WO2012111882A1

WO2012111882A1 - Strain of novel white flammulina velutipes

Info

Publication number: WO2012111882A1
Application number: PCT/KR2011/003287
Authority: WO
Inventors: Won Sik Kong; Young Bok Yoo; Kab Yeul Jang; Chang Sung Jhune; Pyung Gyun Shin; Jae Gun Kweon
Original assignee: Republic Of Korea(Management:Rural Development Administration)
Priority date: 2011-02-17
Filing date: 2011-05-03
Publication date: 2012-08-23
Also published as: KR20120094785A; KR101239641B1

Abstract

A novel white Flammulina velutipes strain (KCCM11134P) and a novel white Flammulina velutipes fruiting body formed from the novel white Flammulina velutipes strain. The novel white Flammulina velutipes strain is a novel strain showing a distinct genetic difference from the known white Flammulina velutipes strain. It is expected that the novel white Flammulina velutipes strain can reduce royalty fees and generate agricultural revenue, thereby improving national export competitiveness.

Description

STRAIN OF NOVEL WHITE FLAMMULINA VELUTIPES

The present invention relates to a novel white Flammulina velutipes strain and a fruiting body of the novel white Flammulina velutipes strain.

Flammulina velutipes is taxonomically classified as belonging to the phylum Basidiomycota, order Agaricales, family Tricholomataceae, genus Flammulina. Wild Flammulina velutipes occurs in bundles on broad-leaved stumps or old trees in temperate or frigid zones during the low-temperature period from late fall to early spring. It has a yellowish brown to brown fruiting body, a thick cap, and a thick short stipe (see Won-Sik Kong, Doctoral Thesis, Graduate School of Konkuk University, 1997).

The Flammulina velutipes that is currently being cultivated for table use was artificially cultivated using persimmon tree wood under natural atmospheric conditions in 1899 in Japan, and a method of inoculating Flammulina velutipes spores was developed in 1926, after which the log cultivation of Flammulina velutipes became prevalent. Additionally, box cultivation of Flammulina velutipes in a sawdust medium was first attempted in 1928, and since then methods of cultivating Flammulina velutipes using rice bran and nutrient additives have been developed. In Korea, the log cultivation method was first introduced in 1936, and was then studied in the National Institute of Agricultural Science and Technology, the Rural Development Administration, and has been widely spread to farmers since the end of the 1980s (Chang-Ye Yoo, Master’s Thesis, Graduate School of Gyeongsang National University, 1997). Unlike wild Flammulina velutipes, cultivated Flammulina velutipes has a cap size of about 10 mm or less, a stipe length of 100-140 mm, and a stipe diameter of about 2-4 mm (see Won-Sik Kong, Kor. J. Mycol., 25(2), 111-120, 1997).

Methods of producing Flammulina velutipes are divided into sexual reproduction and asexual reproduction. In the sexual reproduction process, a fruiting body grows from the mycelium to form four basidiospores which then germinate and grow. In the asexual reproduction process, asexual spores (oidia) produced by segmentation of the formed dikaryotic mycelium germinate and grow. In the mycelial growth of Flammulina velutipes, the asexual reproduction process occurs more frequently, so the formation of monokaryotic oidia from monokaryotic mycelia and dikaryotic mycelia easily occurs (Takemaru, T., Trans. Mycol. Soc. Japan, 36, 152-157, 1995).

In the breeding of Flammulina velutipes, economic efficiency, high productivity, easy cultivation, and genetic safety in the production of spores are considered important factors. Particularly, studies on the development of white Flammulina velutipes to satisfy consumers’ preference have been prominent.

However, the white Flammulina velutipes species that are being cultivated worldwide are all bred in Japan and share very close kinship, thus having similar genetic structures. Accordingly, Korean farmers who cultivate and export Flammulina velutipes are obliged to pay significant royalty fees, which impose a serious burden on the farmers as the plant variety protection system is regularly enforced. For this reason, the development of a native Korean white Flammulina velutipes species is urgently required.

The breeding methods that have been used for the development of new Flammulina velutipes species include: a vegetative propagation/selection method (Senbatsu) of selecting a mushroom species that shows excellent characteristics during cultivation, collecting spores from the mushroom species, and culturing the spores; and a breeding method of mating monokaryotic strains formed during mycelial growth. The former method has problems in that the stable heritability of good traits is low and a small change in the cultivation environment leads to a rapid decrease in productivity. Meanwhile, the mating method has a shortcoming in that it requires a process of selecting and isolating monokaryotic strains having desired traits from wild species or cultivated species, and thus it is difficult to apply the mating method when monokaryotic strains do not exist (see Kitamoto, Y., In Genetics and Breeding of Edible Mushrooms, Gordon and Breach Science Publishers, pp 65-86, 1993).

Accordingly, there has been a continued need to develop a breeding method that overcomes the above-described problems, to breed a Native Korean white Flammulina velutipes species, and to spread the white Flammulina velutipes species to farms.

A number of publications and patent documents are cited through the specification in order to more clearly describe the present invention and the state of the art to which the present invention pertains. The entire disclosure of the cited publications and patent documents is incorporated herein by reference.

The information disclosed in this Background of the Invention section is only for the enhancement of understanding of the background of the invention, and should not be taken as an acknowledgment or any form of suggestion that this information forms a prior art that would already be known to a person skilled in the art.

The present inventors have made many efforts to develop a novel white Flammulina velutipes strain which can contribute to agricultural revenue while avoiding royalty fees. As a result, the present inventors have found that, when white Flammulina velutipes strains are isolated by a multi-sporous random mating method, a novel white Flammulina velutipes strain showing a significant genetic difference from existing white Flammulina velutipes strains can be isolated, thereby completing the present invention relating to a novel white Flammulina velutipes strain.

Therefore, it is an object of the present invention to provide a novel white Flammulina velutipes strain.

According to one aspect of the present invention, there is provided a novel white Flammulina velutipes strain. The strain was deposited with the Korean Culture Center of Microorganisms (KCCM) on November 23, 2010, and assigned accession number KCCM 11134P.

According to another aspect of the present invention, there is provided a white Flammulina velutipes fruiting body formed from the strain.

The methods and apparatuses of the present invention have other features and advantages which will be apparent from, or are set forth in more detail in the accompanying drawings, which are incorporated herein, and in the following Detailed Description of the Invention, which together serve to explain certain principles of the present invention.

FIG. 1 shows the results of examination of the cultural and morphological characteristics of Flammulina velutipes fruiting bodies by evaluating the characteristics of Flammulina velutipes genetic resources;

FIG. 2 is a photograph showing a general method of collecting spores from a mushroom;

FIG. 3 is a schematic view showing a step of isolating a monokaryotic strain;

FIG. 4 is a photograph showing the tools required for crossing;

FIG. 5 is a photograph showing the results of pairing culture for crossing;

FIG. 6 is a photograph showing that a clamp was formed as a result of crossing;

FIG. 7 is a photograph showing a process of culturing a spore suspension by a multi-sporous random mating method;

FIG. 8 is a photograph showing the results of breeding a native white strain by the crossing between brown wild strains;

FIG. 9 shows the genetic difference between a white strain of the present invention and a native Japanese pure strain; and

FIG. 10 is a picture showing novel white Flammulina velutipes fruiting bodies.

Reference will now be made in detail to various embodiments of the present invention, examples of which are illustrated in the accompanying drawings and described below. While the invention will be described in conjunction with exemplary embodiments, it will be understood that the present description is not intended to limit the invention to those exemplary embodiments. On the contrary, the invention is intended to cover not only the exemplary embodiments, but also various alternatives, modifications, equivalents and other embodiments that may be included within the spirit and scope of the invention as defined by the appended claims.

Examples

Evaluation of genetic resources of Flammulina velutipes

The present inventors attempted to construct a novel white Flammulina velutipes strain, which is different from native Japanese white strains, by crossing between only brown Flammulina velutipes species other than the native Japanese brown species that are currently cultivated worldwide.

A. Test strains

In this study, 102 Flammulina velutipes strains, including Flammulina velutipes 4001, collected and archived in the Mushroom Research Division, the National Institute of Horticultural & Herbal Science, the Korean Rural Association, were tested (Table 1).

Table 1

B. Examination of cultural characteristics

Test of medium type

In order to examine the type of medium suitable for mycelial growth and the degree of mycelial growth according to each Flammulina velutipes strain, the following media were prepared: PDA (39 g Gellix PDA and 1000 ㎖ water); YM (3 g yeast extract, 3 g malt extract, 5 g peptone, 10 g dextrose, 20 g agar 20 g, and 1000 ㎖ water); MEA (20 g malt extract, 5 g peptone, 20 g agar, and 1000 ㎖ water); and MCM (20 g dextrose, 0.46 g MgSO4, 1 g K2HPO4, 2 g yeast extract, 2 g peptone, 20 g agar 20 g, and 1000 ㎖ water. Each of the media was placed in an 87-mm disposable Petri dish, and mushroom mycelia were inoculated therein and cultured for 7 days, after which the diameter of the flora was measured.

Test of culture temperature

Each mushroom strain was inoculated in a DA medium-containing glass dish and cultured at each of 10°C, 15°C, 20°C, 25°C, 30°C and 35°C for 7 days, after which the diameter of the flora was measured.

C. Examination of cultural characteristics

Pine sawdust and rice bran were mixed with each other at a volume ratio of 8:2 and adjusted to have a water content of about 65%. Then, the mixture was placed in an 850-ml polypropylene (PP) bottle and sterilized under high pressure. Then, each Flammulina velutipes strain was inoculated into the mixture and cultured at 25°C in an environment-controlled culture room according to a standard culture method. Whether the mushroom was developed was examined, and the weight, shape, color, size and the like of the mushroom were examined.

Culture method:

Standard temperature conditions: Fruiting 14°C, Cold room 4°C, and Growth 7°C.

Breeding of spore-collecting monokaryotic strains in brown fruiting body-forming strains (16 strains)

A. Spore collection

The collection of spores was performed as follows using some modification of the general method of collecting spores (FIG. 2).

(1) A mature fresh mushroom was selected on a clean bench, and the stipe was cut off. Then, the remaining mushroom cap was placed in a glass dish in such a manner that the gills of the cap faced the bottom of the glass dish. Then, the glass dish was covered with a cap in order to prevent air from flowing into the dish.

(2) The glass dish containing the mushroom was kept in a clean place at a temperature of 15~20°C for 6-15 hours so that the spores dropped. Then, the mushroom was removed, and the glass dish containing the spores was stored in a refrigerator at 4°C.

B. Isolation of monokaryotic strain

Basidiospores received from one fruiting body (mushroom) were stored at 4°C and were used in tests as required. The basidiospores were serially diluted in distilled water and applied on a MCM-containing plate medium at a density of about 1 x 104 basidiospores, after which they were cultured at 25°C for 3 to 6 days. Then, the flora were observed with a microscope, and those having no clamp were determined to be monokaryotic strains and transferred to a fresh PDA (potato dextrose agar) medium and used in the test (FIG. 3).

Breeding of hybrid strain by crossing between monokaryotic strains

A. Experimental materials

As shown in FIG. 4, the tools required to cross spores (alcohol lamp, spatula, and punch) were prepared, and the monokaryotic strains isolated as described above were prepared. Before crossing, the monokaryotic strains were punched with a cork borer or a punch to make a disc (circular inocula). All the operations were performed under aseptic conditions (FIG. 4).

B. Crossing method

For crossing, two monokaryotic strains were placed in the same Petri dish in such a manner that they were spaced apart from the bottom of the dish by about 10 mm, after which the strains were cultured at 25°C for 7-12 days. After the culture, a mycelium was separated from the place where the two mycelia met each other, and was observed with a microscope in order to confirm whether a clamp was present in the separated mycelium. After 7 days, both ends of the flora were observed, and those having a clamp were regarded as dikaryotic mycelia.

The crossing was simpler than expected and was based on pairing culture, and the number of the hybrid strain and the date of the experiment were noted (FIG. 5 a).

The mushroom strains were mostly cultured at 25°C, and as shown in FIG. 5(b), a boundary line occurred at the center with the passage of time while the strains were partially crossed. Whether the strains have been crossed was determined by microscopically observing whether a clamp was produced (FIG. 6). As a result of the experiment, 454 dikaryotic strains were bred (Table 2).

Table 2

In Table 2 above, the top row indicates the reference numbers of the strains that are archived in the Mushroom Research Division, the Korean Rural Association (for example, 4004 is ASI4004, and 4005 is ASI4005), and the data expressed as fractions indicate (the number of strains determined to be dikaryotic / the number of hybrid strains between monokaryotic strains).

C. Acquisition of spores

The dikaryotic strains obtained as described above all formed brown fruiting bodies, and among them, light-brown dikaryotic strains (expressed as (52(22) and 23(16)) were selected, and spores were collected therefrom (FIG. 8B).

Breeding of white Flammulina velutipes by multi-sporous random mating

A. Experimental method

By multi-sporous random mating, a white Flammulina velutipes strain was obtained. A method of breeding a white Flammulina velutipes by multi-sporous random mating is as follows.

Spore collection: The 52(22) species was selected, and only the mushroom cap was separated therefrom and placed in a disposable Petri dish in such a manner that the gills faced downward. The lid of the dish was kept slightly open. The next morning, a sufficient amount of spores of the 52(22) species had been obtained.

Preparation of spore suspension: A small glass bottle (20-30 ㎖) was filled about 2/3 full with water which was then sterilized. After the sterile water has been cooled, the tip of a pipette was dipped into the sterile water on a clean bench and stained with the above-collected spores of the 52(22) species. The spores were then released into the sterile water-containing bottle, thereby preparing a spore suspension of the 55(22) species.

Plating of spore suspension: Several drops (50-200μl) of the prepared spore suspension were added to a PDA medium, and were then evenly spread on the medium. The medium thus plated was incubated in an incubator for 7 to 14 days, so that random mating between the germinated monokaryotic mycelia was achieved (FIG. 7).

Selection of floras: Because the cultured floras grew separately from each other, suitable amounts of the floras were picked and transferred to different Petri dishes in which they were then cultured.

Selection and maintenance of strains: After each strain had been cultured, the strains were inoculated in different bottles to produce normal mushrooms. From the produced mushrooms, five white strains (23(16)-52, 52(22)-46, 52(22)-42, 52(22)-41, and 52(22)-45) were selected. To maintain the selected strains, tissues were isolated from the mushrooms for use as stocks.

B. Results of experiment

In the case of the line breeding method, employing the multi-sporous random mating method, a period of 30 days, corresponding to about 1/3 of the conventional breeding period (90 days), was required. Through the line breeding method, five strains (expressed as 23(16)-52, 52(22)-46, 52(22)-42, 52(22)-41, and 52(22)-45) were obtained (FIG. 8C)). Among the five strains, it was possible to obtain a number of Flammulina velutipes bodies from multiple spores (F1) of the strain 52(22), and for this reason, the parental line 52(22) was named the “SS line.” The obtained strain 52(22)-41 was named “SS-41.” The SS-line strain was deposited with the Korean Culture Center of Microorganisms (KCCM) on November 9, 2010 and assigned accession number KCCM 11129P, and the SS-41 strain line was deposited with the Korean Culture Center of Microorganisms (KCCM) on November 23, 2010 and assigned accession number KCCM11134P.

Comparison of DNA pattern between the above bred Native Korean line and Native Japanese species

A. Extraction of genomic DNA

As controls, the Japanese white strain Gosa (C), which is currently most frequently cultivated in farms, and the SS line were used. Also, 2 strains (B) producing brown Flammulina velutipes and 5 strains (W) producing white Flammulina velutipes were used. For extraction of genomic DNA, the mycelium of each strain was inoculated on a PDA plate and cultured in an incubator at 26°C for 7 days. Then, the mycelium grown on the PDA plate was transferred to an MCM liquid medium and cultured for 7 days. Then, the mycelium was freeze-dried and used in this experiment. DNA was extracted using a Mag extractor (plant DNA purification kit; TOYOBO). The freeze-dried mycelium was finely crushed with liquid nitrogen, and 600μl of a lysis buffer was added to 500 mg of the mycelium and allowed to react at 65°C for 10 minutes. Then, 600μl of phenol: chloroform: isoamyl alcohol (25: 24: 1) was added thereto, the mixture was vortexed and centrifuged at 13,000 rpm for 15 minutes, and the supernatant was collected. 600μl of adsorbent liquid and 40μl of magnetic beads were added to the supernatant, and the tube was inserted into a trapper so that the DNA was attached to the magnetic beads. The magnetic beads were washed twice with each of washing liquid and 70% ethanol and dried at room temperature for 10 minutes, after which the beads were dissolved in 100μl of triple-distilled water. The tube was inserted into a trapper to separate the DNA-containing solution from the magnetic beads, and then the DNA alone was transferred into a fresh tube, stored at 4°C and used as a template DNA for PCR.

B. URP-PCR conditions

For URP-PCR, a PCR premix kit (Bioneer) was used. To the premix kit, 2μl of genomic DNA 50 ng, 1μl of primer 100 ng and 17μl of DDW were added. The PCR amplification was performed using ABI PCR SYSTEM 9700 under the following conditions: initial DNA denaturation at 94°C for 5 min; 35 cycles of 1 min at 94°C, 1 min at 55°C and 2 min at 72°C; and final DNA extension at 72°C for 10 min. The amplified PCR product was subjected to 1.5% agarose gel electrophoresis in 1× TAE buffer (40 mM Tris; pH 8.0, 20 mM acetic acid, 1 mM EDTA), and then stained with 1 ㎍/㎖ ethidium bromide solution. The DNA band appearing in a UV transillumanator was observed. As the primer, primer No. 9 of a commercial primer kit (SRILS UniPrimer Kit) consisting of 12 primers was used.

C. Results of experiment

When the DNA pattern of the native Japanese strain was compared with the DNA patterns of the native Korean strains of the present invention, it could be seen that the strains of the present invention showed a distinct genetic difference from the native Japanese strain, indicating that the strains of the present invention differ completely from the native Japanese strain (FIG. 9).

In addition, the fruiting bodies formed from the novel white Flammulina velutipes strain of the invention are shown in FIG. 10.

The features and advantages of the present invention are summarized as follows:

The novel white Flammulina velutipes strain of the present invention is a novel strain showing a distinct genetic difference from the known Japanese white Flammulina velutipes strain. It is expected that the novel white Flammulina velutipes strain can reduce royalty fees and generate agricultural revenue, thereby improving national export competitiveness.

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for the purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teachings. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and their practical application, to thereby enable others skilled in the art to make and utilize various exemplary embodiments of the present invention, as well as various alternatives and modifications thereof. It is intended that the scope of the invention be defined by the Claims appended hereto and their equivalents.

Claims

A novel white strain KCCM 11134P of Flammulina velutipes.
A novel white Flammulina velutipes fruiting body formed from the strain of claim 1.