KR102499480B1 - Novel white Hypsizygus marmoreus BW80(KACC93351P) strain that can be co-cultivated with brown beech mushroom - Google Patents

Abstract

The present invention relates to a novel white zelkova zelkova strain BW80 (KACC93351P) that can be cultivated by mixed inoculation with brown zelkova mushrooms in one container, and white fruiting bodies and brown and white mixed fruiting bodies derived therefrom will be. The strain BW80 (KACC93351P) according to the present invention is a new strain with different physiological characteristics and morphology from the existing zelkova zelkova strain, which is mixed with the brown zelkova strain at the same time to prepare a spawn and inoculate the medium, but do not inhibit each other It has a characteristic that it can be harvested simultaneously in one container because the cultivation period is similar. The brown and white mixed fruiting bodies produced simultaneously in one container can be provided as a new mushroom packaging item for zelkova mushrooms because of their excellent yield and commercial species.

Description

Novel white Hypsizygus marmoreus BW80(KACC93351P) strain that can be co-cultivated with brown beech mushroom}

The present invention relates to a novel white zelkova mushroom strain BW80 (KACC93351P) that can be produced through co-culture and growth with brown zelkova mushrooms and a fruiting body (mushroom) derived therefrom. It also relates to fruiting bodies (mushrooms) produced by cultivating brown and white zelkova mushrooms in one container using strains not used in the present invention.

Zelkova mushroom ( Hypsizygus marmoreus (Peck) HE Bigel.) is also known as Beech mushroom and is a representative edible mushroom called Bunashimeji in Japan. In Korea, the cultivation of zelkova mushroom first started in the late 1980s, and stable production and sales began in earnest in the early 2000s. As of 2018, the number of zelkova mushrooms traded through the domestic agricultural wholesale market is about 2,121 tons, reaching 6 billion won, and the transaction volume is increasing every year. As of 2019, the production of zelkova mushrooms in Japan was 118,597 tons, accounting for 26.1% of the total mushroom production, and it is the most consumed mushroom after 129,104 tons of enoki mushrooms. Zelkova mushroom is known to have a high content of glutamic acid, which is nutritionally equivalent to a seasoning component, and its main functional ingredients are antifungal activity and antitumor effect by Hypsin, collagen binding protein HM23, hypsiziprenol A9, anticancer effect by mushroom polysaccharide, etc. this has been reported

And as an example of the prior patent technology, published Patent Publication No. 10-2008-0043816 A method for preparing an extract derived from Lyophyllum Ulmarium fruiting body or mycelium, using an alkaline aqueous solvent as an extraction solvent A method for preparing a zelkova mushroom-derived extract has been disclosed. In addition, Registered Patent Publication Registration No. 10-1479553 discloses a method for manufacturing cosmetics using zelkova mushrooms and cosmetics thereof.

Despite these excellent nutritional and functional values, zelkova mushrooms have a cultivation characteristic that takes more than 100 days to grow, unlike oyster mushrooms, enoki mushrooms, and large oyster mushrooms. The life cycle of the mushroom is that basidiospores (mononuclear) are formed in the basidiomycetes in the folds of the fruiting bodies, which germinate to become mononuclear hyphae, and mononuclear hyphae that match the karyotype are fused and hybridize or hybridize. becomes Dinucleated mycelia, which have two nuclei in one cell, grow when nutrients necessary for mycelial growth are supplied. This leads to the process of fruiting body formation. After a suicide body (mushroom) is made and grown for a certain period of time, a basidia is formed in the folds made at the lower part of the cap, and mononuclear basidiospores are formed. However, unlike king oyster mushrooms, enoki mushrooms, and oyster mushrooms, zelkova mushrooms require a long cultivation period of about 105 days (85 days in culture, 25 days in growth), making cultivation difficult. In order to solve these disadvantages, many studies have been conducted to create varieties that can shorten the cultivation period. In the breeding of the mango mushroom, first, a parent strain with the desired trait is selected, then mononuclear basidiospores are collected, and artificially cultivated in a designated environment through artificial hybridization or crossing to select a strain with the trait desired by the grower. It is completed by naming the systemic name. As of October 2020, according to the data posted on the National Seed Resources website, there are 18 applications for variety protection of the Manga mushroom, and among them, 11 cases have been registered for variety protection rights. Among the varieties registered in the National Seed Resources, Sunshine (variety registration number 6377) is a brown variety with a much shorter cultivation period of 60-63 days compared to existing varieties. In the case of white zelkova mushrooms, those that take more than 110 days are cultivated, and no application for a variety that can be cultivated jointly with the existing brown variety has been made. Currently, brown and white zelkova mushrooms are separately packaged and auctioned or sold in domestic agricultural wholesale markets or large marts, but there has been no case where brown and white mushrooms are packaged and sold in one container. This can be said to be the biggest reason why brown and white cannot be produced in a single container.

An object of the present invention is to separate the existing brown and white zelkova mushrooms into individual mushroom containers, inoculate and grow spawn, and brown and white zelkova in one container for the existing brown zelkova mushroom varieties. The object of the present invention is to provide a novel white zelkova zelkova strain to simultaneously produce zelkova mushrooms. In addition, it is also an object of the present invention to provide a fruiting body (mushroom) formed from the novel white zelkova mushroom strain.

In order to achieve the above object, the present invention, as a first aspect, provides a novel white zelkova zelkova strain BW80 (KACC93351P) that can be cultivated by inoculation in one and the same container together with the existing brown zelkova zelkova strain. . The strain was deposited at the Agricultural Genetic Resource Center (KACC) of the National Academy of Agricultural Sciences of the Rural Development Administration as of March 2020 (Accession Number: KACC93351P). In another aspect of the present invention, a fruiting body derived from the white zelkova zelkova mushroom strain BW80 (KACC93351P) and a mixed fruiting body of brown and white zelkova mushrooms are provided.

The novel white zelkova zelkova strain of the present invention is a new variety showing genetic and phenotypic differences different from the existing zelkova zelkova strain, and has good mushroom generation and growth in bottle container cultivation, short cultivation period, and color It is white and has excellent quality characteristics. When the strains cultivated through the present invention are used for mushroom production, it is possible to simultaneously produce brown and white zelkova mushrooms in one container, instead of separately producing brown and white zelkova mushrooms, increasing production efficiency. In addition, it is expected that it will be possible to develop a niche consumer market by completing visually excellent marketability. In addition, brown and white mixed mushrooms can be produced, which can be used as new consumption items and export items.

1 is a photograph of the fruiting body of the white zelkova mushroom strain BW80 (KACC93351P) according to the present invention.
Figure 2 is a photograph of the appearance of a fruiting body grown by inoculating a white zelkova zelkova strain BW80 (KACC93351P) and a brown zelkova mushroom in one container according to the present invention.
3 is a photograph of a whole view of a fruiting body of brown and white zelkova mushrooms generated in one cultivation container.
Figure 4 shows the formation of a substitution line showing the fact that the substitution culture between the zelkova mushroom strain BW80 (KACC93351P) and the brown variety "Sunshine" according to the present invention and the substitution culture are different from each other.
Figure 5 shows the polymorphism of the gene amplification product by the nucleic acid amplification method (PCR) method to show the difference in nucleic acid level between the zelkova mushroom strain BW80 (KACC93351P) according to the present invention and the existing brown variety "Hassal".

Hereinafter, preferred embodiments are presented to aid understanding of the present invention, but the following embodiments do not limit the scope of the present invention.

(1) Zelkova Strand Mushroom Strains and Culture

The breeding parent strain used in this experiment was collected from the Biotechnology Department of the Gyeongsangnam-do Agricultural Research and Extension Services. "Sunshine" was used as a brown control variety for the simultaneous production of brown and white mixed mushrooms. Mushroom complete embryos (MCM) were cultured at 25 ° C and used for experiments, and stored at 4 ° C if necessary. For long-term preservation, the mycelia-infested PDA medium was cut into 1 × 1 cm, put in sterilized mineral oil, and stored at 4 ° C.

(2) Collection and mating of single spores

For the strains collected from the zelkova mushroom, fruiting bodies are generated for each strain, harvested before the caps are completely cleaved, and placed on a sterilized petri dish with the wrinkles of the fruiting body facing down for 48 hours. It was allowed to stand for a while to allow single spores to fall from the folds of the fruiting body. The monospores dropped on the Petri dish were smeared on PDA medium using a sequential dilution method, and germinated mononuclear mycelia were collected while culturing at 25 ° C for 7-10 days. Randomly select 20 single spores from each mother and parent line, inoculate and culture them on MCM medium, cut them into 1 × 1 cm (width × length) using a sterilized mallet, and place them in contact with each other in the center of the pet dish. After dentition, it was incubated at 25 ° C. Afterwards, when the hyphae of the two mononuclear strains that faced each other were sufficiently grown, the edges of the medium where the mushroom mycelia did not grow were cut off. When the culture medium is cut in a thermostat at 25℃ for 2-3 days, wait for the mushroom mycelium to grow to the bottom of the pet dish from which the medium was cut, and observe the mushroom mycelia at 200-400 times using a microscope. connection) was confirmed. The results observed through the microscope were written in the mating table, and it was confirmed whether the mated mycelia had normal hyphal fusion with each other. Mycelia with clamp connections were cut into 1 × 1 cm (width × length) using a sterilized mallet, transferred to a new MCM medium, and cultured in an incubator at 25 ° C.

(3) Culture and growth investigation

The medium for cultivating zelkova zelkova is mixed using cottonwood sawdust, wheat bran, rice bran, and dried bean curd, and after adjusting the moisture to around 65% and mixing well, put 550g each in an 850cc PP (polypropylene) bottle, and put the middle part of the medium through a hole (Perforated hole) I gave it away. The bottle containing the medium was sterilized at 121 ° C. and high pressure for 100 minutes using a steam sterilization pot. The sterilized medium was cooled to around 20 ° C. in a cooling room and then inoculated with mushroom spawn. Spawn inoculation was inoculated in MCM liquid medium in advance and cultured brown and white mycelium were mixed in one container and rotted well for about 1 hour. After confirming that the mushroom mycelium was well mixed, it was inoculated by carefully dispensing on the surface of the mushroom medium. The medium inoculated with the spawn was cultured for 50 days in a culture room set to a temperature of 25° C., relative humidity of 65%, and CO ₂ of 2,000 ppm or less. After incubation was completed, the bottle cap was removed for mushroom development, and bacteria were scraped, moved to a growth room, and maintained in an upside-down state at a humidity of 98% and a temperature of 16 ° C until germination was completed. After the hair removal was completed, the growth environment was created by adjusting the relative humidity of 96-988%, temperature of 16 ℃, and CO ₂ of 1,000 ppm or less after erection. Illuminance was maintained at 100-200 lux during the entire growth period. The fruiting body was harvested before the entire cap was completely cleaved as the lower part of the cap was separated from the cap. For quality evaluation, the characteristics of the large length were investigated by referring to the quality characteristics investigation method of zelkova zelkova.

(4) Selection of novel zelkova zelkova strains that can be simultaneously produced in one container with brown zelkova zelkova mushrooms

Through the above cultivation experiment, the possibility of simultaneous growth of brown and white mushrooms, quantity, weight, number of days to harvest, etc. were investigated for fruiting bodies generated in bottle cultivation. For the simultaneous production of mixed mushrooms of zelkova zelkova, it should not compete with the existing brown zelkova mushroom varieties, and it is possible only when the time of germination and the period required for harvest are within a similar range. After analyzing the characteristics of the fruiting bodies selected based on these criteria, a suitable strain BW80 ( ) capable of simultaneous production of brown and white mixed mushrooms was selected.

(5) Characteristics of novel white Zelkova strain BW80 ( ) that can be co-produced with brown Zelkova

The length of the fruiting bodies derived from the novel white zelkova mushroom strain BW80 (KACC93351P), which can be grown in one container to produce mushrooms, was 55.1 mm and the quantity was 225.8 g (/ 850 cc p.p container). The lightness of the shade was 85.4, and it had a bright white color. The total cultivation period from inoculating and culturing the spawn to harvesting the mushrooms was 60.8 days. The average length of the mushrooms produced in the medium inoculated by mixing with the brown Zelkova mushroom was 58.4mm, and the quantity was 122.1g (/850cc p.p container). was 59.0 days, which was almost similar to the number of days required for harvesting when inoculated alone. These results make it possible to produce brown and white mixed mushrooms at once more efficiently than the method of independently inoculating spawn to produce brown and white zelkova mushrooms. In addition, natural production will be possible due to random mushroom generation by seed fungus mixing rather than artificial mixing of brown and white, and it will be able to be used as a mushroom sales item by realizing visual effects to consumers who purchase mushrooms. Table 1 shows the characteristics of the fruiting bodies derived from the existing brown zelkova zelkova cultivar "Sunshine" and the new white zelkova zelkova mushroom strain BW80 (KACC93351P). Brown and white mixed fruit bodies grown in one container with brown zelkova mushrooms using the new strain and fruiting derived from the novel white zelkova mushroom strain BW80 (KACC93351P) of the present invention are shown in Figures 1 and 2 and shown in Figure 3.

division candle
feet
(0-4) after
feet
(0-4) big
length
(mm) Yield (g/bottle)
chromaticity number of days on foot
(Day) Number of harvest days (days) L a b BW80 3.5 3.3 55.1 125.8 85.4 1.3 13.9 9.0 20.8 sunlight 3.0 3.0 55.2 104.6 61.2 5.4 21.0 8.7 20.3 BW80+sunlight 3.5 3.5 58.4 122.1 - - - 8.0 18.0

Table 1. Comparison of cultivation characteristics according to mixed mushroom production using brown Zelkova (Haesat) and new white Zelkova BW80 strain

(6) Uniqueness test

In order to examine the uniqueness of the cultivated strain, the sunlight and the novel white zelkova mushroom strain BW80 () were cut into 1 × 1 cm horizontally and vertically and transferred to a location 2-3 cm apart on the MCM medium, respectively, and mutually incubated at 25 ° C. was cultured until the contact surface grew and observed whether a line of inhibition (line of substitution) was formed. In the uniqueness test using nucleic acid fingerprinting, the gDNA of Sunshine and a new zelkova mushroom strain BW80() was extracted using a DNeasy plant mini kit (Qiagen, USA), a PCR Premix kit was used, and genomic DNA 10 4 μl of ng, 5 μl of primer 1 pmol, and 11 μl of DDW were added. At this time, #8010 (5'CTGACACGGA-3' and #8030 (5'GCCACCTCCT-3') were used as primers (Bioneer, Korea), and Eppendorf proS (Germany) was used for the PCR amplification reaction. 1 cycle of 5 minutes, 94 ℃ for 1 minute, 37 ℃ for 1 minute, and 72 ℃ for 2 minutes were carried out a total of 45 times, and the final DNA synthesis was 10 minutes at 72 ℃. DNA polymorphism was observed by loading and staining with SafeView Classic. Through the above method, the inhibitory line ( Daechi line) was confirmed, and the two varieties were determined to be different varieties (Fig. 4). As a result of comparing the DNA patterns of the existing variety, Sunshine, and the white zelkova mushroom strain BW80 ( ) cultivated in the present invention, It was confirmed that they corresponded to completely different breeds by showing different aspects of genetic differences (Fig. 5A (#8010), Fig. 5B (#8030)).

As a result of the above, by the method of the present invention, the novel white zelkova zelkova strain BW80 ( ) can be cultivated by mixing and inoculating with brown zelkova zelkova mushrooms using a container that can be sterilized at high temperature and high pressure. In addition, brown and white mushrooms can grow and harvest at the same time in one container, so if applied to existing mushroom growers, it will not only create a new source of income, but also give newness to consumers who purchase mushrooms. As described above, in the detailed description of the present invention, specific embodiments have been described, but various modifications are possible without departing from the scope of the present invention. Therefore, it is natural that the practical scope of the present invention should not be limited by the above-described embodiments, and should be defined by not only the claims to be described later but also the claims and equivalent configurations.

Name of depository institution: Center for Agricultural Genetic Resources (KACC)

Accession number: KACC93351P

Entrusted date: 2020. 09. 17.

<110> Gyeongsangnam-do Agricultural Research & Extension Services <120> Novel white Hysizygus marmoreus BW80(KACC P) strain that can be co-cultivated with brown beech mushroom <130> 00-000000 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 10 <212> DNA <213> artificial sequence <220> <223> PCR Forward primer to detect the differentiation of Hysizygus marmoreus strains, Primer sequence (#8010) <400> 1 ctgacacgga 10 <210> 2 <211> 10 <212> DNA <213> artificial sequence <220> <223> PCR Forward primer to detect the differentiation of Hysizygus marmoreus strains, Primer sequence (#8030) <400> 2 gccacctcct 10

Claims (4)

New strain BW80 (KACC93351P) that can be cultivated by simultaneously inoculating brown zelkova mushrooms and white zelkova mushrooms in one container White zelkova mushroom fruiting body formed by the new strain of claim 1 Mixed brown and white fruiting bodies formed together with brown zelkova mushrooms by inoculation in one container using the white zelkova mushroom strain BW80 (KACC93351P) of claim 1 Producing brown and white mixed fruiting bodies (mushrooms) cultivated together with brown zelkova mushrooms by inoculating in one container using other zelkova mushroom strains other than the white zelkova mushroom strain BW80 (KACC93351P) of claim 1 method

Images