein the length of each side is 3cm, and the cut opening is upward.

Preferably, the harvesting time in step 2) is to harvest the sporocarp to seven maturity, i.e. the pileus is in a fan shape and forms a tube hole.

The invention has the advantages of

Compared with the prior art, the novel grifola frondosa strain 86(T5Q9) has the advantages that:

1. the single yield is high, and the average single yield of the novel grifola frondosa strain 86(T5Q9) is improved by 74.50 percent compared with the parent strain Tigri No.1 and is improved by 20.53 percent compared with the parent strain Genghdust 151.

2. The biological efficiency is high, and the biological efficiency of the new strain 86(T5Q9) of the grifola frondosa is improved by 22% compared with that of the parent strain Tigri No.1 and is improved by 8.78% compared with that of the parent strain Genggri 151.

3. The polysaccharide content of the fruit body is high, and the polysaccharide content of the new grifola frondosa strain 86(T5Q9) is improved by 33.52 percent compared with the parent strain Tigri No.1 and is improved by 11.52 percent compared with the parent strain Genggri 151.

4. High concentration CO tolerance₂Poor ventilation in the fruiting room，CO₂Under the environment condition with high concentration, the primordium of the new strain 86(T5Q9) of the grifola frondosa is better differentiated than that of the parent strain Tigri No.1 and Qing Hui 151, and the average single yield of the new strain 86(T5Q9) of the grifola frondosa is 2 times that of the parent strain Tigri No.1 and Qing Hui 151 respectively.

Drawings

FIG. 1 is a photograph of a plate colony of a novel strain 86 of Grifola frondosa (T5Q 9);

FIG. 2 photo of a plate colony of the parent strain Qinghui 151;

FIG. 3 is a photograph of a plate colony of parent strain Tiash No. 1;

FIG. 4 is a photograph of fruiting bodies of the novel strain of Grifola frondosa 86(T5Q 9);

FIG. 5 is a photograph of fruiting bodies of parent strain, Taigray No. 1;

FIG. 6 is a photograph of fruiting bodies of Qinghui 151 of parent strains;

FIG. 7 high concentration CO₂Next, the new strain 86(T5Q9) of grifola frondosa was compared with the parent strain Qinghui 151 and Tilia taiwanensis No.1 fruiting bodies to obtain photographs;

FIG. 8 is a cluster map based on ISSR and SRAP molecular markers.

Detailed Description

The invention is further illustrated by the following specific embodiments, without limiting the scope of protection thereto.

Main source of material

Parental strain information is shown in table 1:

TABLE 1

Strain name	The source of the strain
		Qinghui 151	Main cultivated variety of Qingyuan county of Zhejiang province
Ladder ash number 1	Wild strain collected and separated from ladder mountain in ancient city

DNA polymerase, dNTP, DL5000DNA marker purchased from Takara Bio engineering (Dalian) Ltd;

GelStain was purchased from Beijing Quanjin Biotechnology Ltd;

the UNIQ-10 column type fungal genome DNA extraction kit is purchased from Biotechnology engineering (Shanghai) GmbH.

Culture medium

The PDA enriched medium is: 200g/L of potato, 10g/L of bran, 2g/L of yeast powder, 1g/L of monopotassium phosphate, 1g/L of anhydrous magnesium sulfate, 3g/L of peptone, 20g/L of glucose, 20g/L of agar and the balance of water.

The liquid culture medium is: the PDA without agar was enriched with medium as described above.

The culture material comprises the following components: 42 parts of cottonseed hulls, 40 parts of chestnut sawdust, 16 parts of bran, 1 part of brown sugar and 1 part of gypsum, and water is supplemented until the water content is 65 percent and the pH is natural.

Example 1

The hybrid strain is obtained by the following specific steps:

(1) basidiospore collection and acquisition of mononuclear strains

1) Adopting seven mature parent strains (pileus is in a sector shape and forms tube holes) Qinghui 151 and Taihu No.1 fruiting body pileus,

and (5) putting the self-sealing bag and writing a label.

2) Placing the pileus collected in the step 1) in a sterilization culture dish on an ultra-clean operation table, cutting off stipe, and dividing into a plurality of blocks with proper sizes for later use.

3) Taking a sterilized PDA culture medium, pouring a small amount of sterilized culture dish cover when the sterilized PDA culture medium is quickly solidified, taking the Grifola frondosa pileus prepared in the step 2), burying the upper surface of the Grifola frondosa pileus into the culture medium, fixing the fruiting body pileus when the culture medium is solidified, covering the lower half sleeve of the culture dish with the fungus tube facing downwards, sealing the opening of a Parafilm sealing film, drawing a contour along the outer edge of the fruiting body pileus at the bottom of the culture dish by using a marking pen, and marking the ejection range of basidiospores.

4) Placing the plate prepared in the step 3) at a constant temperature of 25 ℃ in a dark place for 24h, and ejecting the basidiospores to the bottom of the culture dish.

5) 0.5ml of basidiospores at the bottom of the culture dish in the step 4) is absorbed by a pipette gun, is absorbed into a sterilized EP tube, is subjected to vortex oscillation, and is subjected to microscopic counting by using a 25 multiplied by 16 blood counting plate.

6) According to the concentration of the spore suspension in the step 5), the spore suspension is diluted to 10 basidiospores/100 μ L by using sterile water.

7) Sucking 100 μ L of spore suspension prepared in step 6), coating PDA plate, and culturing at constant temperature of 25 deg.C in dark.

8) And (5) transferring to a new PDA culture medium in time after the microcolonies grow out, and storing the strains.

9) Adopting a sheet sticking method, namely, transparently sticking hypha at the edge of a colony, dyeing by using a cotton blue dye solution, and carrying out microscopic examination to determine whether the combination is in a locked state or not, wherein the combination without the locked state is primarily determined to be a mononuclear bacterial strain, and 56 mononuclear bacterial strains and 26 mononuclear bacterial strains are respectively obtained from parent bacterial strains Qinghui 151 and Taihui 1.

(2) Hybridization assembly

Selecting 10 mononuclear strains of the Qinghui 151 and the Tiaoli No.1 prepared in the step (1), pairwise matching the mononuclear strains, combining the mononuclear strains in a total of 100, inoculating the mononuclear strains to a plate culture medium, culturing the mononuclear strains at a constant temperature of 25 ℃ at a distance of 1.5cm, picking up hyphae at a junction after the hyphae of the two strains contact, primarily judging the hyphae with lock joint as hybrid strains, transferring the hyphae with lock joint at the junction to a PDA (personal digital assistant) plate culture, and performing microscopic examination on the combination of 100 pairs of hybrids to obtain 73 hybrid strains.

(3) Identification of hybrid strains by antagonistic experiments

Further identifying hybrid strains by using an antagonistic test, inoculating 73 hybrid strains preliminarily identified in the step (2), parent strains Qinghui 151 and gradient ash 1 into a 9cm plate PDA enriched medium, culturing for 10 days in a dark place at 25 ℃, perforating activated strains by using a perforator with the diameter of 5mm, inoculating bacterium blocks into a plate solid medium with the diameter of 9cm, drawing a cross by taking the circle center of the plate as the center, respectively taking 4 points along the positions 2cm away from the circle center along the cross, namely inoculating positions of 4 different hybrid strains, inoculating 1 parent strain at the circle center of the plate, culturing for 15 days in a dark place at 25 ℃, repeating 3 times for each combination, and observing whether antagonistic reactions exist among different strains and the antagonistic types and degrees of the different strains to obtain 65 hybrid strains with obvious antagonism with the parent strains.

Example 2

The hybrid strain obtained in example 1 was screened by the following specific steps:

1) preparation of liquid spawn

Adding 300mL of liquid culture medium into a 500mL triangular flask, activating the hybrid strain prepared in the step (3) on a PDA rich culture medium plate (the activation condition is 25 ℃ and dark culture is 10d), preparing the bacterial blocks with the same quality and quantity with a perforator with the diameter of 5mm into the bacterial blocks with the same quantity of the parent strain Qing grey 151 and Tai grey 1, inoculating the bacterial blocks into the liquid culture medium, inoculating 8 bacterial blocks/bottle, sealing a breathable sealing film, and culturing at the temperature of 25 ℃ for 160r min^-1And (5) culturing for 14d to obtain liquid strains.

2) Cultivation method

Uniformly mixing the culture materials by a material mixer, then loading the mixture into polypropylene bags (18cm multiplied by 35cm) by an automatic bagging machine, wherein each bag is filled with 1.2kg of wet materials, the height of each bag is 19cm, the bag is sleeved, sterilizing is carried out at 121 ℃ for 2h, inoculation is carried out when the temperature of the culture materials is reduced to below 25 ℃, 10mL of liquid strains prepared in the step 1) are inoculated into each cultivation bag, the bag opening is sealed, the cultivation bags are placed in a cultivation room for constant-temperature dark cultivation at 23 ℃, the relative humidity is 65%, after cultivation for 31d, hypha grows to be full of the cultivation bags, cultivation is continued until the spawn growing day 43 days, the hypha becomes thick and white after physiological maturity, the hypha is transferred into a fruiting room for fruiting management, a thick part is selected on the wall of the fungus bag, a V-shaped opening is cut, each side is 3cm, the cut opening is upward, the fungus bag is placed on a fruiting frame, the temperature in the fruiting room is 18-20₂The concentration is 1000mL/m³The following sporocarp is harvested at the right time until the sporocarp grows to medium maturity (the pileus is fan-shaped and forms a tube hole).

3) Agronomic trait survey

Measuring and calculating various agronomic characters of the 1 st tide fresh mushroom, including the color of the fruiting body, the length of a single fruiting body, the width of the single fruiting body, the height of the single fruiting body, the length of a fungus tube, the average yield per unit (g/bag) and the average full bag time (day), calculating the biological efficiency, and measuring the polysaccharide content in the fruiting body according to NY/T1676-. Biological efficiency is the fresh weight of the fruiting body (g) divided by the dry weight of the compost (g) multiplied by 100%.

4) Statistical analysis of the measurements from step 3) using SPSS, results of screening out the most effective grifola frondosa 86(T5Q9) are shown in table 2:

TABLE 2

Note: capital letters indicate significant differences at the 0.01 level and lowercase letters indicate significant differences at the 0.05 level

Average yield per unit (g/bag): the average yield of the novel grifola frondosa strain 86(T5Q9) is higher than that of 2 parent strains, and the average yield is very different from that of the 2 parent strains (P is less than 0.01), is improved by 74.50 percent compared with the parent strain Tai Hui No.1 and is improved by 20.53 percent compared with the parent strain Qing Hui 151.

Biological efficiency (%): the biological efficiency of the novel grifola frondosa strain 86(T5Q9) is higher than that of 2 parent strains, and the biological efficiency is very different from that of the 2 parent strains (P is less than 0.01), is improved by 22% compared with the parent strain ladder ash No.1, and is improved by 8.78% compared with the parent strain Qing ash 151.

Fruiting body polysaccharide content (%): the polysaccharide content of the fruit body of the novel grifola frondosa strain 86(T5Q9) is higher than that of 2 parent strains, has a very significant difference (P <0.01) with the parent strain Tai gray No.1 and a significant difference (P <0.05) with the parent strain Qing gray No. 151, and the polysaccharide content of the fruit body of the hybrid strain 86 is improved by 33.52 percent compared with the parent strain Tai gray No.1 and is improved by 11.52 percent compared with the parent strain Qing gray No. 151.

The length (mm) of the fungal tube of the new strain 86(T5Q9) of the grifola frondosa is shorter than that of 2 parent strains, and has a significant difference (P <0.05) with the parent strain Taili No.1 and no significant difference with the parent strain Qingli No. 151.

The morphological characteristics of the novel strain of Grifola frondosa 86(T5Q9) are as follows:

Example 3

The novel strain 86 of Grifola frondosa (T5Q9) was identified using ISSR and SRAP molecular markers as follows.

1) Mycelium genome DNA extraction

Inoculating the stored Grifola frondosa strain to a PDA enriched culture medium for activation for 10 days, inoculating to a dish culture medium paved with sterilized glass paper, culturing at 25 ℃ in a dark place for 10 days, slightly scraping hyphae with forceps, putting into a 1.5mL centrifuge tube, and weighing for later use; extracting hypha genome DNA by using a UNIQ-10 column type fungal genome DNA extraction kit, and detecting the purity and concentration of the DNA by using an agarose gel electrophoresis method and a biological spectrophotometer method.

2) Primer and method for producing the same

The 10 ISSR and 7 pairs of SRAP primers used are shown in tables 3 and 4:

the primer combination of the SRAP is as follows: me1/em2, me3/em1, me3/em3, me3/em6, me5/em1, me5/em3, me5/em 5.

TABLE 3 ISSR tagged primer sequences

TABLE 4 SRAP-tagged primer sequences

3) ISSR and SRAP amplification

The ISSR amplification reaction system is 25 μ L: 2.5. mu.L 10 XPCR buffer, 2. mu. LMgCl₂(25mmol/L)，2μLdNTPs(2.5mmol/L)，0.25μLDNAPolymerase (5U/. mu.L), 2.5. mu.L of LDNA template (20 ng/. mu.L), 2. mu.L of primer (20. mu. mol/L) and 13.75. mu.L of ddH₂And O. ISSR amplification reaction procedure: 5min at 94 ℃; 1min at 94 ℃, 1min at 46 ℃, 1min at 72 ℃ and 35 cycles; preserving at 72 deg.C for 10min and 4 deg.C.

The SRAP amplification reaction system is 25 mu L: 2.5. mu.L of 10 XPCR b buffer, 2. mu.L of MgCl₂(25mmol/L), 2. mu.L dNTPs (2.5mmol/L), 2.5. mu.L DNA template (20 ng/. mu.L), 1. mu.L forward primer (10. mu. mol/L), 1. mu.L reverse primer (10. mu. mol/L), 0.25. mu.L LDNA polymerase (5U/. mu.L) and 13.75. mu.L ddH₂And O. SRAP amplification reaction procedure: 5min at 94 ℃; 1min at 94 ℃, 1min at 35 ℃, 1min at 72 ℃ and 5 cycles; 1min at 94 ℃, 1min at 50 ℃, 1min at 72 ℃ and 35 cycles; preserving at 72 deg.C for 10min and 4 deg.C.

The PCR product was electrophoresed through 1.5% agarose gel, stained with GelStain and the results recorded with a gel imager.

4) Cluster analysis

Analyzing the electrophoresis strip by adopting Quantity One 4.6.9 software, counting the strips of 200-3000 bp, and respectively marking the existence of amplified strips as 1 or 0 to form a 0/1 matrix. Genetic similarity cluster analysis was performed using the arithmetic mean unweighted matching method (unweighted pair group method with arithmetric mean, UPGMA) of the software NTSYS-pc 2.1, and a cluster map was generated as shown in FIG. 8.

5) Identification results

The molecular identification based on ISSR and SRAP is carried out on 9 strains in total, namely a novel strain 86(T5Q9) of the grifola frondosa, parent strain terra grey No.1 and Qing grey No. 151 and

hybrid strains

54, 55, 57, 61, 62 and 88, and the new strain 86(T5Q9) of the grifola frondosa has obvious genetic difference with the parent strain terra grey No.1 and the Qing grey No. 151 and is a real new strain, namely the novel strain 86(T5Q9) of the grifola frondosa is a real hybrid strain which is preserved in China general microbiological culture collection management center in 7-11 th in 2019, and the addresses are as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, with the deposit number: CGMCC No. 18143.

Example 4

The method of example 2 is used for culturing and cultivating the new strain 86(T5Q9) of the grifola frondosa, the parent strain Tigri No.1 and the Qing Hui 151, and the method is similar to the method of example 2The process differs in that CO₂The concentration is controlled at 1500mL/m³The fruiting body comparison result is shown in FIG. 7.

CO₂Under the environment condition with high concentration, the primordium of the new strain 86(T5Q9) of the Grifola frondosa is better differentiated than that of the parent strain Tigri No.1 and Geng Ash 151, and the average single yield of the new strain 86(T5Q9) of the Grifola frondosa is 2 times that of the parent strain Tigri No.1 and Geng Ash 151 respectively, which indicates that the new strain 86(T5Q9) of the Grifola frondosa has high concentration CO resistance₂Has certain growth advantage under the condition of poor ventilation of the fruiting room, and is statistically analyzed in CO by SPSS₂The agronomic performance statistics of the cultured Grifola frondosa under high concentration environmental conditions are shown in Table 5.

TABLE 5

Note: capital letters indicate significant differences at the 0.01 level and lowercase letters indicate significant differences at the 0.05 level.

Example 5

1) preparation of liquid spawn

Adding 300mL of liquid culture medium into 500mL triangular flask, activating (culturing at 25 deg.C in dark for 10 days) Grifola frondosa new strain 86(T5Q9) on PDA rich culture medium plate, making into equal mass of bacterial blocks with a hole puncher having a diameter of 5mm, inoculating into liquid culture medium, inoculating 8 bacterial blocks/bottle, sealing air-permeable sealing film, and culturing at 25 deg.C for 160 r.min^-1And (5) culturing for 14d to obtain liquid strains.

2) Cultivation method

Mixing the culture materials, bagging in polypropylene bags (18cm × 35cm) with automatic bagging machine, wherein each bag contains wet material 1.2kg, material height 19cm, bag opening lantern ring, sterilizing at 121 deg.C for 2 hr, inoculating 10mL of liquid strain obtained in step 1) when culture material temperature is reduced to below 25 deg.C, sealing bag opening, culturing at 23 deg.C in dark at relative humidity of 65%, culturing for 31d, and collecting myceliaGrowing full fungus bags, continuously culturing till the fungus growing day is 43 days, transferring the fungus bags into a fruiting room for fruiting after the fungus grows to be thick and white, selecting a place with thick hypha on the walls of the fungus bags, cutting into V-shaped openings for fruiting, wherein each side is 3cm, the cut opening is upward, placing the V-shaped openings on a fruiting frame, the temperature in the fruiting room is 18-20 ℃, the humidity is 90-95%, the illumination intensity in daytime is 200-500 lx, no illumination is provided at night, and CO is used for preventing the growth of the fungus and the growth of the fungus₂The concentration is 1000mL/m³The following sporocarp is harvested at the right time until the sporocarp grows to medium maturity (the pileus is fan-shaped and forms a tube hole).

Example 6

The method for cultivating the novel strain 86 of Grifola frondosa (T5Q9) was the same as in example 5, and the strain 86 of Grifola frondosa (T5Q9) was cultivated after passage to 7 generations.

Example of effects:

example 5 the primary novel strain of Grifola frondosa 86(T5Q9) cultivated in example 6 the novel strain of Grifola frondosa 86(T5Q9) cultivated in generations from 1 to 7 had the cultivation results shown in Table 6 below:

TABLE 6

The fruiting body of the new Grifola frondosa strain 86(T5Q9) which passes through 1 generation to 7 generation is dark in color, is gray black, has high average yield of 289.8-301.4 g/bag, has high biological efficiency of more than 50 percent, has average bag filling time of 30-31.85 days, has a production period from inoculation to collection of 68 days, is 22 days shorter than that of the traditional agricultural cultivation, has short fungus tube with the length of 0.59-0.66 mm, and has high fruiting body polysaccharide content of 4.607-5.017 percent; the new strain 86 of maitake mushrooms from 1 generation to 7 generations (T5Q9) has no significant difference in agronomic traits such as average yield per unit, biological efficiency, average full-bag time, length of fungal tube and content of fruiting body polysaccharide, and the like, and the new strain 86 of maitake mushrooms (T5Q9) has genetic stability and no degeneration.

Claims

1. A Grifola frondosa gronodosa (Dicks.) Gray new strain 86(T5Q9) deposited at the china general microbiological culture collection center on 7/11 th 2019, address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, with the deposit number: CGMCC No. 18143.

2. The method for cultivating the novel strain of Grifola frondosa 86(T5Q9) according to claim 1, comprising the steps of:

1) preparing liquid strains: inoculating activated maitake mushroom 86(T5Q9) into fungus liquid culture medium at 25 deg.C for 140-160 r.min^-1Culturing for 14-16 days to obtain liquid strains;

2) and (3) mushroom cultivation: preparing a culture medium bag, sterilizing, inoculating liquid strains prepared in the step 1) when the temperature of the culture medium bag is reduced to below 25 ℃, wherein the inoculation amount is (8-11) mL/kg, sealing the opening, placing the culture medium bag in a culture room for 20-25 ℃ constant-temperature dark culture, the relative humidity is 60-70%, after culturing for 40-50 days, transferring the culture medium bag to a fruiting room for fruiting management after hyphae grow over the fungus bag and become thick and white due to physiological maturity, cutting the fungus bag, and culturing the fungus bag in the fruiting room at 18-20 ℃, the humidity is 90-95%, the daytime illumination intensity is 200-500 lx, no illumination is provided at night, and CO is not used for illumination, and the temperature is 90-95%₂The concentration is 1000mL/m³Culturing to harvest;

3. The cultivation method as claimed in claim 2, wherein the activation medium in step 1) is PDA enriched medium consisting of: 200g/L of potato, 10g/L of bran, 2g/L of yeast powder, 1g/L of monopotassium phosphate, 1g/L of anhydrous magnesium sulfate, 3g/L of peptone, 20g/L of glucose, 20g/L of agar and the balance of water; the fungus liquid culture medium in the step 1) is a PDA enriched culture medium without agar.

4. The cultivation method according to claim 2, wherein the activation cultivation temperature in step 1) is 25 to 28 ℃ and the cultivation time is 14 to 16 days.

5. The cultivation method as claimed in claim 2, wherein the composition of the culture medium in step 2) is as follows in parts by weight: 42 parts of cottonseed hulls, 40 parts of chestnut sawdust, 16 parts of bran, 1 part of brown sugar and 1 part of gypsum, and supplementing water until the water content reaches 65 percent and the pH is natural.

6. The cultivation method according to claim 2, wherein the bags of the culture medium in step 2) are made of polypropylene bags (18cm x 35cm) filled with 1.2kg of culture medium per bag and having a height of 19cm, and the bags are sterilized at 121 ℃ for 2 hours.

7. The method according to claim 6, wherein the amount of the medium bag inoculated in step 2) is 8.5 mL/kg.

8. The cultivation method as claimed in claim 6, wherein the cutting in step 2) is performed by cutting a V-shaped cut at a position where the density of the hypha is selected on the wall of the fungus bag, each side is 3cm long, and the cut is upward.

9. The cultivation method as claimed in any one of claims 2 to 7, wherein the harvesting time in step 2) is from the time when the sporophore grows to seven degrees of maturity, i.e. the pileus is in the shape of a sector, forming a tube hole.