XU Ning , JIANG Xing-jian *, HUANG Feng-lan , WANG Xiao-yan , FENG Li-guo ,ZHANG Rui-qin , GUO Hong-mei , ZHOU Li-wen

1. Hunan Provincial Institute of Edible Fungus, Changsha 410013, PRC;

2. Yizhang Fucheng Biological Technology Company, Yizhang 424200, PRC

Abstract Comparative test of 4 Ganoderma lucidum varieties from different sources showed that the mycelium of Chizhi 1 grew fast with thick and dense hyphae, round and solid cap, and high spore powder yield. Chizhi 1 was proved to be an excellent variety because of its strong resistance and high spore powder yield. Different cultivation materials were chosen and combined to form 3 cultivation formulations.The results showed that Formulation (3), in which basswood was soaked in nutrient solution for 24 h, presented fast mycelial growth and high spore powder output, and therefore was proved to be a high-yield formulation of Ganoderma lucidum spore powder.

Key words Ganoderma lucidum spore powder, High quality and high yield variety,Cultivation techniques

1. Introduction

Ganoderma lucidum

Karst, commonly known as Hongzhi, Chizhi,Danzhi, Immortal grass, and Auspicious grass in China, is a medicinal fungus of

Ganoderma

genus and Ganodermataceae family (Polyporales order and Basidiomycetes class).

Ganoderma lucidum

spore powder is the seed of

Ganoderma lucidum

, mainly composed of ganoderan,triterpenoid, polypeptide and adenosine. According to the research of modern medicine,

Ganoderma lucidum

spore powder is considered to have therapeutic efficacy on cancer and boosting effects on immunity.There are more than 10 varieties of

Ganoderma lucidum

in northeast,southwest, eastern, and southern China. The yields of spore powder vary greatly according to the variety of

Ganoderma lucidum

and cultivation materials. In this research, 4

Ganoderma lucidum

varieties and 3 cultivation formulations were selected, and the growth of mycelium, the shape of sporophore, and the dry weight of spore powder per bag were measured in order to figure out the optimal variety and the best cultivation formulation to improve the economic benefits of

Ganoderma lucidum

. The findings provided theoretical basis for the cultivation of quality

Ganoderma lucidum

with high spore powder yield.

2. Materials and Methods

2.1. Testing materials

Chizhi 1 and Chizhi 2 were reserved in Hunan Provincial Institute of Edible Fungus; Chizhi 3 and Chizhi 4 were introduced from Hunan Institute of Microbiology.

Culture medium for maternal seeds was formed by potato 200 g, glucose 20 g, peptone 2 g,magnesium sulfate 0.1 g, monopotassium phosphate 0.1 g, agar 20 g, and water 1 000 mL.

Culture medium for basic seeds was formed by miscellaneous wood dust 79%, bran 20%, and gypsum 1%.

2.2. Testing management

2.2.1. Preparation of fungus bag

The fungus bags were made from polypropylene bags with the dimension of 18 cm×33 cm×0.005 cm;each bag contained 500 g of culture medium (drying material); different-sized basswoods were selected and tied (as tight as possible) together by plastic rope into a bundle 10 cm in diameter; these wood bundles were sterilized with steam at 126℃ for 120 min.

2.2.2. Fungus cultivation and growth management in fungus bag

Fungus cultivation: place the inoculated fungus bags under dark light for cultivation at a constant temperature of 24±2℃ (55%～70% relative air humidity)until the bags were overgrown with hyphae.

Ganoderma lucidum

growth management: in order to accelerate budding, the temperature should be controlled to 25℃～28℃, the relative air humidity to 85%, and illumination intensity to 100～200 lx; at the growth stage, the temperature should be 25℃～32℃,the illumination intensity should be 200～400 lx, the COconcentration in the air should be 800～1000 ppm,and the relative air humidity should be 85%～90%;at the sporulation stage, the temperature should be≦32℃, the soil water content should be about 65%,and the film should be removed 3～4 times per day for ventilation.

2.3. Experiment design

2.3.1. Screening of good-quality variety with high spore powder yield

The testing varieties were Chizhi 1, Chizhi 2,Chizhi 3 and Chizhi 4; 200 bags for each treatment;3 replications. Screen out the optimal variety by comparing the growth of hyphae, the anti-mold contamination rate, and the yield of spore powder.

2.3.2. Screening of cultivation materials

Wood dust, corncob, and basswood were chosen as the main cultivation materials and Chizhi 1 as the testing variety to evaluate the optimal cultivation formulation. Formulation 1 was formed by miscellaneous wood dust 80%, bran 18%, lime 1%, and land plaster 1%. Formulation 2 consisted of corncob 80%, bran 18%, lime 1%, and land plaster 1%; Formulation 3 was prepared by the following procedures: soak the basswood in nutrient solution(white sugar 1%, land plaster 1%, compound fertilizer 0.5%, V10 mg/L, V10 mg/L, and pH 7.5) for 24 h; add miscellaneous wood dust 78%, bran 20%, lime 1%, and land plaster 1%. The water contents in these formulations were 60%. There were 200 testing bags for each treatment of each formulation; 3 replications.

2.4. Testing items and methods

Observe the temperature, relative air humidity,illumination, COconcentration, budding, and spore powder yield; record the number of primordium differentiation and biological traits of sporophore,including the color and roundness of cap, the length of stipe, and the yield of

Ganoderma lucidum

.2.4.1. Growth rate of hyphae in fungus bags

Measure the growth rate of hyphae in fungus bags by decussation method and use PDA to determine the colony diameter. Use a puncher (inner diameter:7 mm) to pick fungus blocks evenly; inoculate these blocks in the center of culture plates; cultivate fungi at 25℃. Observe the growth of fungal colony; measure the diameter of fungal colony by decussation method 6 d later; calculate the growth rate of hyphae in the plate as follows:

Growth rate of hyphae (cm/d) = Fungal colony radius (cm) / Growing days of hyphae (d)2.4.2. Yield of spore powder

Ganoderma lucidum

is mainly planted for its spore powder. In this research, only the first-wave spore powder was collected to evaluate the yield. Dry the fresh spore powder to constant weight. Take 100 fungal bags and measure the total release amount of spores; calculate the average release amount of spores;perform significant analysis to determine the spore powder yield.

2.4.3. Determination of contamination rate

Measure the percentage of fungus bags that have been contaminated by microbes (e.g. Trichoderma viride and Aspergillus flavus) at bud-forcing stage.

Contamination rate (%)= The number of fungus bags contaminated by microbes / Total number of fungus bags

3. Results and Analysis

3.1. Comparison of Ganoderma lucidum varieties with high spore powder yield

According to Table 1, growth rates varied with fungal strains: Chizhi 1 had the fastest growth rate and was followed by Chizhi 2; there was no significant difference between the growth rates of Chizhi 2 and Chizhi 3. In the culture media for both the maternal seeds and basic seeds, there were no significant differences among the 4 fungal strains in respect to the density, color, and edge uniformity of hyphae and mycelia. Chizhi 1 was the best; the other three strains were also vigorous with white and dense hyphae. The resistance of four fungal strains, ranked in descending order are as follows: Chizhi 1, Chizhi 2, Chizhi 3, and Chizhi 4. Chizhi 1 showed the lowest contamination rate and the highest mold resistance; significant differences were found between Chizhi 1 and the other three strains. Among the four fungal strains, Chizhi 1 showed the optimal sporophore—the cap was round,regular, thick, and solid, and it was brown in color and uniform in size. The yields of spore powder ranked in descending order were as follows: Chizhi 1, Chizhi 2,Chizhi 4, and Chizhi 3; there were highly significant differences between the spore powder yields of Chizhi 1 and the other three fungal strains.

3.2. Cultivation formulation for high spore powder yield

According to Table 2, the growth of hyphae varied with the formulation. The growth rate of hyphae peaked in Formulation 2, followed by Formulation 3 and 1. The hyphae cultivated by Formulation 3 were dense, thick, and white in color, which were superior to those grown by Formulation 1 and 2. The shape of sporophore also varied with the formulation: the cap of Formulation 3 was round, thick, and uniformin size; the tissue was compact and solid. The yields of spore powder, ranked in descending order were as follows: Formulation 3, Formulation 1, and Formulation 2. To conclude, Formulation 3 was better than Formulation 1 and 2. Basswood soaked with lime water was conducive to the growth of hyphae and sporophore;

Ganoderma lucidum

cultivated by this formulation had round and compact cap, fine quality,and high yield of spore powder.

Table 1 Hyphae growth, sporophore shape and spore powder yield of 4 Ganoderma lucidum strains

Table 2 Hyphae growth and spore powder yield by different cultivation formulation

4. Conclusions

In this research, 4 different

Ganoderma lucidum

varieties were cultured to find out the optimal variety with high spore powder yield. Significant differences were observed in the results. The hyphae of Chizhi 1 showed the highest growth rate; the hyphae were dense and thick, with strong mold resistance. The sporophore of Chizhi 1 was round, regular, and uniform in size,and the spore powder yield was very high. Therefore,Chizhi 1 was no doubt the superior variety with high yield of spore powder. Miscellaneous wood dust, corncob, and basswood soaked with nutrient solution were selected to test the optimal cultivation formulation. The results showed that Formulation 3,in which basswood was soaked in nutrient solution for 24 h and added with miscellaneous wood dust, could produce quality

Ganoderma lucidum

with high spore powder yield.