Abstract:
The present variety of mushroom plant named ‘marmo shiro-2’ was cultivated by the gathering and repeated breeding of Bunashimeji mushrooms having dominant traits, which has good qualitative characteristics and appearance, less adhesion of the stem, excellent keeping qualities and storage life, and improved after-storage taste. This edible mushroom is exquisite in stability, reproducibility and uniformity when being produced.

Description:
BACKGROUND OF THE INVENTION 
     This invention relates to a new and distinct variety of mushroom plant of Bunashimeji mushroom,  Hypsizygus marmoreus  (Peck) Bigelow. This new variety named ‘marmo shiro-2’ cultivated by repeated breeding of Bunashimeji mushrooms having dominant traits, which has less adhesion of stem, excellent keeping quality and improved after-storage taste and ensure presentable stability, reproducibility and uniformity. 
     Bunashimeji ( Hypsizygus marmoreus ) now boasts of being the second most consumed edible mushroom after enokitake mushroom. Ever since bunashimeji was cultivated and began to be sold in large quantities in the market, its characteristic bitter taste had been a matter of concern. Consequently, ‘Hokuto-8 strain’, whose bitterness has been reduced to an imperceptible level, was developed to develop a tasty Bunashimeji mushroom. Investigation on tasty bunashimeji was continued even after the development of ‘Hokuto-8’, and a variety with better taste, quality and keeping quality named ‘Hokuto-18 strain’ was developed, which contributed to increase in consumption. However, most of the naturally growing mushrooms that are popular in Japan from the olden days are collected at the beginning of autumn, due to which one generally forms an image of autumn and winter when the word mushroom is heard. We started working on the development of a new variety of mushroom that can be used with bright-colored vegetables of summer in salad and other recipes since we can stably supply them throughout the year by growing under protected conditions. As a result, the ‘Hokuto shiro-1 strain’ mushroom that has refreshing whiteness and jellylike food texture was developed and patented in US (U.S. Plant Pat. No. 16,294 P3). 
     In order to further improve the stability of cultivation and quality of mushrooms, selective breeding was repeatedly carried out by cross-breeding. As a result, the ‘marmo shiro-2 strain’ that has the whiteness and food-texture of ‘Hokuto shiro-1’, as well as the quality and keeping quality of ‘Hokuto-18’ was developed. Subsequently, the stability, reproducibility and uniformity of the variety were verified, and cultivation was completed. 
     SUMMARY OF THE INVENTION 
     The present invention is a new and distinct variety of mushroom characterized particularly by its good qualitative character and appearance, good keeping quality and storage life and delicious tasting, which can be cultivated by gathering and repeated breeding of fungal strains having dominant traits and is exquisite in stability, reproducibility and uniformity when being produced. This novel and distinct variety of mushroom is identified as ‘marmo shiro-2’. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS. 1A and 1B  respectively show front and back images of a dual-culture of ‘marmo shiro-2’ colony. 
         FIGS. 2A and 2B  respectively show front and back images of a dual-culture of ‘marmo shiro-2’ and ‘Hokuto shiro-1’ strains. 
         FIGS. 3A and 3B  respectively show front and back images of a dual-culture of ‘marmo shiro-2’ and ‘Hokuto-18’ strains. 
         FIG. 4  shows an image of a fruit body of ‘marmo shiro-2’. 
         FIG. 5  shows an image of a fruit body of ‘Hokuto shiro-1’. 
         FIG. 6  shows an image of a fruit body of ‘Hokuto-18’. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The history of the ‘marmo shiro-2’ mushroom in terms of improvement period and the like are set forth in the following chronological list of each stage of variety improvement: 
     October 2001: Cultivation of ‘Hokuto shiro-1 strain’ 
     October 2003: Cultivation of ‘Hokuto 18 strain’ 
     November 2006: Cultivation of ‘MH025464’ 
     September 2009: ‘Hokuto shiro-1’ and ‘MH025464’ were crossed and the strains with white cap which are to be kept were picked from the obtained strains, and considered as ‘MH025465’. 
     September 2010: ‘Hokuto-18 strain’ and ‘MH025465’ were crossed and an excellent strain that fulfill the cultivation objective (test number 10HM395) was obtained. Then, growing test was repeatedly conducted and the distinguishability, stability and uniformity were confirmed, upon which the strain was named ‘marmo shiro-2’ and cultivation was completed. 
     March 2012: Applied for registration of new variety to Ministry of Agriculture, Forestry and Fisheries of Japan. 
     The ‘marmo shiro-2’ mushroom has the following characteristics: less adhesion of stem, excellent keeping quality and improved after-storage taste. 
     (1) Comparison with Existing Varieties by Dual Culture 
     Formation of zone line in the varieties similar to ‘marmo shiro-2’ and parental varieties were studied by conducting dual culture. 
     Study Method: 
     The ‘marmo shiro-2’ and another strain were co-inoculated with an interval of 3 cm by using potato dextrose agar culture medium. Then, the formation of zone line was determined by culturing for 28 days at 25° C. 
     Strain Used: 
     ‘marmo shiro-2’: Present variety 
     ‘Hokuto shiro-1 strain’: Varieties similar to the present variety 
     ‘Hokuto-18 strain’: Parental variety of the present variety 
     Results: 
     Zone lines were formed between ‘marmo shiro-2’ and all other co-cultured varieties (Table 1,  FIGS. 1 to 3 ). These results show that this strain is a new variety. 
                                             TABLE 1                   Results of dual culture                Similar variety                Hokuto shiro-1 strain    Hokuto-18 strain               marmo shiro-2   +   +               * No zone line was seen in the dual culture of ‘marmo shiro-2 strains’.            
(2) Culture Characteristics of ‘marmo shiro-2’
 
     After inoculating a piece of agar of ‘marmo shiro-2’ with 5 mm diameter in potato dextrose agar, preliminary culture was carried out for 4 days at 25° C. and hyphae were regenerated (about 10 mm diameter). Then, it was cultured for 7 days at 5-30° C. with 5° C. interval. When the mean hyphae growth speed per day was calculated from the hyphae growth speed of 7-day culture, it was found that the mean hyphae growth rate was somewhat slow at 20° C. and 25° C. (See Table 2). 
     (3) Morphological Characteristics Based on an Example of Cultivating ‘marmo shiro-2’ 
     Cultivation Method: 
     Container: An 850 polypropylene bottle (Capacity: 850 ml, mouth diameter: 58 mm) was used. 
     Culture medium: Conifer sawdust, corn-cob, rice bran and wheat bran were mixed in the dry-weight ratio of 7:3:8:2, and the mixture was adjusted to 65% water content. The culture medium was filled up to the brim of bottle at the rate of 540±20 g per bottle, and was sterilized at high pressure. 
     Starter culture: 20 ml of sawdust culture was inoculated per bottle. 
     Culture: 50-90 days culturing was conducted under 22° C. temperature and 70% humidity. 
     Development: After incubation, the cultivar was shifted to a growing room after Kinkaki (removing the original inoculum mechanically). Development was conducted under 15±1° C. temperature and 95% or more humidity. The cultivar is not exposed to light particularly in the first 14 days. After the 14th day, about 500-1,000 Lx is taken and development is conducted at about 2,000 ppm CO 2  density. The mushroom is harvested when the cap in the center of the stump has fully opened. 
     Cultivation Results: 
     The characteristics of ‘marmo shiro-2’ cultivated under the conditions mentioned above, and the specific difference in characteristics with the most similar variety are explained in Table 2 below. 
     Also, the images of the respective fruit bodies have also been attached. (Refer to  FIGS. 4 to 6 ). 
     (4) Comparison with ‘MH025465’ by Dual Culture 
     Formation of zone line in ‘marmo shiro-2’ and ‘MH025465’ were studied by conducting dual culture. 
     Study Method: 
     The ‘marmo shiro-2’ and ‘MH025465’ were co-inoculated with an interval of 3 cm by using potato dextrose agar culture medium. Then, the formation of zone line was determined by culturing for 28 days at 25° C. 
     Strain Used: 
     ‘marmo shiro-2’: Present variety 
     ‘MH025465’: Parental variety to the present variety 
     Results: 
     Zone lines were formed between ‘marmo shiro-2’ and ‘MH025465.’ This result shows that ‘marmo shiro-2’ is different from ‘MH025465.’ No zone line was seen in the dual culture of ‘MH025465 strains.’ 
     (5) Comparison with ‘MH025465’ by SSR Marker 
     Applicant verified that ‘marmo shiro-2’ is different from ‘MH025465’ by using SSR marker. ‘MH025465’ do not have the same alleles as ‘marmo shiro-2.’ This result also shows that ‘marmo shiro-2’ is different from ‘MH025465.’ 
     
       
         
               
             
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 Fungus characteristics Table of  H. marmoreus  (Peck) 
               
               
                 Bigelow of Recording and Registration 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Present variety 
                 Similar variety 
               
             
          
           
               
                   
                 marmo shiro-2 
                 Hokuto shiro-1 
                 Hokuto-18 
               
               
                   
               
               
                 Physiological property 
                   
                   
                   
               
               
                 Dual culture 
                   
                   
                   
               
               
                 Zone line formation 
                 observed 
                 observed 
                 observed 
               
               
                 Dislike-touch reaction 
                   
                   
                   
               
               
                 Density of hyphae 
                 medium 
                 medium 
                 medium 
               
               
                 Mycelial growth 
                 medium 
                 medium 
                 medium 
               
               
                 Color of surface of colony 
                 white 
                 white 
                 white 
               
               
                 Color of back of colony 
                 pale yellow 
                 pale yellow 
                 pale yellow 
               
               
                 Accommodativeness for  
                   
                   
                   
               
               
                 temperature 
                   
                   
                   
               
               
                 Optimal temperature for  
                 24° C. 
                 24° C. 
                 24° C. 
               
               
                 hyphal growth (° C.) 
                   
                   
                   
               
               
                 Hyphal growth rate 
                   
                   
                   
               
               
                 5° C./mm 
                 0.34 mm 
                 0.41 mm 
                 0.39 mm 
               
               
                 10° C./mm 
                 1.04 mm 
                 1.00 mm 
                 1.14 mm 
               
               
                 15° C./mm 
                 1.87 mm 
                 1.98 mm 
                 1.99 mm 
               
               
                 20° C./mm 
                 2.51 mm 
                 3.07 mm 
                 2.82 mm 
               
               
                 25° C./mm 
                 2.80 mm 
                 3.58 mm 
                 3.13 mm 
               
               
                 30° C./mm 
                 0.28 mm 
                 0.38 mm 
                 0.56 mm 
               
               
                 Morphological property 
                   
                   
                   
               
               
                 Cap 
                   
                   
                   
               
               
                 Cross sectional shape 
                 rounding 
                 rounding 
                 rounding 
               
               
                   
                 mound 
                 mound 
                 mound 
               
               
                 Mottle of the surface 
                 few 
                 few 
                 much 
               
               
                 Size of mottle 
                 small 
                 small 
                 much 
               
               
                 Distribution of mottle 
                 center 
                 center 
                 whole 
               
               
                 Clarity of mottle 
                 not clear 
                 not clear 
                 clear 
               
               
                 Diameter of cap 
                 17.6 mm 
                 16.3 mm 
                 16.9 mm 
               
               
                 Thickness of cap 
                 7.8 mm 
                 7.2 mm 
                 6.1 mm 
               
               
                 Color of central area 
                 white 
                 white 
                 grayed- 
               
               
                   
                 155B 
                 155B 
                 orange 177B 
               
               
                   
               
               
                   
                   
                   
                 grey-brown 
               
               
                   
                 Present variety 
                 Similar variety 
                 199D 
               
               
                   
                 marmo shiro-2 
                 Hokuto shiro-1 
                 Hokuto-18 
               
               
                   
               
               
                 Fleshy 
                 medium 
                 medium 
                 medium 
               
               
                 Gill 
                   
                   
                   
               
               
                 Color 
                 white 
                 white 
                 yellow- 
               
               
                   
                 155B 
                 155B 
                 white 158C 
               
               
                 Alignment 
                 normal 
                 normal 
                 normal 
               
               
                 Width 
                 medium 
                 medium 
                 medium 
               
               
                 Density 
                 medium 
                 medium 
                 medium 
               
               
                 Stipe 
                   
                   
                   
               
               
                 Shape 
                 medium-thick 
                 medium-thick 
                 medium-thick 
               
               
                 Length 
                 45.0 mm 
                 41.8 mm 
                 50.0 mm 
               
               
                 Maximum diameter of 
                 9.0 mm 
                 9.3 mm 
                 8.4 mm 
               
               
                 stipe 
                   
                   
                   
               
               
                 Color 
                 white 
                 white 
                 white 
               
               
                   
                 NN155C 
                 NN155C 
                 NN155C 
               
               
                 Hair 
                 absent 
                 absent 
                 absent 
               
               
                 Fleshy 
                 medium 
                 medium 
                 medium 
               
               
                 Ratio of maximum 
                 1.75 mm 
                 1.64 mm 
                 1.56 mm 
               
               
                 diameter of stem to 
                   
                   
                   
               
               
                 diameter just below cap 
                   
                   
                   
               
               
                 Cultural property 
                   
                   
                   
               
               
                 Development of fruit body 
                   
                   
                   
               
               
                 Development 
                 stock 
                 stock 
                 stock 
               
               
                 Optimal culture period 
                 81-90 days 
                 91-100 days 
                 81-90 days 
               
               
                 Length of time from fruit  
                 21.5 days 
                 21.3 days 
                 21.6 days 
               
               
                 body-formation 
                   
                   
                   
               
               
                 promotion to fruit body 
                   
                   
                   
               
               
                 harvesting at optimal 
                   
                   
                   
               
               
                 temperature 
                   
                   
                   
               
               
                 Optimal temperature for 
                 14-16° C. 
                 14-16° C. 
                 14-16° C. 
               
               
                 primordial development 
                   
                   
                   
               
               
                 Optimal temperature for  
                 14-16° C. 
                 16-18° C. 
                 14-16° C. 
               
               
                 fruit body growth 
                   
                   
                   
               
               
                 Adaptivity for illuminance 
                   
                   
                   
               
               
                 Adaptivity for culture 
                   
                   
                   
               
               
                 Yield 
                   
                   
                   
               
               
                 Yield of fruit body 
                 139.3 g 
                 129.8 g 
                 141.4 g 
               
               
                 The number of productive  
                 52.0 
                 48.1 
                 61.4 
               
               
                 stem 
                   
                   
                   
               
               
                 Other 
                   
                   
                   
               
               
                 Bitterness component 
                   
                   
                   
               
               
                 Disease resistance 
                   
                   
                   
               
               
                 Contains 
               
               
                   
               
               
                 *The employed color chart is R.H.S. Colour Chart, 2007, Fifth edition, prescribed by the Royal Horticultural Society, England.