阅读说明：本技术 一种真姬菇菌株 (Hypsizigus marmoreus strain ) 是由 黄清华 陈长青 许喜佳 江萍 蒋小庆 尹军 王志学 严会 李挺 丁祥 于 2020-04-27 设计创作，主要内容包括：本发明公开了一种真姬菇菌株,所述真姬菇菌株为真姬菇XCXH-2,保藏编号为CCTCC M 2020068,保藏日期为2020年4月17日,保藏地址为湖北省武汉市武昌珞珈山,保藏单位为中国典型培养物保藏中心。本发明提供的真姬菇XCXH-2,发菌速度快于亲本菌株,单产高于亲本,品质优良,菇帽不易开伞、花纹清晰,菇柄白且粗壮保质期长、外形美观、味道鲜美,可集中栽培和重复栽培,稳定性强,生长整齐,可作为真姬菇工厂化生产企业的生产种源和科研单位的育种的优势种源。(The invention discloses a hypsizigus marmoreus strain, wherein the hypsizigus marmoreus strain is hypsizigus marmoreus XCXH-2, the preservation number is CCTCC M2020068, the preservation date is 2020, 4, 17 days, the preservation address is Wuchang Lojia mountain in Wuhan city, Hubei province, and the preservation unit is China center for type culture preservation. The hypsizigus marmoreus XCXH-2 provided by the invention has the advantages of higher spawning speed than that of parent strains, higher yield per unit than that of the parent strains, excellent quality, difficult umbrella opening of mushroom caps, clear patterns, white mushroom stems, sturdiness, long shelf life, attractive appearance, delicious taste, capability of centralized cultivation and repeated cultivation, strong stability and regular growth, and can be used as a production provenance of hypsizigus marmoreus industrial production enterprises and a breeding advantage of scientific research units.)

1. A hypsizigus marmoreus strain is characterized in that the hypsizigus marmoreus strain is XCXH-2, the preservation number is CCTCC NO: M2020068, the preservation date is 2020, 4 and 17 days, the preservation address is Wuchang Loojia mountain in Wuhan city, Hubei province, and the preservation unit is the China center for type culture preservation.

2. The strain of hypsizigus marmoreus of claim 1, wherein the hypsizigus marmoreus XCXH-2 pileus is semicircular, the middle area of the pileus is a grayish brown stripe, and the periphery of the pileus is grayish yellow; the streaks on the mushroom cap are clear, the color of the mushroom stem is white, and the diameter range of the mushroom stem is 1.5-2.7 cm.

3. The strain of hypsizigus marmoreus according to claim 1, wherein the average yield per hypsizigus marmoreus XCXH-2 is not less than 300 g/strain.

4. The strain of hypsizigus marmoreus of claim 1, wherein the hypsizigus marmoreus XCXH-2 has a hyphal growth rate of 0.6 cm/day on average.

5. The strain of hypsizigus marmoreus of claim 4, wherein the hypsizigus marmoreus XCXH-2 has a shelf life of up to 60 days under refrigeration.

Technical Field

The invention belongs to the technical field of edible fungus strains, and particularly relates to a hypsizigus marmoreus strain.

Background

Hypsizigus marmoreus is a rare edible fungus introduced from Japan, also known as Hypsizygus marmoreus, belonging to Basidiomycotina, Hymenomycetes, Agaricales, Tricholomataceae, Lyophyllum, Hypsizygus. As reported in Yanghua, wild Hypsizygus marmoreus is also present in Yunnan province, but Hypsizygus marmoreus cultivated in various places at present is derived from Japan.

In 1972, artificial cultivation of Hypsizigus marmoreus was first succeeded and patented by Nippon Baojiu Kaisha. Since 1973, the method is put into production in Changyun county, Hypsizigus marmoreus is introduced in 80 s in China, small-scale cultivation is mainly carried out in Shanxi, Hebei, Henan, Shandong and Fujian, and Japanese advanced complete set of automatic mechanical equipment is introduced in Shanghai, Guangdong and the like in 2001, and industrial whole-year production of Hypsizigus marmoreus is carried out.

In recent 20 years, the production capacity and the technical level of various domestic hypsizigus marmoreus manufacturers are greatly improved, but the used strains are basically from Japan. Therefore, the selection of a new hypsizigus marmoreus strain with remarkable characteristics, high quality and high yield and the improvement of the research and development capacity of hypsizigus marmoreus strains are of great importance to various manufacturers at present.

The strains used by the domestic hypsizigus marmoreus manufacturers at present are basically from Japan, and the dominant strains of the hypsizigus marmoreus currently used are numbered wz-29, wz-65 and wz-77; the fruiting performance of the strain wz-29 is good in quality and relatively low in yield per unit; wz-65 and wz-77 fruiting show that the yield per unit is relatively high, the mushroom cap is provided with a tumor cover, the quality guarantee period is short, and the quality is relatively poor; therefore, there is a need to breed a new strain of hypsizigus marmoreus having proprietary intellectual property rights, high quality and high yield.

Disclosure of Invention

The invention aims to provide a hypsizigus marmoreus strain, which is high in quality, attractive in appearance, delicious in taste, high in stability and regular in growth and can be cultivated intensively and repeatedly, and a crossbreeding method is adopted.

In order to achieve the technical purpose, the technical scheme provided by the invention is as follows:

the invention provides a hypsizigus marmoreus strain, wherein the hypsizigus marmoreus strain is hypsizigus marmoreus XCXH-2(Hypsizygus marmoreus XCXH-2), the preservation number is CCTCC M2020068, the preservation date is 2020, 4 and 17 days, the preservation address is Wuchang Loa Jia mountain in Wuhan city, Hubei province, and the preservation unit is China center for type culture preservation.

The pileus of Hypsizigus marmoreus XCXH-2 is semicircular, the middle area of the pileus is a grey brown stripe, and the periphery of the pileus is grey yellow; the streaks on the mushroom cap are clear, the color of the mushroom stem is white, and the diameter range of the mushroom stem is 1.5-2.7 cm.

The average yield per unit of the hypsizigus marmoreus XCXH-2 is not less than 300 g/strain.

The average growth rate of hypsizigus marmoreus XCXH-2 mycelium is 0.6 cm/day.

The Hypsizigus marmoreus XCXH-2 has a shelf life of up to 60 days under refrigeration.

Hypsizigus marmoreus XCXH-2 has better morphological characters: has plump stipe and cap, beautiful stripe and excellent quality, long storage time, pure color of the stipe, and is obviously superior to the parent strain.

The cultivation characteristics of hypsizigus marmoreus XHXC-2 are as follows: the pileus is semicircular, the middle area of the pileus is tawny stripes, and the periphery of the pileus is gray yellow; except for the edges of the pileus, the speckles are distributed with the pileus, the pileus grows in a standard way, and the color is white; the stipe is white, middle-sized, without hair, middle-sized and coarse.

According to the invention, a hypsizigus marmoreus XCXH-2 strain with various excellent properties is artificially bred by a crossbreeding method, has the advantages of high yield compared with parents, excellent quality and long quality guarantee period, attractive appearance, delicious taste, capability of centralized cultivation and repeated cultivation, strong stability and regular growth, and capability of being used as a production seed source of hypsizigus marmoreus industrial production enterprises and a breeding seed source of scientific research units.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 is an electrophoretogram of the primer ISSR10, wherein M is a 2000bp DNA ladder, 1 is WB-86, 2 is WB-96, and 3 is XCXH-2;

FIG. 2 is an electrophoretogram of the primer ISSR23, wherein M is a 2000bp DNA ladder, 1 is WB-86, 2 is WB-96, and 3 is XCXH-2;

FIG. 3 is an amplification electropherogram of primer S42, wherein M is a 2000bp DNA ladder, 1 is WB-86, 2 is WB-96, and 3 is XCXH-2;

FIG. 4 is an amplification electropherogram of primer S338, M being a 2000bp DNA ladder, 1 being WB-86, 2 being WB-96, 3 being XCXH-2;

FIG. 5 shows the results of the antagonistic experiments on the test strains;

FIG. 6 is a photograph of harvest time of Hypsizigus marmoreus XCXH-2.

Detailed Description

The following examples are provided to further illustrate embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the respective embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and biomaterials, if not specifically indicated, are commercially available.

Example 1

(1) Monospore hybridization

Six domestic hypsizigus marmoreus strains (wz-29, wz-65, wz-77, WB-19, WB-86 and XCHW-1, which can be purchased from commercial channels) are selected for spore collection, are diluted and coated for single spore picking, and are determined to be single spores through non-locked combination observation by microscopic examination. And (3) carrying out cross breeding by taking monospore wz-29 as a male parent and monospore wz-65, wz-77, WB-19, WB-86 and XCHW-1 as a female parent, and finally selecting a new hypsizigus marmoreus strain which is not provided with a tumor cover and has high yield and is hybridized with wz-29 and WB-86 by taking the fruiting results of each heterozygote which is successfully bred, wherein the number of the hypsizigus marmoreus strain is XCXH-2.

(2) Characterization of Hypsizigus marmoreus XCXH-2

ISSR and RAPD identification

The preparation method of the experimental culture medium comprises the following steps: 200g of potatoes, 20g of glucose and 15g of agar powder, and adding water to a constant volume of 1L; CYM medium: 200g of potato, 20g of glucose, 15g of agar powder, 1.5g of magnesium sulfate, 3g of monopotassium phosphate and 10.1g of vitamin B, and adding water to a constant volume of 1L.

Hypha culture: transferring a test strain (shown in Table 1) from a test tube to a flat plate for activation, placing the test strain in a constant-temperature incubator at 20 ℃ for culture for 13-15d, picking hyphae after the hyphae are activated, avoiding picking a culture medium as much as possible in the picking process so as to avoid causing experimental errors, and placing the extracted hyphae in a 1.5mL centrifuge tube for later use.

TABLE 1 test strains

Extracting strain DNA: refer to the HiPure Fungal DNA Kit (100mg) Fungal DNA extraction Kit (Kyoto Meyji Biotech Co., Ltd., Guangzhou, No. D3171-02) instructions from magenta.

ISSR and RAPD marker amplification: the primers used are shown in Table 2, the reaction system is shown in Table 3, and the ISSR-PCR amplification reaction program: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 42-45 ℃ for 1min, extension at 72 ℃ for 1.5min, and 45 cycles; finally, filling at 72 ℃ for 10min, and stopping at 22 ℃. RAPD-PCR amplification reaction program: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 35-38 ℃ for 1min, extension at 72 ℃ for 1.5min, and 45 cycles; finally, filling at 72 ℃ for 10min, and stopping at 22 ℃.

TABLE 2 ISSR-PCR and RAPD-PCR primer sequences

TABLE 3 PCR reaction System for ISSR and RAPD

Detection of amplification products: spotting 5 mu L of amplification products on 1.2% agar gel, taking DL2000 Marker as an indication strip, setting the voltage of an electrophoresis apparatus to be 120-150V and the current to be 100A, carrying out electrophoresis for 20-40 min, and taking a picture on a gel imaging system and storing.

ISSR and RAPD result analysis, namely hybridizing parent strains WZ-29 and WB-86 to obtain a strain XCXH-2, identifying the strain XCXH-2 by using ISSR and RAPD molecular marker methods, screening primers used by two molecular markers, and amplifying to obtain a primer with stability and polymorphism and an electrophoresis chart, which are shown in figures 1-4; as can be seen from FIG. 1, the unique bands of the strains WB-86 and WZ-29 at the primer ISSR10 are A-1 and A-2, respectively; in the strain XCXH-2, neither A-1 nor A-2 are present. In FIG. 2, the unique bands of strains WB-86 and WZ-29 at primer ISSR23 are B-1 and B-2, respectively; in the strain XCXH-2, however, a band B-3 identical to that of B-2 was present, but no band identical to that of B-1 was present. In FIG. 3, the unique bands of the strains WB-86 and WZ-29 at primer S42 are C-1 and C-2, respectively; no C-1, C-2 and another band C-3 appeared in strain XCXH-2, demonstrating that the hybrid strain XCXH-2 is distinct from strains WB-86 and WZ-29. In FIG. 4, the unique bands of the strains WB-86 and WZ-29 at primer S388 are D-1 and D-2, respectively; in strain XCXH-2 there is a D-3 band identical to D-2, but not a specific band for WZ-29.

Conclusion of ISSR and RAPD detection analysis: the comprehensive analysis of the 4 screened primers ISSR10, ISSR23, S42 and S388 shows that the hybrid strain XCXH-2 has genetic difference from the parent strains WB-86 and WZ-29, and the hybrid strain XCXH-2 belongs to different strains compared with the parent strains WB-86 and WZ-29.

Example 2

(1) Growth of hyphae

Within the sterile zone formed by the alcohol burner, weak hyphae and groveling wall hyphae at the position of 1 cm at the front end of the test tube are scraped by an inoculation needle after burning and cooling, parts with the upper front end and the upper rear end are selected and cut into small blocks with the size of 3 x 3mm, the small blocks are placed into a flat dish, the distance between the two fungus blocks is 4cm, after inoculation is finished, a batch is marked on the transferred flat dish, the flat dish is wrapped by a freshness protection bag and placed into a basket, the flat dish is covered by newspaper and placed into a biochemical culture box for culture, and the culture temperature is 21 ℃. In the culture process, measuring and recording the diameter of a bacterial colony every day, and observing the shape, color and growth of hyphae; and (5) finishing the culture when hyphae grow over the plate. And (3) calculating the growth speed of hyphae according to the following calculation formula: hypha growth rate (cm/d) is colony diameter (cm)/growth days (d);

the hyphal growth is shown in Table 4:

TABLE 4 hyphal growth of different strains

Remarking: the average (cm) is the hyphal growth rate.

As can be seen from Table 4, the XCXH-2 strain has a significantly better hyphal growth rate than the parent WB-86 and the parent WZ-29.

(2) Antagonism experiment

Within the sterile area formed by the alcohol lamp, weak hyphae and groveling wall hyphae at the position of 1 cm at the front end of the test tube are scraped by an inoculation needle after burning and cooling, the part of the upper section of the front end and the rear end is selected and cut into small blocks with the uniform size of 3 x 3mm, the two fungus blocks are separated by 4cm and placed on a flat dish, after inoculation is completed, a batch is marked on the switched flat dish, the inoculated flat dish is wrapped by a freshness protection bag and placed into a basket, the flat dish is covered by newspaper and placed into a biochemical culture box for culture, the temperature is 21 ℃, the antagonistic condition is observed, and the result is shown in figure 5.

As can be seen from fig. 5: the XCXH-2 strains and parent strains have antagonistic reactions, which indicates that the XCXH-2 strains are new varieties, and compared with the parents, the XCXH-2 strains have obvious advantages in growth vigor and vitality compared with the parent strains.

(3) Bottle cultivation characteristics

The culture medium is mixed wood chip culture medium, and the bottle volume is 1170 cc. The culture medium is prepared from broad-leaved tree sawdust, corncobs, cottonseed hulls, rice bran and wheat bran in a ratio of 4:1:1:1:1, and has a water content of 65%. Sterilizing the prepared culture medium at 121 ℃ for 120 minutes, cooling to 20 ℃, inoculating, culturing at 20-25 ℃ for 70-90 days, removing fungi, performing bud forcing in an environment with the temperature of 15-18 ℃ and the humidity of 90-98%, culturing at the temperature of 15-17 ℃, the humidity of 90-95% and the light intensity of 100Lx after bud forming, and harvesting when the cap is about to open.

The cultivation characteristics of the XHXC-2 strain were as follows: the pileus is semicircular, the middle is tawny stripe, and the periphery is gray yellow; the speckles are distributed except the pileus edges, the growth of the pileus is standard, and the color is white; the stipe is white, middle-sized, without hair and thick; the average yield per unit harvest was 323.1 g.

Appearance characteristics of XHXC-2 strain are shown in FIG. 6, and the strain characteristic pairs of parental and heterozygote are shown in Table 5:

TABLE 5 appearance characteristics of fruiting bodies of XCXH-2 strain, WZ-29 and WB-86

As can be seen from fig. 6 and table 5, the XCXH-2 strain has better morphological properties, full stipe and cap, more beautiful streaks and superior quality compared to the parent.

Example 3

Preservation test

Collecting 24 XCXH-2 bacterial strains on 23 th day, cutting into roots and boxes in a packaging workshop, weighing by an electronic scale, controlling the weight of the boxes to be 150g-160g, and performing plastic packaging by a box packaging machine, wherein the temperature in the packaging workshop is 14-16 ℃ and the packaging time is 1 hour.

And packaging the packaged boxed mushroom foam boxes, sealing the boxes by using transparent adhesive tapes, and placing the boxes in a cold storage sample reserving frame, wherein the temperature of the cold storage is set to be 2-4 ℃. The color, freshness, smell and quality of the cap and stem were recorded every other week, and the results are shown in Table 6.

TABLE 6 preservation of XCXH-2 on different days of preservation

Example 4

Sensory testing

Collecting 200g of XCXH-2, WZ-29 and WB-86 in harvesting period, treating with boiling water to remove mushroom flavor, taking out, draining water, parching with flos Allii Fistulosi and vegetable oil, and flavoring with salt to obtain parched Agaricus campestris. The 40 persons were asked to eat blindly (without informing the eating subjects), and the delicacy, the storehouse position, and the firmness of the pan-fried mushroom were evaluated organoleptically and scored, and the results are shown in table 7.

TABLE 7 sensory testing of different strains

As can be seen from Table 7, the XCXH-2 strain had a better mouthfeel after stir-frying, was tasteful and crisp, and had no bitterness, compared to the parental WZ-29 and WB-86.

In conclusion, the XCXH-2 strain is obviously different from the parent strain, is a new hypsizigus marmoreus strain, has unique morphological characteristics (the color stripes of the cap of the hypsizigus marmoreus are aesthetic and the stem of the hypsizigus marmoreus is white), has stable strain properties, can be repeatedly cultivated, and has high yield per unit and good quality.

The embodiments of the present invention have been described in detail with reference to experimental data, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.