CN110964645B - A kind of high temperature resistant mushroom strain and cultivation method thereof - Google Patents
A kind of high temperature resistant mushroom strain and cultivation method thereof Download PDFInfo
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Abstract
本发明公开了一种耐高温香菇菌株及其栽培方法。该耐高温香菇菌株中国微生物菌种保藏管理委员会普通微生物中心的登记入册编号为CGMCC No.18565。香菇高16×扣46是以香菇高温8号和香菇扣香作为亲本进行杂交选育出的一株可在高温环境下出菇的农艺性状优于香菇高温8号和香菇扣香的杂交菌株：其出菇时间早于香菇高温8号和香菇扣香，其子实体单朵重高于香菇高温8号和香菇扣香，其子实体菌盖厚度高于香菇高温8号和香菇扣香，其子实体的碳水化合物含量高于香菇高温8号和香菇扣香，其子实体的必需氨基酸含量高于香菇高温8号和香菇扣香，其子实体产量高于香菇高温8号和香菇扣香。The invention discloses a high temperature resistant mushroom strain and a cultivation method thereof. The registration number of the high temperature resistant mushroom strain of the General Microbiology Center of the China Microorganism Culture Collection Management Committee is CGMCC No.18565. Mushroom Gao 16×Kou 46 is a hybrid strain which was bred by using Mushroom Gaojiao No. 8 and Mushroom Kouxiang as parents. Its fruiting time is earlier than that of Shiitake High Temperature No. 8 and Shiitake Kouxiang, its fruiting body weight is higher than that of Shiitake High Temperature No. 8 and Shiitake Kouxiang, and its cap thickness is higher than that of Shiitake High Temperature No. The carbohydrate content of the fruiting body was higher than that of Shiitake High-temperature No.8 and Shiitake Kouxiang, the essential amino acid content of its fruiting body was higher than that of Shiitake High-temperature No.
Description
Technical Field
The invention relates to a high-temperature resistant lentinus edodes strain and a cultivation method thereof in the field of biological breeding.
Background
Lentinus edodes (Lentinula edodes) is a saprophytic fungus produced in northern hemisphere temperate zone and subtropical zone, is rich in nutrition, has high health care value, and is popular with people since ancient times. However, in the medium-low temperature variable temperature fruiting mushrooms, the optimal growth temperature of hyphae is 24-27 ℃, the optimal development temperature of fruiting bodies is 8-20 ℃, and the hyphae are dead and fungus sticks are rotten when the mushrooms meet high environmental temperature in production. The cultivation season of shiitake mushrooms in most areas of China is from autumn to spring of the second year, so that market supply slack seasons of 3-4 months often appear in high-temperature seasons of summer, during which the price of shiitake mushrooms is usually 2-3 times that of other months. The cultivation of mushrooms in high-temperature seasons is mainly concentrated in provincial cold and cool regions such as northeast, northriver, inner Mongolia and Shanxi of China, but due to global warming, high-temperature stress has influenced the production of mushrooms in many regions in summer, and the cultivation of high-temperature resistant mushroom strains is urgently needed.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a mushroom hybrid strain with at least one of the following excellent properties: the fruiting body yield is higher than that of the parent, the carbohydrate content of the fruiting body is higher than that of the parent, the essential amino acid content of the fruiting body is higher than that of the parent, the fruiting time is earlier than that of the parent, the hypha full bag time is earlier than that of the parent, and the single-flower weight of the fruiting body is higher than that of the parent; the parent is shiitake mushroom high temperature No. 8 and shiitake mushroom button fragrance.
In order to solve the technical problems, the invention provides a high-temperature-resistant mushroom hybrid strain.
The mushroom hybrid strain provided by the invention is mushroom (Lentinula edodes) with the height of 16 multiplied by 46, and the registration number of the mushroom hybrid strain in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 18565. The strain has been preserved in China general microbiological culture Collection center (CGMCC for short) in 2019, 09 and 04 months. Hereinafter, it is abbreviated as shiitake mushroom height 16 x button 46.
The height of the mushroom is 16 multiplied by 46 times, the spore is white, the mushroom is elliptical, and the size is (4-6 multiplied by 3-5) mu m; the mycelium is thick white, the optimal growth temperature of the mycelium is 18-28 ℃, and the optimal pH value is 5.5; the fruiting body grows singly, the color of the pileus is light brown, the mushroom shape is round, the flower shape is medium-large, the optimum development temperature of the fruiting body is 15-28 ℃, and the fruiting can be normally carried out in a high-temperature environment of 30-34 ℃.
The mushroom high 16 x button 46 has at least one of the following characteristics:
1) the fruiting body yield of the shiitake is higher than that of the shiitake high-temperature No. 8 and the shiitake is fragrant,
2) the content of essential amino acids in the fruiting body of Lentinus Edodes is higher than that in the high temperature No. 8 Lentinus Edodes and the Lentinus Edodes fragrance,
3) the carbohydrate content of the fruiting body of the shiitake is higher than that of the high-temperature No. 8 shiitake and shiitake buckle fragrance,
4) the fruiting time of the mushrooms is earlier than the high-temperature No. 8 mushrooms and the mushroom buckling fragrance,
5) the weight of the single fruit body of the mushroom is higher than that of the high-temperature No. 8 mushroom and the mushroom fragrance,
6) the bag filling time of the hypha of the mushroom is earlier than the high-temperature No. 8 mushroom and the mushroom fragrance.
The 6) is that the bag filling time of the hypha with the height of 16 times 46 times of that of the mushroom is 1 to 5 days earlier than that of the parent strain.
The shiitake mushroom 16 × shiitake mushroom 46 has at least one of the characteristics 1) to 6) described above in both a high temperature environment and a normal temperature environment.
The temperature of the high temperature environment may be 30-34 deg.c, and the temperature of the normal temperature environment may be 15 deg.c to less than 30 deg.c. The high-temperature environment and the normal-temperature environment both refer to the environment for the development of the fruiting body.
The fruiting body, mycelium and/or spore of mushroom with height of 16X deduct 46 also belong to the protection scope of the invention.
The protoplast of mushroom with height of 16X deduction 46 also belongs to the protection scope of the invention.
The shiitake stick containing the shiitake mushroom 16X buttons 46 also belongs to the protection scope of the invention.
The application of the shiitake mushroom 16X deduction 46 in the preparation of shiitake mushroom fruiting bodies and/or shiitake mushroom mycelia and/or shiitake mushroom spores also belongs to the protection scope of the invention.
In the application, the preparation of the mushroom fruiting body comprises the step of fruiting culture in a high-temperature environment, wherein the temperature of the high-temperature environment is 30-34 ℃.
The application of the shiitake mushroom 16X deduction 46 in the shiitake mushroom breeding also belongs to the protection scope of the invention.
In the application, the breeding of the shiitake mushrooms can be the cultivation of the shiitake mushrooms resistant to high temperature stress.
The invention also provides a cultivation method of mushroom with the height of 16 multiplied by 46.
The cultivation method of the mushroom high 16 x button 46 provided by the invention comprises the step of fruiting and cultivating the mushroom high 16 x button 46 in a high-temperature environment.
In the above method, the temperature of the high temperature environment may be 30 to 34 ℃.
In this context, the fruiting mode of three-dimensional frame fruiting, ground vertical fruiting or soil-covered fruiting can be adopted for fruiting.
The shiitake mushroom high 16 x shiitake mushroom 46 is a hybrid strain which is bred by taking the high-temperature No. 8 shiitake mushroom of a main cultivar in summer and the shiitake mushroom with the fragrance as parents through hybridization and has superior agronomic characters to the high-temperature No. 8 shiitake mushroom with the fragrance. The mushroom height 16 x deduction 46 has the following agronomic characters: m1) the yield (fruiting body yield) of the shiitake mushroom with 16 times higher shiitake mushroom and 46 times higher yield is remarkably higher than that of 2 parent strains, is increased by 25.48 percent compared with the shiitake mushroom with fragrant, and is increased by 28.47 percent compared with the shiitake mushroom with high temperature No. 8. M2) although the highest temperature in the second mushroom wetting period reaches 34 ℃, the second mushroom wetting yield (fruiting body yield) of the mushroom with the height of 16 multiplied by 46 is obviously higher than 2 parent strains, and the yield is improved by 24.34% compared with the mushroom capping yield and 21.72% compared with the mushroom high temperature No. 8 yield. M3) although the highest temperature reached 33 ℃ during the third tide of mushrooms, the yield (fruiting body yield) of the third tide of mushrooms 16 Xkou 46 higher than that of the mushrooms of 2 parent strains was significantly higher, which was 84.53% higher than that of shiitake kou and 24.37% higher than that of shiitake high temperature No. 8 (Table 4-5). M4) the carbohydrate content of the shiitake mushroom 16X deduction 46 is obviously higher than that of 2 parent strains, and the carbohydrate content of the shiitake mushroom 16X deduction 46 is 2.3 times of that of the shiitake mushroom high temperature No. 8 and 1.8 times of that of the shiitake mushroom deduction. M5) the content of the essential amino acid of the shiitake mushroom 16 Xkou 46 is obviously higher than that of 2 parent strains, and the content of the essential amino acid of the shiitake mushroom 16 Xkou 46 is 1.3 times of the high temperature No. 8 of the shiitake mushroom and 1.7 times of the shiitake mushroom kou (Table 6). M6) the bag filling time of the hypha with the height of 16 times that of the button 46 of the mushroom is earlier than that of the mushroom with the high temperature of No. 8 for 5 days. M7) the fruiting time of the mushroom with the height of 16 times that of the mushroom is higher than that of the parent for 3-5 days, the fruiting time of the mushroom is earlier than that of the mushroom with the high temperature of No. 8 for 5 days, and the fruiting time of the mushroom is earlier than that of the mushroom with the fragrance of the mushroom for 3 days. M8) the weight of the single fruiting body of the mushroom with the height of 16 multiplied by 46 is higher than that of the high temperature No. 8 mushroom and the fragrant mushroom, and the pileus thickness of the fruiting body of the mushroom with the height of 16 multiplied by 46 is higher than that of the high temperature No. 8 mushroom and the fragrant mushroom. The shiitake mushroom 16X-deducting 46 is a high-temperature resistant shiitake mushroom hybrid strain.
Deposit description
The strain name: mushroom (Lentinula edodes)
The strain number is as follows: high 16X button 46
The preservation organization: china general microbiological culture Collection center
The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)
Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district
The preservation date is as follows: 09 month 04 of 2019
Registration number of the preservation center: CGMCC No.18565
Drawings
FIG. 1 is a photograph of antagonistic lines between a shiitake mushroom 16X kola 46 higher strain and a parent strain in a hybrid progeny of shiitake mushroom high temperature No. 8 and shiitake mushroom kola. In the three strains in the figure, the strain at the right upper part is the high-temperature No. 8 shiitake mushroom, the strain at the right lower part is the shiitake mushroom kouxiang, and the strain at the left lower part is the shiitake mushroom 16 multiplied by kouxiang 46.
FIG. 2 is a photograph of antagonistic lines between three strains numbered 2X deduct 28 high, 17X deduct 4 high and 16X deduct 46 high among hybrid progeny of shiitake mushroom high temperature No. 8 and shiitake mushroom deduction. In the three strains in the figure, the upper strain is the height of shiitake mushroom 16X deducted 46, the lower strain on the left is the height of shiitake mushroom 2X deducted 28, and the lower strain on the left is the height of shiitake mushroom 17X deducted 4.
FIG. 3 shows the ISSR amplification result of the high temperature No. 8 shiitake mushroom and the shiitake mushroom and partial hybrid progeny. In the figure, A is the champignon fragrance; b, high-temperature No. 8 shiitake mushrooms; c is a strain with the number of 16 multiplied by 46 in hybrid filial generation of the high-temperature No. 8 shiitake mushrooms and the shiitake mushroom deduction; d is the strain with the number of 2 multiplied by 28 in the filial generation of the shiitake mushroom high-temperature No. 8 and the shiitake mushroom deduction.
FIG. 4 is a clustering diagram of the genetic relationship between high-temperature No. 8 shiitake mushrooms and shiitake mushroom buckling fragrance and partial hybrid progeny.
FIG. 5 is a photograph showing the generation of primordia after the low temperature stimulation of shiitake mushroom 16X buttons 46. In the figure, the projections indicated by arrows are the primordia generated.
FIG. 6 is a photograph of a hybrid in which no primordia are produced after low-temperature stimulation.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The high temperature No. 8 shiitake mushroom and the shiitake mushroom kouxiang in the following examples are products of Beijing Zhi Ci Xing Biotech Co.
Example 1 Breeding of high temperature resistant Lentinus Edodes Strain, Lentinus Edodes high 16X knot 46
The high-temperature 8 shiitake mushroom (short for high-temperature 8) and shiitake mushroom (short for shiitake mushroom) which are main cultivars in summer are selected as parent strains to be hybridized and bred with the high-temperature resistant shiitake mushroom hybrid strain. The method comprises the following specific steps:
1.1 test strains
Parent strain: the high-temperature No. 8 shiitake mushrooms and the shiitake mushrooms are fragrant, and parent sporocarps with round shapes and mature 7 minutes are selected to collect monospore.
1.2 Single spore isolation and identification
Suspending parent fruiting body in triangular flask containing sterile water for 3-5 days, diluting, coating on PDA plate, culturing at 25 deg.C for 3-5 days, selecting single colony for transfer culture, and observing under microscope to determine whether there is locked combination. Without latticed association, it was initially considered to be a monocytic hyphae (i.e., a single spore strain) produced by single spore germination.
1.3 paired hybridization
And respectively inoculating two parent monospore strains in pairwise matching manner in a PDA culture medium, culturing at 25 ℃ for 7-10 days with the distance between inoculation points being 1-1.5cm, picking up hyphae at a middle contact point after the contact of mononuclear hyphae, culturing in a new PDA plate, and observing whether the locking combination exists or not under a microscope. If the two parent monospore strains are in locked combination, the strain can be preliminarily considered as the hybrid binuclear hyphae generated by the hybridization of the two parent monospore strains.
The results show that 248 strains of the hybrid binuclear strain (Table 1) are obtained after parent monospore separation, monospore mononuclear verification, hybrid pairing and hybrid binuclear verification.
TABLE 1 parental monospore and hybrid combinations obtained
1.4 Primary screening of hybrid progeny strains
1.4.1 antagonistic assays
Inoculating the hybrid and two parent hyphae into a PDA culture dish by a Chinese character Pin-shaped inoculation method, culturing in a constant-temperature incubator at 25 ℃ to ensure that each strain hyphae are pairwise crossed, and judging whether the hybrid is different from the parent strains according to the existence of an antagonistic line between the hybrid and the two parents.
According to the principle that antagonism between strains with a close homology relationship is weak, 83 filial generation strains which are not obvious in antagonism line with parent strains are removed, 68 strains which are not obvious in antagonism line between the filial generation strains are removed, and 97 filial generation strains are primarily screened out. Wherein. Antagonistic lines between the strain named shiitake mushroom height 16 × deduction 46 and the parent strain in the hybrid progeny are shown in fig. 1, and the antagonistic lines between the three strains named shiitake mushroom height 2 × deduction 28, shiitake mushroom height 27 × deduction 12 and shiitake mushroom height 17 × deduction 4 in the hybrid progeny are shown in fig. 2.
1.4.2 molecular marker screening
Activating the hybrids and the two parent hyphae on a PDA plate, and culturing in a constant temperature incubator at 25 ℃ for 10 days. 100mg of hyphae are scraped respectively, genome DNA of each strain is extracted, and ISSR and SRAP molecular marker analysis are carried out respectively. ISSR-PCR: the reaction system was 25. mu.l containing 12.5. mu.l of 2 XTaq PCR Mix, 1. mu.l of ISSR primer (0.4. mu. mol/L), 1. mu.l of DNA (30ng), 10.5. mu.l of sterile double distilled water. Reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 48 ℃ for 30s, extension at 72 ℃ for 1min, and 35 cycles; extending for 10min at 72 ℃, and storing at 4 ℃. SRAP-PCR: the reaction system was 25. mu.l containing 12.5. mu.l of 2 XTaq PCR Mix, 1. mu.l of SRAP forward primer (0.4. mu. mol/L), 1. mu.l of SRAP reverse primer (0.4. mu. mol/L), 1. mu.l of DNA (30ng), 9.5. mu.l of sterile double distilled water. Reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 48 ℃ for 30s, extension at 72 ℃ for 1min, and 35 cycles; extending for 10min at 72 ℃, and storing at 4 ℃.
The amplification product was subjected to 1.5% agarose gel electrophoresis, observed with an ultraviolet imaging system and photographed. And (3) regarding amplified strong bands or low bands with good distinguishability on the same position of each lane as amplification positive, assigning a value of '1', regarding un-amplified bands as amplification negative, assigning a value of '0', performing cluster analysis by using NTSYSpc 2.10 software, and constructing a phylogenetic tree by a non-weighted group average method.
The extracted genome DNA of the parent strain and the hybrid progeny strain is used as a template, ISSR and SRAP primers are used for PCR amplification (figure 3), and NTSYSpc 2.10 software is used for carrying out cluster analysis on the test strains to obtain a genetic relationship cluster map (figure 4) among the strains.
The similarity coefficient between strains reflects the distance of the genetic relationship, strains with genetic similarity coefficient more than 0.85 are selected, and 28 strains with larger genetic difference with the parent strain are screened from 97 filial generations through the index for rescreening (figure 4).
1.5 identification of the fruit setting ability of hybrid progeny strains
And (4) re-screening the primarily screened hybrid progeny through fructification capability identification. Respectively inoculating the 28 preliminarily screened hybrid progeny strains into a comprehensive PDA culture medium, culturing at 25 ℃ until hyphae grow over the whole dish, placing at 5-10 ℃ for low-temperature stimulation culture for 2-3d when the hyphae in the dish start to change color, then placing at 25 ℃ again for culturing for a period of time, repeating the process for 3 times, and screening out hybrids generated by mushroom buds. The results indicated that some of the hybrid progeny hyphae began to turn color in the PDA plates and appeared as raised primordia. As a result, 4 hybrid progenies capable of stably producing primordia were selected and designated as shiitake mushroom height 16X deducted 46, shiitake mushroom height 2X deducted 28, shiitake mushroom height 27X deducted 12 and shiitake mushroom height 17X deducted 4, respectively. FIG. 5 shows a photograph of the shiitake mushroom with 16X shiitake mushroom at a high level and 46 when stimulated at a low temperature, and FIG. 6 shows a photograph of a hybrid with no primordia when stimulated at a low temperature. The height of the mushroom is 16 multiplied by 46 times, the spore is white, the mushroom is elliptical, and the size is (4-6 multiplied by 3-5) mu m; the mycelium is thick white, and the optimal growth temperature of the mycelium in a PDA culture medium is 18-28 ℃, and the optimal pH is 5.5; the fruiting body grows singly, the color of the pileus is light brown, the mushroom shape is round, the flower shape is medium-large, the optimum development temperature of the fruiting body is 15-28 ℃, and the fruiting can be normally carried out in a high-temperature environment of 30-34 ℃.
1.6 evaluation of agronomic traits of hybrids
The parent mushroom 8 at high temperature and the mushroom with the fragrance being deducted, the hybrid offspring mushroom 16X 46, the mushroom 2X 28, the mushroom 27X 12 and the mushroom 17X 4 were subjected to the following cultivation tests, and the agronomic character evaluation of the hybrid was performed.
The following experiments were all carried out in Fengning county, Hebei province.
1.6.1 preparation of cultivated strains
And a three-level strain breeding system is adopted. First-stage strain: using comprehensive PDA culture medium (peeled potato 200g, decocted juice, glucose 20g, agar 20g, KH)2PO4 3g，MgSO41.5g, peptone 5g, vitamin B110mg, adding distilled water to reach the constant volume of 1000mL, sterilizing at 121 ℃ for 20min), culturing at 25 ℃ for 10d after inoculation, and selecting the strain with good colony growth as a first-level strain. Secondary strain: using polypropylene bags of 17cm multiplied by 35cm multiplied by 0.05cm, uniformly stirring culture materials (79% of fine sawdust, 20% of bran and 1% of gypsum, the water content is adjusted to about 62%, the pH is natural), sterilizing the bags under high pressure for 2 hours, cooling the bags to room temperature, inoculating a first-class strain under the aseptic condition, placing the bags in a culture room of 22-26 ℃ for culture until the bags are full, and periodically checking for pollution. And (3) cultivating strains: using polypropylene bags of 17cm multiplied by 58cm, uniformly stirring culture materials (79% sawdust, 20% bran, 1% gypsum, water content adjusted to about 55%, pH natural), 1kg of dry materials in each bag, sterilizing at 103-105 ℃ for 10h, stewing for 3h,cooling to room temperature, inoculating the second-level strain under aseptic condition, inoculating 3 inoculation holes per rod, and culturing at 22-26 deg.C until the rod is full of mycelia to obtain the cultured strain.
1.6.2 inoculation and spawn running
The test adopts a random block design, each strain is treated by 1000 fruiting bars, 3 times of repetition are set, the date of inoculating the fruiting bars and the date of full bag of hyphae of different strains are recorded, and the time of full bag of hyphae is calculated.
Uniformly stirring culture materials (79% of sawdust, 20% of bran and 1% of gypsum, the water content is adjusted to about 55%, the pH is natural) by using polypropylene bags of 15cm multiplied by 60cm multiplied by 0.05cm, 1.1kg of dry materials are packaged in each bag, the bag is sterilized at 103-105 ℃ for 10h, and is stewed for 3h, and the bag is cooled to the room temperature, so that the mushroom sticks are obtained. And (3) moving the fruiting rods into an inoculation workshop, cleaning the cultivation strain potassium permanganate, then putting the cleaned cultivation strain potassium permanganate on a superclean bench, sticking 4 inoculation holes on each fruiting rod by using a plastic adhesive tape, allowing the inoculation holes to grow in a germination room at 22 ℃, and ventilating regularly. When hyphae around the inoculation holes grow and are connected together, the mixture is stacked upside down to a fungus growing shed for fungus cultivation, the temperature of the stick is maintained at 10-15 ℃, when the hyphae grow over about 95 percent of the fungus sticks, the iron nail is punctured for oxygenation, the relative air humidity of the fungus growing shed is 60-70 percent, and the culture is carried out on a culture frame when the hyphae grow over the fungus sticks
The results show that in 2-4 days of 2 months, the mushroom sticks are inoculated from the parent mushroom 8-th high-temperature and mushroom deduction and the hybrid filial generation mushroom thereof, namely the mushroom 16 multiplied by deduction 46, the mushroom 2 multiplied by deduction 28, the mushroom 27 multiplied by deduction 12 and the mushroom 17 multiplied by deduction 4, and the bag filling time of the mushroom 16 multiplied by deduction 46 is the shortest in 4 hybrid filial generation strains, namely 65 days, earlier than the mushroom 8-th high-temperature and 5 days and later than the mushroom deduction 1 day (Table 2).
TABLE 2 growth period of each strain
| Bacterial strains | Date of mushroom stick inoculation | Date of bag full of hypha | Time (sky) full of bags | Date of harvesting of first tide of mushroom | Fruiting time (Tian) |
| Incense button | 2 months and 2 days | 4 month and 7 days | 64±2.8aA | 6 months and 2 days | 120±2.3aA |
| High temperature No. 8 | 2 months and 2 days | 4 month and 13 days | 70±3.5cB | 6 months and 4 days | 122±3.9aA |
| High 16X button 46 | 2 month and 4 days | 4 month and 10 days | 65±2.1abA | 6 months and 1 day | 117±1.3aA |
| High 2X button 28 | 2 month and 4 days | 4 month and 14 days | 69±2.9cB | 6 months and 3 days | 119±3.7aA |
| High 27X button 12 | 2 month and 3 days | 4 month and 10 days | 66±1.7bA | 6 months and 10 days | 127±4.9aA |
| High 17X button 4 | 2 month and 3 days | 4 month and 10 days | 66±2.7bA | 7 month and 2 days | 149±11.3bB |
Note: different lower case letters following the same column of data indicate significant difference (P <0.05) and different upper case letters indicate very significant difference (P < 0.01).
1.6.3 fruiting culture
And (4) fruiting in high-temperature seasons by adopting high-shed layer frames. The specific culture method is as follows:
and (3) transferring the mushroom sticks full of hyphae to an environment with the temperature of 15-28 ℃, the relative humidity of air of 85-95%, a proper amount of scattered light and good ventilation, and harvesting the first tide of mushrooms until pileus is unfolded but spores are not ejected. Stopping spraying water after picking, keeping the ambient temperature at 15-28 deg.C, the relative humidity of air at 80-90%, scattering light in proper amount, and ventilating well to recover hypha for 10 days; then culturing for 20 days under the conditions that the ambient temperature is 20-34 ℃, the relative humidity of air is 85-95% and a proper amount of scattered light is adopted (the average temperature at night is 23 ℃, the lowest temperature is 20 ℃, the average temperature in daytime is 30 ℃, the highest temperature is 34 ℃, and the highest temperature in daytime reaches 34 ℃ in 6 days during harvesting), and harvesting the second tide of mushrooms. Stopping spraying water after the second tide of mushrooms are picked, keeping the ambient temperature at 20-30 ℃, the relative humidity of air at 80-90%, scattering light in a proper amount, well ventilating, recovering hyphae for 10 days, then culturing for 20 days under the conditions that the ambient temperature is 18-30 ℃, the relative humidity of air is 85-95% and scattering light in a proper amount (the average temperature at night is 21 ℃, the minimum temperature is 18 ℃, the average temperature in the daytime is 30 ℃, the maximum temperature is 33 ℃, the maximum temperature in 3 days and the maximum temperature in the daytime reaches 33 ℃) and picking a third tide of mushrooms. Stopping spraying water after the third tide of mushrooms are picked, keeping the ambient temperature at 15-28 ℃, the relative humidity of air at 80-90%, scattering light in a proper amount, ventilating well, recovering hyphae for 10 days, then culturing for 20 days under the conditions that the ambient temperature is 15-25 ℃, the relative humidity of air at 85-95% and scattering light in a proper amount, and picking fourth tide of mushrooms.
Observing and recording the fruiting date of the first tide of the different strains, and subtracting the date of inoculating the fruiting stick to obtain the fruiting time; the fruiting body single weight, pili diameter, pili thickness, stipe length and pili hardness, yield and biological efficiency, carbohydrate content, protein content, essential amino acid content, fat content, dietary fiber content and crude polysaccharide content of the 1-4 th tide mushroom are determined. The method for measuring the hardness of the pileus comprises the following steps: measured using a fruit hardness tester of type FR-5120 of Leichang Taiwan. The method for determining the content of the carbohydrate comprises the following steps: the determination is carried out according to the national standard GB/Z21922-2008.
The protein content determination method comprises the following steps: the determination is carried out according to the first method of national standard GB 5009.5-2016.
The method for measuring the content of the essential amino acid comprises the following steps: the test is carried out according to the national standard GB 5009.124-2016.
The fat content determination method comprises the following steps: the test is carried out according to the second method of national standard GB 5009.6-2016.
The method for measuring the content of the dietary fiber comprises the following steps: measured according to national standard GB 5009.88-2014.
The method for measuring the content of the crude polysaccharide comprises the following steps: the determination is carried out according to the standards NY/T1676 and 2008 of the ministry of agriculture.
The biological efficiency is the ratio of the fresh weight of the edible fungi to the dry weight of the used culture material (other components except water in the culture medium of the fruiting body), and is expressed by the common percentage. If 80kg of fresh edible fungi is produced by 100kg of dry culture medium, the biological efficiency of the edible fungi is 80 percent.
Among 6 strains of parent mushroom high-temperature No. 8 and mushroom and hybrid offspring thereof, 16 Xdeduct 46, 2 Xdeduct 28, 27 Xdeduct 12 and 17 Xdeduct 4, the fruiting time of 16 Xdeduct 46 is the earliest, 117 days, 3-5 days earlier than two parents, 5 days earlier than 8 high-temperature No. 8 mushroom and 3 days earlier than mushroom deduction (Table 2).
The parent mushroom high-temperature No. 8 and the mushroom deduction incense and the filial generation thereof are 6 strains with the height of 16 multiplied by deduction 46, the height of 2 multiplied by deduction 28, the height of 27 multiplied by deduction 12 and the height of 17 multiplied by deduction 4, and in the fruit body of the four-tide mushroom, the single weight with the height of 16 multiplied by deduction 46 is the largest and is higher than that of other strains; the pileus thickness was maximal at 16 × clasps 46, up to 19.24mm (table 3).
As can be seen from tables 3-5, 4 hybrids can form fruiting bodies in high temperature seasons, but the fruiting bodies are high by 17 Xdeduct 4, are deformed, have extremely low yield and are eliminated. The yield of the mushroom with 27 multiplied by 12 and four tides is obviously lower than that of the parent strain, and the mushroom is eliminated.
Although the maximum temperature of the second-tide mushroom reaches 34 ℃ in 6 days in the day and reaches 33 ℃ in 3 days in the third-tide mushroom harvesting period, the mushroom is 16 multiplied by 46 in height, 2 parent primordia are differentiated, and the sporocarp is normally developed.
The height of the mushroom is 16 multiplied by 46, the fruiting is neat, the consistency is high, the shape is round, the single fruit body of the mushroom is 30.17g, and the weight is higher than 2 parent strains; the cap hardness of the fruiting body with the height of the mushroom being 16 times that of the mushroom being buckled 46 is 1.76N, which is lower than the high temperature No. 8 mushroom and the mushroom being buckled.
The yield of the shiitake mushroom in a single bag of the four-tide shiitake mushroom with the height of 16 Xkou 46 reaches 610.96g, is extremely higher than that of 2 parent strains, and is increased by 25.48 percent (610.96/486.89-1.2548) and 28.47 percent (610.96/475.55-1.2847) compared with the shiitake mushroom with the high temperature 8 (Table 4-5). Although the highest temperature reached 34 ℃ during the second tide of mushrooms, the yield of the second tide of mushrooms 16 × deducted 46 higher than that of the 2 parent strains was significantly higher than that of the mushrooms, which was 24.34% higher than that of the mushrooms with deducted aroma (182.47/146.75 ═ 1.2434) and 21.72% higher than that of the mushrooms with high temperature No. 8 (182.47/149.90 ═ 1.2172). Although the highest temperature in the third tide mushroom period reaches 33 ℃, the yield of the third tide mushroom of 16 × shiitake mushroom is remarkably higher than 2 parent strains, and is increased by 84.53% (132.73/71.93 ═ 1.8453) and 24.37% (132.73/132.73 ═ 1.2437) compared with shiitake high temperature 8. The carbohydrate content of the shiitake mushroom 16 Xdeduct 46 is 6.30g/100g fresh products, which is remarkably higher than that of 2 parent strains, and the carbohydrate content of the shiitake mushroom 16 Xdeduct 46 is 2.3 times (6.30/2.70-2.3) of the shiitake mushroom high temperature No. 8 and 1.8 times (6.30/3.60-1.8) of the shiitake mushroom deducting; the content of the essential amino acid of the shiitake mushroom high 16 Xdeduct 46 reaches 2.47g/100g fresh products, is remarkably higher than that of 2 parent strains, and the content of the essential amino acid of the shiitake mushroom high 16 Xdeduct 46 is 1.3 times of that of the shiitake mushroom high temperature No. 8 (2.47/1.87 ═ 1.3) and 1.7 times of that of the shiitake mushroom deducting (2.47/1.44 ═ 1.7) (Table 6). Therefore, the high 16X deduction 46 is selected as a new hybrid strain with good heat resistance and named as the high 16X deduction 46 of the mushroom.
The shiitake mushroom 16 × shiitake mushroom 46 is already preserved in the China general microbiological culture Collection center (CGMCC) in 04.09.2019, and the accession number of the shiitake mushroom in the CGMCC is CGMCC No. 18565.
The height of the mushroom is 16 multiplied by 46 times, the spore is white, the mushroom is elliptical, and the size is (4-6 multiplied by 3-5) mu m; the mycelium is thick white, the optimal growth temperature of the mycelium is 18-28 ℃, and the optimal pH value is 5.5; the fruiting body grows singly, the color of the pileus is light brown, the mushroom shape is round, the flower shape is medium-large, the optimum development temperature of the fruiting body is 15-28 ℃, and the fruiting can be normally carried out in a high-temperature environment of 30-34 ℃.
Table 3. fruiting part of agronomic characters of each strain in high temperature season
| Bacterial strains | Weight (g) of single piece | Diameter of mushroom cap (mm) | Pileus thickness (mm) | Length of fungus stalk (mm) | Diameter of mushroom stalk (mm) | Hardness of pileus (N) |
| Incense button | 19.94±2.90ab | 60.51±2.85a | 16.14±3.65a | 34.08±3.40bc | 12.04±1.49a | 2.06±1.06ab |
| High temperature No. 8 | 27.53±8.95ab | 61.63±5.27a | 19.08±2.52a | 41.61±5.78a | 15.17±2.09a | 2.09±0.43ab |
| High 16X button 46 | 30.17±8.46a | 61.09±8.21a | 19.24±2.61a | 41.08±7.17a | 14.29±2.60a | 1.76±0.46b |
| High 2X button 28 | 16.51±3.97b | 55.71±4.12a | 15.24±1.94a | 32.46±5.41c | 12.82±2.56a | 1.79±0.59b |
| High 27X button 12 | 21.41±13.29ab | 51.38±13.63a | 18.33±3.08a | 39.32±6.12ab | 16.19±6.34a | 2.73±1.15a |
| High 17X button 4 | 5.10±1.47c | 3.01±0.84b | 3.33±2.51b | 10.67±0.39d | 11.01±1.00a | 2.36±0.86a |
Note: different lower case letters following the same column of data indicate significant difference (P <0.05) and different upper case letters indicate very significant difference (P < 0.01).
TABLE 4. yield of fruiting in different tide times in high temperature season for each strain
Note: different lower case letters following the same column of data indicate significant difference (P <0.05) and different upper case letters indicate very significant difference (P < 0.01).
TABLE 5 Total average yield and Total average biological efficiency of four-tide mushrooms of each strain
| Bacterial strains | Total average yield of four tide mushrooms (g/bar) | Total average biological efficiency of four-tide mushroom (%) |
| Incense button | 486.89±40.32bB | 44.26±3.67bB |
| High temperature No. 8 | 475.55±18.77bB | 43.23±1.71bB |
| High 16X button 46 | 610.96±30.35aA | 55.54±2.76aA |
| High 2X button 28 | 412.84±34.70cC | 37.53±3.16cC |
| High 27X button 12 | 263.34±37.25dD | 23.94±3.39dD |
| High 17X button 4 | 10.11±0.58eE | 0.92±0.05eE |
Note: different lower case letters following the same column of data indicate significant difference (P <0.05) and different upper case letters indicate very significant difference (P < 0.01).
TABLE 6 nutrient composition of fruiting body of each strain
Note: "-" indicates no detection; different lower case letters following the same column of data indicate significant difference (P <0.05) and different upper case letters indicate very significant difference (P < 0.01).
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (8)
1. Mushroom (Lentinula edodes), its characterized in that: the strain number of the mushroom is 16 multiplied by 46, and the registration number of the mushroom in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 18565.
2. Fruiting body, mycelium and/or spore of shiitake mushroom according to claim 1.
3. Protoplasts of shiitake mushroom according to claim 1.
4. A shiitake mushroom stick comprising the shiitake mushroom according to claim 1.
5. Use of the shiitake mushroom according to claim 1 for preparing a shiitake mushroom fruiting body and/or a shiitake mushroom mycelium and/or a shiitake mushroom spore.
6. Use according to claim 5, characterized in that: the preparation of the mushroom fruiting body comprises the step of fruiting culture in a high-temperature environment, wherein the temperature of the high-temperature environment is 30-34 ℃.
7. Use of the shiitake mushroom according to claim 1 in breeding of shiitake mushroom.
8. A method for cultivating shiitake mushroom according to claim 1, comprising the step of subjecting the shiitake mushroom according to claim 1 to fruiting culture in a high temperature environment having a temperature of 30 to 34 ℃.
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