A cold region hydrangea strain and a high-yield cultivation method thereof

Abstract

The present invention discloses a cold-region hydrangea strain and a high-yield cultivation method thereof, which relates to the field of biotechnology and aims to solve the problem that there is a lack of high-yield hydrangea varieties suitable for cold-region cultivation in the prior art. Sparassis crispa ) Haxiu 3 was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on June 20, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 41342. The hydrangea fungus Haxiu 3 obtained by separation and domestication in the present invention has the advantages of short mycelium growth time, higher primordium formation rate, and high yield. It can have a higher mushroom production rate in the temperature range of 10~25℃, has stronger adaptability to the environment, has good low temperature resistance, and is more suitable for cultivation in cold areas. Haxiu 3 of the present invention is highly sensitive to light. Irradiation with yellow light during the primordium differentiation period can promote primordium formation, shorten the primordium formation time, and improve the uniformity of mushroom production.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a cold region hydrangea strain and a high-yield cultivation method thereof. Background Art

[0002] Hydrangea Sparassis crispa ], belonging to the phylum Basidiomycetes, class Hymenomycetes, order Aphyllopodiaces, family Hydrangeaceae, genus Hydrangea, also known as Hydrangea, flower fungus, etc. It has a high immune activation ability and contains a large amount of β-glucan, antioxidants, vitamin C, vitamin E, etc. The flesh of Hydrangea is crisp and tender, with a pleasant aroma, unique flavor and delicious taste. It is recognized by gourmets at home and abroad as a "treasure among mushrooms" and a rare wild mushroom.

[0003] Hydrangea is usually a wild edible and medicinal mushroom, which is rare and difficult to collect, making it difficult to meet people's daily needs, so artificial cultivation is imperative. Although there are artificial domestication and cultivation of Hydrangea, there are few reported varieties of Hydrangea suitable for artificial cultivation. Most of them have low yields and a narrow fruiting temperature range (15-20℃), which is not suitable for cultivation in cold areas. In addition, the existing cultivation methods have a low degree of standardization and a long production cycle. At the same time, traditional culture media lead to slow mycelial growth, which is not conducive to the cultivation of Hydrangea. Summary of the invention

[0004] The technical problems to be solved by the present invention are:

[0005] The prior art lacks hydrangea varieties with high yield and suitable for cultivation in cold regions.

[0006] The present invention adopts the following technical solutions to solve the above technical problems:

[0007] The present invention provides a strain of Hydrangea, which is Hydrangea ( Sparassis crispa ) Ha Xiu 3 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 20, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.41342.

[0008] The present invention provides a hydrangea fruiting body for cultivating the hydrangea ( Sparassis crispa ) The fruiting body obtained by Ha Xiu 3.

[0009] The present invention provides a method for cultivating the fruiting body of Hydrangea sphaerocephala, comprising the following steps:

[0010] (1) inoculating the hydrangea into a sawdust culture medium for rejuvenation, culturing again to obtain a strain, and then inoculating the strain into a liquid culture medium to obtain a liquid strain;

[0011] (2) The cultivation material is placed in a mushroom bag, sterilized, inoculated with the liquid fungus, and then subjected to fermentation and yellow light irradiation to promote primordium formation and fruiting management, and finally harvested to obtain the finished hydrangea mushroom product.

[0012] Furthermore, during the cultivation of the liquid bacteria in step (1), 500 lx red light is irradiated for 12 h / day.

[0013] Furthermore, the cultivation material in step (2) comprises, by mass fraction, 83% pretreated pine sawdust, 10% wheat bran, 5% soybean powder, 1% gypsum powder and 1% sugar; the pretreated pine sawdust is obtained by placing the pine sawdust in lime water and heating it to 80-85° C., then washing the pine sawdust with clean water until the pH is neutral, and exposing it to the sun.

[0014] Furthermore, in step (2), the yellow light irradiation for promoting primordium formation has an illumination intensity of 500 lx and an irradiation time of 12 h / day.

[0015] Furthermore, the temperature for mushroom production management in step (2) is 10-25°C.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The hydrangea 3 obtained by separation and domestication of the present invention has the advantages of short mycelial growth time, higher primordium formation rate, and high yield. It can have a higher mushroom production rate in the temperature range of 10-25°C, has stronger environmental adaptability, has good low temperature resistance, and is more suitable for cold region cultivation. The hydrangea 3 of the present invention is highly sensitive to light, and yellow light irradiation during the primordium differentiation period can promote primordium formation, shorten the primordium formation time, and improve the uniformity of mushroom production.

[0018] The method for cultivating the strain of Harxiu 3 of the present invention prepares a liquid culture medium through rejuvenation of sawdust culture medium, so that the mycelium grows more robustly, the growth rate of the fungus ball is accelerated, and the shaking time is shortened. The cultivation culture medium is prepared by pre-treating pine sawdust in the manner of lime and steaming, so that the cultivation culture medium is more suitable for the growth of hydrangea, and the contamination rate is reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a growth diagram of the primordium of the hydrangea 3 in the embodiment of the present invention;

[0020] Figure 2 This is a fruiting body diagram of the hydrangea mushroom Haxiu 3 in an embodiment of the present invention.

[0021]

Preservation Information

[0022] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0023] Embodiment 1:

[0024] 1. Strain Isolation

[0025] The present invention collects 15 wild hydrangea strains from dead trees in Changbai Mountain, Jilin Province, and obtains a strain with robust mycelium growth and suitable for bag cultivation by domesticating and screening the 15 wild hydrangea strains, which is named Haxiu 3. The specific method is as follows:

[0026] 1.1. Culture medium preparation

[0027] Culture medium formula: 200 g potato (peeled), 20 g glucose, 2 g peptone, 2 g yeast powder, 20 g agar and 1000 ml larch extract, natural pH.

[0028] Weigh 200g of peeled potatoes, cut into thin slices, wash, add 1000ml of larch extract and boil. After the water boils, boil for another 20 minutes, then filter with gauze, take the filtrate, if the filtrate is less than 1000ml, add 1000ml, then add 20g of agar and cook until melted, then add 20g of glucose, 2g of peptone and 2g of yeast powder to melt, divide into test tubes for sterilization while hot, the test tubes should be filled with about one-fifth to one-quarter of the height of the test tubes, seal the test tubes with cotton plugs, and autoclave at 121℃ for 20min. After sterilization, place them on a slant for later use.

[0029] 1.2. Tissue Isolation

[0030] Cut off the base of the collected wild hydrangea fruiting bodies, rinse them clean, rinse them several times with sterile water in a sterile room, and use sterile paper to fully absorb the surface moisture, and then soak them in 0.1% mercuric chloride for 5 minutes. After taking them out, rinse them several times with sterile water (you can also directly apply 75% alcohol to the surface of the fruiting body) to disinfect the surface. After disinfection, use a clean knife above the flame of an alcohol lamp to cut off the thorns, and quickly use an inoculation needle to pick about 0.5cm from the middle of the wild hydrangea fruiting body. 2 The fruiting body tissue blocks were inoculated on the test tube slant culture medium, and 5 test tubes were separated for each fruiting body.

[0031] 1.3. Cultivation

[0032] The inoculated test tubes were placed in a constant temperature incubator at 25°C for culture and screening to obtain strains X001-X007.

[0033] 2. Mycelial Growth Test

[0034] 2.1. Culture medium preparation

[0035] Plate culture medium formula: 200g potato, 20g glucose, 20g agar and 1000ml larch extract.

[0036] Weigh 200g of peeled potatoes, cut into thin slices, wash, add 1000ml of larch extract and boil, boil for 20min after the water boils, then filter with gauze, take the filtrate, if the filtrate is less than 1000ml, add 1000ml, then add 20g of agar and boil until melted, then add 20g of glucose and melt, autoclave at 121℃ for 20min, after sterilization, pour into a sterile plate with a diameter of 9cm in the clean bench for use.

[0037] 2.2. Inoculation and culture

[0038] Take 0.5cm on the slope of X001-X007 test tube 2 The mother culture blocks were inoculated on the plate culture medium (d=9cm) and cultured at a constant temperature of 25℃. When the mycelium covered the plate, a 0.7cm diameter puncher was used to take out the uncontaminated blocks of the same age and size and transferred them to the center of the plate culture medium (d=9cm). The plate was cultured at a constant temperature of 25℃. After the mycelium germinated, the cross-line method was used to mark the scale every 24h until the mycelium covered the plate. The mycelium growth rate was calculated and the mycelium growth potential was observed. Each strain was repeated 3 times. The results are shown in Table 1.

[0039] Table 1

[0040]

[0041] Among them, "+" means that the mycelium is sparse and weak, "++" means that the mycelium is relatively sparse and grows well, "+++" means that the mycelium is dense and grows vigorously, and "++++" means that the mycelium is dense and strong.

[0042] 3. Preparation of Liquid Bacteria

[0043] 3.1. Preparation of liquid culture medium

[0044] Culture medium formula: 100 g potato (peeled), 20 g glucose, 3 g potassium dihydrogen phosphate and 1000 ml larch extract; culture medium pH is 5-6.

[0045] Weigh 100g of peeled potatoes, cut into thin slices, wash, add 1000ml of larch extract and boil. After the water boils, boil for 20 minutes, then filter with gauze, take the filtrate. If the filtrate is less than 1000ml, add 1000ml, then add 20g of glucose and 3g of potassium dihydrogen phosphate to melt, and divide it into Erlenmeyer flasks while hot, with the filling amount being about two-fifths of the volume of the Erlenmeyer flask. Sterilize under high pressure at 121℃ for 20min, and set aside after cooling.

[0046] 3.2. Inoculation and culture

[0047] Use a hole punch to take 5-8 pieces of X001-X007 0.5cm 2 The mother culture block is inoculated into a liquid culture medium, and cultured at 20-25° C. and 140-160 r / min with shaking to obtain a liquid culture medium.

[0048] 4. Cultivar Preparation

[0049] 4.1. Preparation of culture medium

[0050] The culture medium for the cultivated species includes: 83% pine sawdust, 10% wheat bran, 5% soybean powder, 1% gypsum powder and 1% sugar.

[0051] Select fresh, mold-free culture medium, put wheat bran, soybean powder and gypsum powder together in proportion and mix them evenly, then add pine sawdust in proportion, put sucrose into water to melt, stir the mixed dry material and sucrose water evenly, prepare the cultivation medium (water content 60wt%), put it into 17x35cm bags, put 1.5kg of medium into each bag, sterilize it at normal pressure for 12h, and let it stand until the temperature drops to 25℃.

[0052] 4.2. Inoculation and culture

[0053] Under sterile conditions, X001-X007 liquid strains were inoculated into the culture medium, and after inoculation, the culture medium was moved into a greenhouse for uniform cultivation; the culture conditions were: temperature 22-26°C, relative air humidity 60-65%, and good ventilation and light avoidance were ensured.

[0054] 5. Mushroom production test

[0055] The X001-X007 bags were managed for fruiting under the same culture conditions. During the fruiting period, the temperature was controlled at 18-23℃, the humidity was controlled at 80-95%, and the scattered light was 500-800Lx. The fruiting bodies of the hydrangea mushrooms were harvested when the leaves were unfolded, the edges were wavy, the back was slightly white "fluffy", and the fruiting bodies were white, strong and beautiful. Figure 2 As shown in Table 2, it is found that the strain numbered X003 has the earliest fruiting rate and the fruiting is neat, and each bag can produce 410g of fresh mushrooms.

[0056] Table 2

[0057] Strain number Full bag time (d) Mycelium density Mycelium growth Pollution rate (%) Output (g / bag) X001 67 Dense ++ 17 377 X002 71 Sparse + 25 228 X003 61 Thick +++ 5 410 X004 66 Dense ++ 16 369 X005 69 Sparse + 23 254 X006 64 Thick +++ 7 396 X007 70 Sparse + 29 235

[0058] Among them, "+" means that the mycelium is sparse and weak, "++" means that the mycelium is relatively sparse and grows well, "+++" means that the mycelium is dense and grows vigorously, and "++++" means that the mycelium is dense and strong.

[0059] By comparing the mycelial growth and yield of 7 different wild hydrangea strains, the strain numbered X003 was screened out.

[0060] 6. Molecular Biological Identification

[0061] ITS sequencing was performed on strain X003. Sequence 1 was obtained through amplification and DNA sequencing. Molecular biological identification showed that strain X003 belonged to the Fungi, Hymenophores, Polyporales, Hydrangeaceae, and Hydrangea. Comprehensive morphological identification showed that strain X003 belonged to Hydrangea Sparassis crispa .

[0062] 7. Preservation

[0063] On June 20, 2024, the wild domesticated Hydrangea strain X003 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It was classified and named Hydrangea, and the strain was named Ha Xiu 3. The deposit number is CGMCC No.41342.

[0064] Embodiment 2:

[0065] 1. Optimization of liquid strains

[0066] PDA medium formula: 200 g potato (peeled), 20 g glucose, 20 g agar and 1000 ml water; natural pH.

[0067] Weigh 200g of peeled potatoes, cut into thin slices, wash, add 1000ml of water, boil, boil for another 20min after the water boils, then filter with gauze, take the filtrate, if the filtrate is less than 1000ml, add more to make up, then add agar and cook until melted, add glucose, and sterilize in test tubes while hot. The test tubes should be filled with about one-fifth to one-quarter of the height of the test tubes, and autoclave at 121°C for 20min. Finally, place on a slant to obtain PDA culture medium.

[0068] The sawdust culture medium includes: 70% oak sawdust and 30% pine sawdust, with a water content of 60wt%.

[0069] Mix 70% oak sawdust and 30% pine sawdust evenly, stir the mixed dry materials with water to a water content of 60wt%, divide into test tubes for sterilization, the test tube filling amount is about two-thirds of the test tube height, and sterilize under high pressure at 121°C for 90min to obtain sawdust culture medium.

[0070] Liquid culture medium formula: 100g potato, 50g cottonseed hull, 15g glucose, 2g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 0.3ml glycerol and 1000ml larch extract; pH is 5-6.

[0071] Weigh 100g of peeled potatoes, cut into thin slices, wash, add 1000ml of larch extract and boil. After the water boils, boil for 5min. Add 50g of cottonseed hulls and boil for another 15min. Then filter with gauze and take the filtrate. If the filtrate is less than 1000ml, add 1000ml to make up. Then add 15g of glucose, 2g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate and 0.3ml of glycerol to melt. Divide them into Erlenmeyer flasks while hot, with the filling amount being about two-fifths of the volume of the Erlenmeyer flask. Autoclave at 121℃ for 20min to obtain liquid culture medium.

[0072] Inoculation method 1: Inoculate the hydrangea fungus Harxiu 3 into the PDA culture medium, and after the mycelium has grown all over the test tube, inoculate it into the liquid culture medium for shaking; culture it in a dark place with shaking at 20-25℃ and 140-160r / min.

[0073] Inoculation method 2: inoculate the hydrangea 3 into the PDA medium, transfer it to the sawdust medium for cultivation after the mycelium fills the test tube, transfer it to the PDA medium after it grows well, and insert it into the liquid culture medium for shaking after the mycelium grows well again; culture with shaking at 20-25℃ and 140-160r / min, and supplement with 500lx red light during the shaking process, with 12h of light and 12h of darkness every day.

[0074] The results of shaking time and mycelium growth under the two inoculation methods are shown in Table 3.

[0075] Table 3

[0076]

[0077] Among them, "+" means that the mycelium is sparse and weak, "++" means that the mycelium is relatively sparse and grows well, "+++" means that the mycelium is dense and grows vigorously, and "++++" means that the mycelium is dense and strong.

[0078] The results in Table 3 show that after rejuvenation with sawdust culture medium, the mycelium of Haxiu 3 test tube grew more robustly; adding red light treatment during the shaking process accelerated the growth rate of the fungus ball and shortened the shaking time.

[0079] 2. Cultivar Optimization

[0080] Cultivated variety formula: 83% pine sawdust, 10% wheat bran, 5% soybean powder, 1% gypsum powder and 1% sugar.

[0081] Treatment method 1: Choose fresh, mold-free culture medium, dry-mix wheat bran, soybean powder and gypsum powder in proportion, then add sawdust and continue dry-mixing, put sucrose into water to melt, mix the mixed dry material with sucrose water evenly, prepare the cultivation medium (water content 60wt%), put it into 17x35cm bags, put 1.5kg of medium into each bag, sterilize it at normal pressure for 12h, wait until the temperature drops to 25℃, and set aside.

[0082] Treatment method 2: pre-treat the pine sawdust before use, mix the pine sawdust with 0.5%~5% lime water at a mass ratio of 1:2, soak in 80-85℃ hot water, boil for 30 minutes, pick up the pine sawdust and rinse with clean water until the pH is about 7, expose to sunlight to obtain pre-treated pine sawdust. Then prepare the cultivation seed fungus material (water content 60wt%) according to the method of treatment method 1.

[0083] The optimized Haxiu 3 liquid strain was inoculated under sterile conditions and moved into a sterile culture room for uniform culture after inoculation. The results are shown in Table 4.

[0084] Table 4

[0085]

[0086] Among them, "+" means that the mycelium is sparse and weak, "++" means that the mycelium is relatively sparse and grows well, "+++" means that the mycelium is dense and grows vigorously, and "++++" means that the mycelium is dense and strong.

[0087] The results in Table 4 show that pretreatment of pine sawdust can remove harmful compounds in pine sawdust, shorten the time for the culture medium to fill the bag with mycelium, reduce the contamination rate, and ensure the mycelium grows robustly.

[0088] 3. Mushroom production management

[0089] The germinated Haxiu 3 was placed in an environment of 18-23℃ and 80-95% humidity for cultivation, and good ventilation was ensured. During the primordium differentiation period, different types of light (500lx, 12h / d) were given to stimulate and induce primordium formation. The light parameters and primordium formation rate are shown in Table 5.

[0090] Table 5

[0091]

[0092] Among them, "+" means uneven fruiting, "++" means relatively even fruiting, and "+++" means very even fruiting.

[0093] Table 5 shows that in the primordium differentiation period of Haxiu 3, 500 Lux yellow light irradiation can effectively promote the primordium formation ( Figure 1 ), shortening the primordium formation time and improving the uniformity of mushroom production. No primordium is formed in the dark treatment.

[0094] Embodiment 3:

[0095] 1. Mycelium growth rate test

[0096] Take Haxiu 3 and existing varieties of Hydrangea-DB (Northeast Edible Fungi Research Institute) and Hydrangea-SD (Jinxiang Mushroom-Feng Edible Fungi Research Institute, Shandong) as test materials, prepare Haxiu 3, Hydrangea-DB and Hydrangea-SD into test tubes, and take 0.5cm 2 The bacterial blocks were inoculated into the plate culture medium (d=9cm) and cultured at a constant temperature of 25℃. When the mycelium covered the plate, a 0.7cm diameter puncher was used to take out the bacterial blocks of the same age, equal size and no contamination and transferred them to the center of the plate culture medium (d=9cm). The plate was cultured at a constant temperature of 25℃. After the mycelium germinated, the cross-line method was used to mark the scale every 24h until the mycelium covered the plate. The mycelium growth rate was calculated and the mycelium growth potential was observed. The results are shown in Table 6.

[0097] Table 6

[0098]

[0099] Among them, "+" means that the mycelium is sparse and weak, "++" means that the mycelium is relatively sparse and grows well, "+++" means that the mycelium is dense and grows vigorously, and "++++" means that the mycelium is dense and strong.

[0100] 2. Cultivation test

[0101] The test tube seeds of the present invention, Hydrangea 3 and existing varieties Hydrangea-DB and Hydrangea-SD were prepared into cultivated seeds according to the optimized method in Example 2, and cultured in a sterile room away from light, with the temperature controlled at 20-25° C., the humidity controlled at 50-60%, and good ventilation ensured.

[0102] During the primordium differentiation period, yellow light (500lx) was used to stimulate and induce primordium formation. The primordium formation rate was counted after 20 days. The temperature was controlled at 20-23℃, the humidity was controlled at 80-85%, and good ventilation was ensured during the primordium differentiation period. During the fruiting stage, the temperature was controlled at 20-23℃, the relative humidity was 90-95%, and the light was 500-800Lx. When the leaves of the hydrangea fruiting bodies were unfolded, the edges were wavy, the back was slightly white "fluff", and the fruiting bodies were white, strong and beautiful, they were harvested. The fruiting body traits and yields of the three kinds of hydrangeas are shown in Table 7.

[0103] Table 7

[0104]

[0105] Among them, in the uniformity of mushroom production, "+" means uneven mushroom production, "++" means relatively uniform mushroom production, and "+++" means very uniform mushroom production.

[0106] Table 7 shows that the Hydrangea spp. Haxiu 3 of the present invention is more sensitive to light treatment than the mainstream varieties Hydrangea-DB and Hydrangea-SD in the prior art, has a higher primordium formation rate, good mushroom uniformity and higher yield.

[0107] 3. Temperature test

[0108] The cultivation of Haxiu 3, Xiuqiu-DB and Xiuqiu-SD was carried out in different temperature environments, with relative air humidity of 80-85% and light of 500Lx; the results of temperature and mushroom production rate are shown in Figure 8. It can be seen that the fruiting temperature range of the hydrangea fungus Haxiu 3 of the present invention is wider, has good low temperature resistance, is more adaptable to the environment, and is more suitable for factory cultivation in cold regions.

[0109] Table 8

[0110]

[0111] Although the present invention is disclosed as above, the protection scope of the present invention is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A strain of Hydrangea, characterized in that: The strain is Hydrangea Sparassis crispa ) Ha Xiu 3 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 20, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.41342.

2. A hydrangea fruiting body, characterized in that: For cultivating the hydrangea described in claim 1 ( Sparassis crispa ) The fruiting body obtained by Ha Xiu 3.

3. A method for cultivating the fruiting body of Hydrangea sphaerocephala according to claim 2, characterized in that: The steps include: (1) inoculating the hydrangea described in claim 1 into a sawdust culture medium for rejuvenation, culturing again to obtain a strain, and then inoculating the strain into a liquid culture medium to obtain a liquid strain; (2) The cultivation material is placed in a mushroom bag, sterilized, inoculated with the liquid fungus, and then subjected to fermentation and yellow light irradiation to promote primordium formation and fruiting management, and finally harvested to obtain the finished hydrangea mushroom product.

4. The method for cultivating hydrangea fruiting bodies according to claim 3, characterized in that: During the cultivation of the liquid bacteria in step (1), 500 lx red light is irradiated for 12 h / day.

5. The method for cultivating hydrangea fruiting bodies according to claim 4, characterized in that: In step (2), the cultivation material comprises, by mass fraction, 83% pretreated pine sawdust, 10% wheat bran, 5% soybean powder, 1% gypsum powder and 1% sugar; the pretreated pine sawdust is obtained by placing the pine sawdust in lime water and heating it to 80-85° C., then washing the pine sawdust with clean water until the pH is neutral, and then exposing it to the sun.

6. The method for cultivating hydrangea fruiting bodies according to claim 5, characterized in that: In step (2), the yellow light irradiation for promoting primordium formation has an illumination intensity of 500 lx and an irradiation time of 12 h / day.

7. The method for cultivating hydrangea fruiting bodies according to claim 6, characterized in that: The temperature for mushroom production in step (2) is 10-25°C.

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