CA1137430A - Method of producing novel monokaryotic mycelium of coriolus versicolor - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
When a dikaryotic mycelium of Coriolus versicolor (Fr.) Quél. (a species of fungi belonging to the genus Coriolus of Polyporaceae) is subjected to a mechanical treatment such as grinding or shearing in a liquid medium, or when such mycelium is subjected to submerged culture while undergoing the mechanical treatment, there is produced a monokaryotic mycelium which is different from the dikaryotic mycelium in morphological and physiological characteristics.
The thus obtained monokaryotic mycelium is a novel product and characterized by its extremely high propagation ??te as compared with the known dikaryotic mycelium. Also, the substance extracted from the culture of the mycelium has excellent physiological activities.

Description

~1374;~) This invention relates to a method of producing novel monokaryotic mycelium from Coriolus versicolor (Fr.) Quél. which is a known Basidiomycete belonging to the genus Coriolus of Polyporaceae.
The usefulness of the polysaccharides obtained by tlle extraction of Coriolus versicolor (Fr.) Quél. or a culture thereof as a base component for the preparation of medical drugs or foods and drinks has come to be acknowledged recently, and various attempts for producing such Basidiomycete in a high yield by artificial culture have been made. Never-theless, there is still no advantageous method capable of propagating the Basidiomycete in a high yield.
In the course of our study aimed at realizing high-yield propagation of Coriolus versicolor (Fr.) Quél., we found that when this Basidiomycete is subjected to submerged culture while performing a mechanical treatment, such as grinding or shearing in a liquid medium, the Basidiomycete loses its clamp connection, which is its intrinsic morphological characteristic, and is changed into a monokaryotic mycelium, and that the thus-formed monokaryotic mycelium is stable and also has a specificcharacteristic in that it is extremely high in its propagation rate as compared with the known dikaryotic mycelium.
An object of this invention is thus to provide a method of producing a monokaryotic mycelium having no clamp connection.
According to one aspect of the invention there is provided a method of producing a monokaryotic mycelium of Coriolus versicolor (Fr.) Quél., characterized in that a dikaryotic mycelium of Coriolus versicolor (Fr.) Quél. is first subjected to a mechanical treatment in a liquld medium and then subjected to a submerged culture, or said dikaryotic

- 2 - ~

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mycelium is subjected to a submerged culture while performing said mechanical treatment on said mycelium.
According to another aspect of the invention there is provided monokaryotic mycelium of Coriolus versicolor (Fr.) Quél.
The mechanical treatment above can be used to convert the dikaryotic mycelium to the monokaryotic form.
Other objects and features of this invention will become apparent from the following detailed description of preferred forms of the invention, with reference to the accompanying drawings, in which:
FIG. 1 is a graph showing comparatively a propagation curve in an aerated and agitated culture of the monokaryotic mycelium derived from Coriolus versicolor (Fr.) Quél. according to one embodiment of the method of this invention and a similar propagation curve of the known dikaryotic mycelium;
FIGS. 2 and 4 are microphotographs of the dikaryotic mycelium obtained from a slant culture of Coriolus versicolor (Fr.) Quél.; and FIG. 3 is a microphotograph of a monokaryotic mycelium obtained according to one embodiment of the present invention.
In the graph of FIG. 1, the mycelium concentration (g/l) in the medium is plotted as the ordinate and the cul-tivation time (hr) as the abscissa. Also, (I) indicates the propagation curve of the dikaryotic mycelium and (II) the propagation curve of the monokaryotic mycelium.
It is generally believed that the fungi constituting a mushroom produce basidiospores and such spores germinate to form a primary mycelium usually consisting of monokaryons, and such a mycelium fuses together to form a secondary ~3743~

mycelium consisting of dikaryons. It is believed that the monokaryotic mycelium has no ability to form the fruit bodies but the dikaryotic mycelium has such ability. No report has been made concerning the generation of the monokaryotic mycelium from the spores in Coriolus versicolor (Fr.) Quél.
Moreover, the white aerial mycelium obtained in our experiments by cultivating the spores was dikaryotic, and this appears to be due to the fact that the monokaryotic mycelium from the spores is rapidly converted into dikaryotic form.
The monokaryotic mycelium obtained from the di-karyotic mycelium according to this invention can be maintained in stable form, presenting a striking difference from the existing dikaryotic mycelium. Table 1 below lists the differences between the monokaryotic mycelium of Coriolus versicolor (Fr.) Quél. and the dikaryotic mycelium.
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Table 1 Monokaryotic mycelium Items Dikaryotic mycelium (this invention) . . .

Appearance 1. Submerged Non-suspended Suspended contition.
culture condition.
2. Plate culture Aerial mycelium Not formed.
is formed.

Microscopic observation 10 3. Formation of Observed. None.
clamp connection 4. Shape of Long and fine. Shorter and far thicker mycelium than dikaryotic mycelium.

Physiological and biochemical properties 5. Propagating Low. High.
rate 6. Cellulose Positive. Slightly positive.
assimilation 7. Potassium No growth. Growth.
nitrate as a sole nitrogen source 8. Thiamine Required. Not required.
9. Litmus milk Acidified. Not acidified.
medium -- --~37~3(~

The followin~ facts are to 'be further noted in connection with the properties of the respective mycelium shown in the above Table. The ordinary dikarvotic mycelium obtained by cultivation of Coriolus versicolor (~r.) Quél.
according to a conventional method are usually in the form of pellets. On the other hand, the monokaryotic mycelium, when cultivated, is not formed into pellets and the culture assumes a turbid condition as if pulp were suspended in water, thus presenting an obvious difference from the ordinary di-karyotic mycelium.
A method for counting nuclei in the cell was completedfor the first time b'y the present inventors, and this method comprises the following means and techniques.
Helly's fixing fluid is first added to the Basidiomycete mycelium, and then this mycelium is allowed to stand usually for about 24 hours and then washed with water until it is decolored. The thus obtained mycelium is immersed in lN hydrochloric acid solution and the solution is heated to a temperature of 60C. After cooling to room temperature and washing with water, it is further immersed in 20 to 50 times diluted nitric acid solution, followed by additional washing with water. The period of immersion is from ten to twenty minutes in the hydrochloric acid solution and a few minutes in the nitric acid solution. The thus obtained fibrous cells are spread on a slide glass and left thereon until moisture evaporates away, and then Giemsa's solution is added dropwise thereto. At the point when staining has been accomplished with the solution (approximately 10 minutes later), the cells are washed lightly with water and then dried. After drying, the cells are examined under a light microscope of 1,000 magnifications and the circular red-stained spots 1~l3~43~}

(considered to be nuclei) are counted. Thus, the number of nuclei can be determined by counting the red-stained spots in one cell.
"Helly's fixing fluid" used herein is a solution of which the base is prepared by dissolving 2.5 gr of potassium bichromate, 1 gr of sodium sulfate and 5 gr of mercuric chloride in 100 ml of water, and immediately before use, such base solution is mixed with formalin in an amount of 5 ml per 100 ml of the solution.
"Giemsa's solution" is a nucleus staining solution prepared by dissolving 3.0 gr of azur II eosine and 0.8 gr of azur in 250 ml of glycerin by heating them to 60C, further adding thereto 250 ml of methyl alcohol, allowing the mixed solution to stand for 24 hours and then filtering the solution.
In use, the thus prepared stock solution is diluted by adding a phosphoric acid buffer solution (pH 6.4 - 6.8) in an amount of 100 ml for 3 ml of the stock solution.
As a result of the measurements by the above-described method, it was found that the number of nuclei in one cell of the ordinary pellet-shaped mycelium is 2 whereas that of the suspension-type mycelium produced by this invention is 1.
The monokaryotic mycelium exhibiting the peculiar ~ -properties, such as those stated above, can be produced by subjecting the dikaryotic mycelium of Coriolus versicolor (Fr.) Quél. to a mechanical (physical) treatment such as grinding or shearing in a liquid medium, or by subjecting the dikaryotic mycelium to submerged culture while practicing the mechanical treatment on the mycelium. More specifically, this method may be accomplished in the following ways.
(1) When subjecting the dikaryotic mycelium of Coriolus ~3~30 versicolor (Fr.) nuél. to a shaking culture, the mycelium can be ground by adding an inactive solid granular material, such as glass beads.
(2) When subjecting the dikaryotic mycelium to a con-tinuous submerged culture, such culture is performed while shearing the mycelium with an agitating element.

(3) When subjecting the dikaryotic mycelium to submerged culture, the mycelium can be sheared or ground by a homogenizer to such an extent that no pellet-shaped mycelium is noted by external observation.
In the formation of the monokaryotic mycelium under the above-mentioned conditions, it is preferable also to use the following techniques.
(i) An enriched nutritious condition can be provided by using a medium with a concentration 1.5 to 3 times as high as the ordinary medium, for example, a glucose-yeast extract medium containing 5% glucose and 0.75% yeast extract.
(ii) The atmosphere above a submerged culture can be maintained under reduced oxygen partial pressure. The "reduced oxygen partial pressure" may be provided by keeping the fermenter airtight, or by flowing an inert gas, such as nitrogen gas or carbon dioxide gas, into the fermenter.
(iii) A submerged culture can be carried out continuously while supplying additional amounts of the liquid medium.
The dikaryotic mycelium of Coriolus versicolor (Fr.) Quél. can easily be converted into the monokaryotic mycelium by employing the above-mentioned methods (1) - (3), either singly or in suitable combination with techniques (i) - (iii). -When a sufficient amount of the monokaryotic mycelium can not be obtained by one run of culture, the above-mentioned operation can be continued after homogenizing the culture ~37431~

until the desired amount of monokaryotic mycelium is obtained.
The culture is usually practiced at a temperature of 25 + 5C
for a period of 3 to 15 days.
It was found that the monokaryotic mycellium of Coriolus versicolor (Fr.) Quél. thus obtained always maintains the same state and the same properties if the cultivation is continued under the above-mentioned conditions. This means that the monokaryotic mycelium comes out as the same mono-karyotic mycelium in the next generation, maintaining the io properties of monokaryotic mycelium shown in Table 1.
In plate culture or surface culture (stationary culture), the monokaryotic mycelium is reluctant to form an aerial mycelium, but an aerial mycelium comes to be produced if cultivation is continued for several months. This aerial mycelium is dikaryotic, and when it is inoculated into a bed log to form fruit bodies, they proved to be Coriolus versicolor .
(Fr.) Quél. itself. This revealed that the mycelium produced is just the same as the original one.
These facts indicate that the monokaryotic mycelium of Coriolus versicolor (Fr.) Quél.is derived from the original dikaryotic mycelium of Coriolus versicolor (Fr.) Quél.
The monokaryotic mycelium is a novel mycelium which was developed for the first time by the method of this invention, and this novel mycelium was named Coriolus versicolor (Fr.) Quél. GX-101-3 and deposited under FERM-P No. 3686 on August 25, 1976 in the Fermentation Research Institute, Agency of Industrial Science and Technology (Chiba-shi, Japan), a Japanese governmental organ.
The characteristic features of the monokaryotic mycelium of Coriolus versicolor (Fr.) Quél. are as shown in Table 1, but the greatest industrial significance of this g ~37~3~f~

fungus is its lligh rate of propagation. The propagation rate of this fungus is 1.5 to 10 times as high as those of the original dikaryotic mycelium, and obviously, such high pro-pagation rate is extremely beneficia:L for industrial production.
This monokaryotic mycelium can be applied for the same purposes of use as the dikaryotic mycelium of Coriolus versicolor (Fr.) Quél. For instance, it is possible to obtain nitrogen-containing polysaccharides from its extraction with an aqueous medium (such as water, a dilute alkaline solution or a dilute acid solution), and such nitrogen-containing poly-saccharide substance can be used for the preparation of drugs such as anti-tumor agents, immunity activating agents, anti-viral drugs, antifungal agents, anti-leprous drugs, appetite promoting drugs, etc.
It is also possible to collect various kinds of enzymes such as protease, amylase, etc., by low-temperature extraction. Further, the mycelium itself or its extracts or residues can be used for foods and drinks, feed for animals and fertilizer for plants.
Additionally, the mycelium of this invention can be employed for all the uses of Coriolus versicolor (Fr.) ~uél.
This invention is now described in further detail by way of some preferred embodiments thereof in the following Examples, but these embodiments are not to be taken as limiting the scope of the invention in any way. In the following description of the embodiments, the term "percent (%)" means percent by weight unless otherwise noted.

Production of monokaryotic mycelium 100 ml of a liquid medium containing 5% of glucose (produced by Showa Sangyo Co., Ltd.) and 0.75% of yeast extract ~37~3(1 (produced by Kyokuto Seiyaku Kogyo Co~, Ltd.) was pipetted into a sno ml conical flask, and after a 20 minute steam sterilization at 120C in an autoclave, the medium was inoculated with 1 ml of a suspension of mycelium prepared by dispersing the mycelium of Coriolus versicolor (Fr.) Quél.
(obtained from a 20 day stationary culture at 25C by using 50 ml of liquid medium containing 3% of glucose and 0.5% of yeast extract in 60 ml of physiological saline solution by breaking up the mycelial mat with a blender at speed of 6,000 r.p.m. for 3 minutes), and then a shaking culture was started at a speed of 200 r.p.m. at 25C. Three days after the start of the cultivation, the cultivated material was transferred aseptically into a 200 ml blender cup (mfd. by Sakuma Seisakujo), and after grinding the material by a homomixer (mfd. by Sakuma Seisakujo) at a speed of 10,000 r.p.m. for 10 minutes, the shaking culture was immediately resumed for a total period of 7 days. The thus cultivated mycelium had no clamp connection and was poor in generation of aerial mycelium in a standard agar plate medium. The result of a microscopic examination after the staining described below showed that the mycelium thus obtained was all monokaryotic.
Staining:
1 ml of the broth containing the mycelium obtained in the manner described above was mixed with 10 ml of water and then subjected to centrifugal separation at 2,000 to 5,000 G for 5 minutes. The supernatant liquid was eliminated and the mycelium was transferred into a test tube, to which Helly's fixing fluid was added. After standing for 24 hours, the separated cells were washed with 10 ml of water until they were decolored. Then the cells were put into 10 ml of lN
hydrochloric acid and heated at 60C for 15 minutes, followed 1137~31r) by cooling to room temperature, washing with 10 ml of water, 2 minute immersion in 10 ml of 20 to 50 times diluted nitric acid solution and 2 to 3 times of washing with 10 ml of water.
The obtained fibrous cells were spread on a slide glass to let moisture evaporate away and then a few drops of Giemsa's solution were added to the cells, and after 15 minutes of standing, they are washed lightly with water and then dried.
When the thus nucleus-stained cells were examined under a microscope of 1,000 magnification, each nucleus was observed as a circular red-stained spot. Therefore, the number of nuclei could be easily determined by counting the circular red-stained spots in one cell of the mycelium. The mycelium obtained did not acidify litmus milk and had no gelatin-liquefying ability.
Propagation of monokaryotic mycellium The monokaryotic mycellium obtained in the manner described above was fed with 12 litres of a liquid medium containing 5% of glucose and 0.75% of yeast extract into a 20-litre jar fermenter (mfd. by Kyoritsu Riko Co., Ltd.), followed by blowing 2 kg/cm of steam directly into the jar fermenter. After the steam sterilization at 120C for 20 minutes and cooling, 1 litre of suspension containing the monokaryotic mycelium was inoculated (at the rate of 0.5 g/l), immediately followed by cultivation at an aeration rate of 0.5 v.v.m. and at an agitating speed of 550 r.p.m. For the sake of comparison, cultivation of the dikaryotic mycelium was carried out under completely the same conditions as those used for the cultivation of the monokaryotic mycelium. When the propagation rates of these mono- and dikaryotic mycelia were compared by way of the time required for attaining a mycelium concentration of 8 g/l, it was noted that the 1~3743~

monokaryotic mycelium required only 1/4 of the cultivation time of the dikaryotic mycelium (see Fig. l). It was also confirmed that the propagation yield of the monokaryotic mycelium increased about 20% over that of the dikaryotic mycelium.

Calculation of the number of nuclei was made, in the manner of Example 1, of the mycelium obtained from a slant culture of Coriolus versicolor (Fr.) Quél. As a result, it was found that, as shown in the photograph of Fig. 2, all of the cells were dikaryotic and no monokaryotic mycelium was detected.
This original fungus is named Coriolus versicolor tFr.) Quél. CM-101 and deposited under FERM-P No. 2412 on December 25, 1973 in the afore-mentioned governmental organ.
100 ml of a liquid medium containing 5% of glucose and 0.75% of yeast extract was put into a 500-ml conical flask and sterilized therein by heating. This medium was then inoculated with the above-stated dikaryotic mycelium by using a platinum loop and was subjected to a 3 day shaking culture (preculture) in a room adjusted to a temperature of 25 + 2C. A pellet-shaped mycelium was consequently produced.
The broth containing these pellet-shaped mycelium was homo-genized by a homo-blender (mfd. by Sakuma Seisakujo) for 5 minutes and then subjected to a shearing treatment, whereby the pellet form substantially disappeared. Cultivation was continued under the above-mentioned conditions, and 4 days later (main culture), the produced pellet-shaped mycelium was again homogeniæed for 5 minutPs and then subjected to a shearing treatment. The concentration of the cells of the fungus at this stage was 11 g/l.

7~n 1 ml of broth containin& the homogenized mycellium was added to the same liquid medium as used in the first run of cultivation and then subjected to a second run of cultivation under the same conditions as in the first run. However, the cultivation period was slightly changed, that is, the pre-culture was performed for 3 days and the main culture also for 3 days. The fungal cell concentration was of the sub-stantially same level as in the first run of shaking culture.
The third run of cultivation was further continued in a similar way, with the fungal cell concentration reaching substantially the same level as that of the first run of shaking culture by 2 days of preculture and 3 days of main culture.
Likewise, a fourth run of cultivation was carried out, obtaining a broth with a fungal cell concentration of 12 g/l, higher than that of the first run of cultivation, by a 2 day preculture and 2 day main culture. The mycelium obtained was not in the form of pellets and stayed dispersed in the form of pulp, requiring no homogenization treatment.
The fungal cell, as observed by a microscope, had no clamp connection as found in the ordinary dikaryotic mycelium, and was about twice as large in width as the dikaryotic mycelium.
From the measurement of the number of nuclei by the method described in Example 1, it was ascertained that these cells were all monokaryotic mycelium as shown ~n the micro-photograph of Fig. 3.
When the mycelium was further cultivated under the same conditions as in the previous run of cultivation, the propagated mycelium was constituted from the monokaryotic mycelium and had all of the properties possessed by the 7~

monokaryotic mycelium shown in Table 1.
It was also found that the monokaryotic mycelium was more than twice as high in propagation rate as the dikaryotic mycelium.
10 gr of the dried product of the monokaryotic mycelium obtained in the above-described method was extracted with 300 ml of hot water at 95 to 100C for 3 hours. The extract solution was concentrated under reduced pressure to 30C and then mixed with pure ethanol to a concentration of 90%, and the produced precipitate was dried, giving 0.2 gr of gray powder.
A chemical analysis of this gray powder revealed that this substance was a nitrogen-containing high-molecular-weight polysaccharide. When this substance was administered to mice transplanted Sarcoma-180 solid type, it demonstrated a high anti-tumor activity.

100 ml of a liquid medium containing 5% of glucose and 0.75~ of yeast extract was fed into a 500-ml-capacity conical flask which already contained 8 gr of glass beads having a diameter of 2 to 5 mm. This mixed medium, after heat sterilization, was inoculated with the dikaryotic mycelium of Coriolus versicolor (Fr.) Quél., the same as employed in Example 1, by using a platinum loop and then subjected to a 7 day shaking culture at 25 + 2C.
1 ml of broth containing the thus obtained mycelium was supplied to the same medium containing glass beads as mentioned above and further subjected to a second 6 day shaking culture.
The resultant product was further subjected to a third run of shaking culture for a period of 5 days.

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The thereby produced mycelium had no clamp connection and was about 1.5 tlmes as large in width as the original dikaryotic mycelium, and the result of counting nuclei in the manner of Example 1 revealed that it was a monokaryotic mycelium.
1 ml of this broth was inoculated into 100 ml of a liquid medium containing 5% of glucose and 0.75~ of yeast extract and not containing any glass beads and cultivated at 25 + 2~C. The mycelium concentration 3 days after the start of the cultivation was 10.5 g/l, while the result of a similar culture of the dikaryotic mycelium showed a 7 g/l concentration 4 days after the start of the cultivation.

100 ml of a liquid medium containing 5~ of glucose and 0.75% of yeast extract was put into a 500-ml conical flask, and this medium, after heat sterilization, was inoculated with the dikaryotic mycelium of Coriolus versicolor (Fr.) Quél., the same as employed in Example 1, by using a platinum loop and then subjected to a shaking culture for 3 days.
After a homogenization treatment, the entirety of the fermenter was covered by a polyethylene bag and sealed against external air, followed by a 4 day cultivation.
At the point of completion of .he cultivation, 5.0 f C2 gas was contained in the fermenter atmosphere.
1 ml of broth containing the thus produced mycelium was inoculated into a medium after the manner of the first run of cultivation and then subjected to a second run of shaking culture in the same way as the first run. Such cultivation was further repeated twice. As a result of a similar measurement as conducted in Example l, it was ascertained that the mycelium produced in this process of ~ 3~4~n cultivation was all monokaryotic.

The mycelium obtained from a slant culture of Coriolus versicolor (Fr.) Quél. was sampled out and subjected to the nucleus determination following the procedure described - in Example 1, whereby it was ascertained that all of the mycelium was dikaryotic and no monokaryotic mycelium was present as seen in the photograph of Fig. 4. This indicates that the mycelium obtained from this run of cultivation was dikaryotic mycelium.
This fungus is Coriolus versicolor (Fr.) 0ue~1. CM-103 and deposited under FERM-P No. 2414 on December 25, 1973 in the afore-mentioned governmental organ.
100 ml of a liquid medium containing 5% of glucose and 0.75% of yeast extract was put into a S00-ml-capacity conical flask and, after heat sterilization, inoculated with the mycelium of the Coriolus versicolor (Fr.) Quél. CM-103 by using a platinum loop, followed by a 7 day shaking culture at 25 + 2"C. The mycellium in this culture was dikaryotic and contained at the concentration of 10.8 g/l.
The thus obtained dikaryotic mycelium was inoculated at 0.01~ into 20 litres of a liquid medium having dissolved therein 10% of glucose and 1.5% of yeast extract and was subjected to submerged culture under agitation in a fermenter adjusted to 25 + 2C by using a paddle type agitator at the speed of 500 r.p.m. After a 7 day cultivation, the mycelium was produced at a concentration of 10.2 g/l in the broth.
20 ml of this broth was inoculated into the same medium and a second run of cultivation was performed under the same conditions for 6 days. A similar cultivation was further repeated for 5 days in a third run and for 4 days in a fourth run.

43~

After completion of the fourth run of cultivation, the broth was in the form of a uniform pulp and contained the produced mycelium at the concentration of 11.5 g/l. The mycelium had no clamp connection and was all monokaryotic.

Claims (10)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method of producing a monokaryotic mycelium of Coriolus versicolor (Fr.) Quél., characterized in that a dikaryotic mycelium of Coriolus versicolor (Fr.) Quél. is first subjected to a mechanical treatment in a liquid medium and then subjected to a submerged culture, or said dikaryotic mycelium is subjected to a submerged culture while performing said mechanical treatment on said mycelium.

2. The method according to claim 1, wherein said mechanical treatment is shearing or grinding by an homogenizer.

3. The method according to claim 1, wherein said mechanical treatment is shearing or grinding by an agitator.

4. The method according to claim 1, wherein said mechanical treatment is grinding by use of an inactive solid granular material.

5. The method according to claim 1, claim 2 or claim 3, wherein said submerged culture is carried out in a highly nutritive state by using a liquid medium with a high con-centration of nutrient.

6. The method according to claim 1, claim 2 or claim 3, wherein said submerged culture is carried out in an atmosphere having a reduced oxygen partial pressure.

7. The method according to claim 1, claim 2 or claim 3, wherein said submerged culture is carried out continuously for a long period of time while additionally supplying the liquid medium.

8. The method according to claim 4, wherein said inactive solid granular material is glass beads.

9. The method according to claim 1, claim 2 or claim 3, wherein said submerged culture is carried out by means of successive transferring.

10. Monokaryotic mycelium of Coriolus versicolor (Fr.) Quél.