Study on biological characteristics and technology of spawn production, cultivation of king oyster mushroom (pleurotus eryngii) and yunzhi mushroom (trametes versicolor) in vietnam

MINISTRY OF EDUCATION MINISTRY OF AGRICULTURE AND TRAINING AND RURAL DEVELOPMENT VIETNAM ACADEMY OF AGRICULTURAL SCIENCES NGUYEN THI BICH THUY STUDY ON BIOLOGICAL CHARACTERISTICS AND TECHNOLOGY OF SPAWN PRODUCTION, CULTIVATION OF KING OYSTER MUSHROOM (PLEUROTUS ERYNGII) AND YUNZHI MUSHROOM (TRAMETES VERSICOLOR) IN VIETNAM Branch of study: Crop Science Code : 62.62.01.10 SUMMARY OF DOCTORAL THESIS Hanoi - 2014 This thesis is completed at: VIETNAM ACADEMY OF AGRICULTURAL SCIENCES Science supervisor: 1. Prof. Ph.D. Trinh Tam Kiet 2. Ass. Dr. Nguyen Trung Thanh Critic 1: ............................................................................ Critic 2: ............................................................................ Critic 3: ............................................................................ The thesis will be defended before Council for thesis assessment at Institutional level held at Vietnam Academy of Agricultural Sciences at ...... hour ..... minute, day .... month ..... year ..... This Thesis can be referred to at: 1. National Library 2. Library of VietnamAcademy of Agricultural Sciences 3. Library of...................................................................... 1 INTRODUCTION 1. The urgency of the topic King Oyster Mushroom (Pleurotus eryngii (DC.) Quél) is a delicious, edible fungus, high nutritional and medicinal values. King oyster mushroom’s fruiting bodies are of large size, pretty shape, so for these advantages, this fungus is called "King Oyster mushroom”. Yunzhi mushroom (Trametes versicolor (L.) Pilat) is a medicinal fungus of very high medicinal value, that has been preferred by consumers in countries like China, Japan, European, American countries,... In Yunzhi mushroom, there are polysaccharide compounds associated with protein, consisting of two main types: PSP (polysaccharide peptide) and PSK (polysaccharide krestin). PSP and PSK have the effect to inhibit various types of cancer cells such as carcinoma cell (carcinoma), cancer of the blood cells (leukemia), stomach cancer, colon cancer, throat cancer, lung cancer and breast cancer …... (P.M.Kidd, 2000). Together with the development of society, the demand for people’s is not only good, but also nutritious, safe and valuable health promoting food. Now, on the market, there are many types of edible and medicinal mushrooms imported from China into Vietnam with quantity of hundreds of tonnes/year, by many different ways, without origins, long time of transportation, using preservatives, thus causing bad psychological attitudes for consumers. Currently, the edible mushroom - medicinal mushrooms cultivated in Vietnam, especially the King Oyster and Yunzhi mushrooms are still interested by consumers. The production of edible - medicinal mushrooms is growing strongly, the units of breeding and cultivating mushrooms in our country are all applying traditional breeding technologies (solid), so there exist some disadvantages as: high rate of disease - infected spawns, long incubation time for one spawn level with an average duration from 10-25 days, the time from implanting spawns into farming materials to commercial mushroom picking is long, within 2 months that lead to high prices for spawn and commercial mushrooms . While research and seed production of liquid spawn in world has reached some remarkable achievements. The application of liquid spawn production show remarkable effectiveness as compared to solid spawn production, that help shortening growth duration of one spawn level down to only 3-5 days, with high purity, good quality, reducing infection rates, appropriate for spawn production and mushroom cultivation in industrial scale. 2 In order to contribute to the overcoming of the above limitations, edible - medicinal mushrooms in general and King Oyster -Yunzhi mushrooms in particular to develop effectively, the research theme: "Study on biological characteristics and technology of spawn production, cultivation of the King Oyster Mushroom (Pleurotus eryngii) and Yunzhi mushroom (Trametes versicolor) in Vietnam" has been conducted. 2. Objective of topic To conduct research on some biological characters of strains of King Oyster and Yunzhi mushrooms to identify best developing strains in climatic condition of Vietnam, and at the same time, to set up spawn production technologies for mushroom strains of liquid spawn in order to improve production efficiency for edible and medicinal mushrooms. 3. Scientific and practical significances 3.1. Scientific significance The findings of the study have provided scientific data on the basic growth indicators of King Oyster mushroom (P. eryngii) and Yunzhi mushroom (T. versicolor), as well as the need for nutrition and optimal environmental conditions for growth and development of two mushroom varieties on liquid cultivation; pointing out the genetic differences of the currently being stored strains and correlation with the biological characteristics in the cultivation, and at the same time, to provide the data observed by the electron microscope. 3.2. Practical significance Through the results of the research subject, two mushroom varieties of King Oyster mushroom (P. eryngii) and Yunzhi mushroom (T.versicolor), suitable for growing conditions in Vietnam, have been released to production; at the same time, propagation technologies in liquid types for these two strains have been set up, improving their growth capacity, shorterning incubation time, increasing productivity of commercial mushrooms. This technology is highly feasible, capable of applicating in mushroom production in industrial scale. 3.3. The novelty of the thesis Having studied the morphological characteristics basidiospores, indicating the growth characteristics and development of mushroom mycelium and fruiting bodies; simultaneously assess the genetic differences between strains of King oyster mushroom and Yunzhi 3 mushroom. On that basis, edible mushrooms and medicinal mushrooms adaptable to production conditions of Vietnam have been selected. Having build up successful propagation processes for liquid spawns of King oyster and Yunzhi mushrooms (from propagation of the first grade spawn → intermediate spawn → commercial spawn and using for cultivation); replacing traditional technologies, shortening incubation time, increasing mushroom yield, enhancing economic efficiency. 4. The layout of the thesis The main content of the thesis are presented in 134 pages (excluding references and appendices). Introduction: 4 pages; Chapter 1: Overview document: 36 pages; Chapter 2: Materials, content and research methodology: 17 pages; Chapter 3: Research Results and Discussion: 75 pages; Conclusions and recommendations: 2 pages. The thesis consists of 26 tables, 65 figures, 18 Vietnamese documents, 78 documents in English. The appendix includes: Process Mapping; Calculation of the economic efficiency of liquid spawn multiplication technologies in comparison with traditional technologies; Images of mushroom spawns and mushroom fruiting bodies in some experiments; Results of statistical analysis and experimental data processing and content related to the thesis. Chapter 1 OVERVIEW 1.1 . Status of research and production of mushrooms in the world and in Vietnam 1.1.1. Status of research and production of mushrooms in the world 1.1.1.1 . Research situation on multiplication of liquid spawns and cultivation of King Oyster and Yunzhi mushrooms As King oyster and Yunzhi mushrooms are of high medicinal value, they are interested to be studied on by many concerned countries. Scientists around the world have conducted further studies in the growth capacity of King oyster and Yunzhi mushroom by the effects of additional nutrient supply such as sources of carbon, nitrogen, mineral as well as their best additional contents. Authors also studied the effect of pH, temperature, nutrients to the growth of King oyster and Yunzhi musrooms. Cultivating materials and nutritional supplement components, nutrient contents were also mentioned. Deep layer nurturing fermentation method (liquid spawn multiplication/propagation) is applied to produce different varieties of 4 edible and medicinal mushrooms (Liu, 2010; Diamantopoulou P, 2012; Jonathan, 2009. At present, technology for liquid spawn propagation has been developed in many countries over the world, there have been many studies to point out optima additional nutrition component, aeration rate, affects of liquid medium pH to the growth of fiber systems, advantages and disadvantages of liquid spawn propagation. 1.1.1.2. Research on the genetic diversity of some strains 1.1.1.3. The situation of mushroom production in the world 1.1.2. Status of research and production of mushroom in the country 1.1.2.1. Research on the genetic diversity of some strains In Vietnam, the RAPD technique has much been applied in the genetic analysis of plants. Some authors have used RAPD technique to analyze genetic diversity in the different objects but genetic research on diversity of edible mushrooms and medicinal mushrooms have not been many. 1.1.1.2. The situation of mushroom research and production in Vietnam In recent years, there have been many research works of authors such as Trinh Tam Kiet, Nguyen Thi Chinh, Phan Huy Duc, Le Xuan Tham, Le Thi Hoang Yen ... These authors focused on the study of different areas on various different objects. There are several studies on liquid spawn but mainly in the laboratory with the aim of extracting biomass, not any research work has been applied in farming sector. Centre for Mushroom Research and development and bioproducts- Reishi Co., Ltd. has successfully produced many products with preventive and curative effects, deeply focuses on scientific research to create food functional products. Along with the studies on the effect of Yunzhi mushroom, our company has also conducted studies on manufacturing processes for products from Yunzhi mushroom. There are 2 main types of products, that are mycelia biomass powder and fruit bodies from Yunzhi mushroom. At present, annually, the company provides about 10 tons of mycelia biomass powder/year and 2-3 tons of fruit bodies /year. Mushroom cultivation is being transferred to multiple production facilities. Materials used for cultivation are sawdust supplemented with cultivating nutritional additives; the most suitable temperature for mushroom fruit bodies to grow and develop is from 20 - 25 0 C and the most appropriate time for fruit bodies to grow is from february to may (Solar calendar) (Nguyen Thi Chinh , 2011 ) . 5 Some new species of mushrooms imported into Vietnam, including King oyster mushroom, and fresh mushroom products currently on the market are mainly imported from China. King oyster mushroom, also called chicken leg mushroom, is now being studied and farmed by the Center for Plant Biotechnology Research by a traditional method, with mixing farming materials of 43 % cotton by-product + 43 % sawdust + 6 % corn starch + 7 % rice bran + 1 % CaCO3. The cultivation stages of King oyster mushroom are described in detail: using plastic bags size 19 x 33 cm as packages, weight of material bag: 0.8 kg / bag. Authors also identified suitable environmental conditions for cultivation of King oyster mushroom: temperature from 12 -15 0 C; light intensity from 800 - 1200lux ; air humidity: 85-95%; good ventilation condition. The findings noted that, with the above growing conditions, the actual yield of King oyster mushrooms reached 35-40 % (Dinh Xuan Linh et al, 2012). In general, a number of scientists in our country focussed deeply on the study of edible mushrooms, especially medicinal mushrooms (Reishi mushroom, Yunzhi mushroom, Pupae cordyceps mushroom ...). This trend of study was performed in liquid medium but intensivly concentrated on mycelia biomass extraction technology to produce functional products; as mushroom cultivating for obtaining fruiting bodies mainly used solid spawn sources (seed spawns), the cultivation time usually lasts longer. 1.2. General introduction of King oyster and Yunzhi mushrooms King oyster mushroom (English name is also King oyster mushroom) has a scientific name as Pleurotus eryngii (DC. Fr.). This is a specy of edible mushroom that has high medicinal and nutritional values. Yunzhi mushroom has the scientific name as Trametes versicolor (L.Fr.) Pilat, with its English name as Yunzhi or Turkey tails. Previously, they had different scientific names as Coriolus versicolor, Polyporus versicolor. Yunzhi is a medicinal mushroom which is used in many Asian countries. During the processes of growth, development, King oyster and Yunzhi mushrooms are influenced by many factors. Agents that affect the formation of fruiting bodies of the fungi can be divided into 3 types: external factors such as temperature, humidity, light, pH, CO2 concentration and wind speed as well as the role played by other microorganisms coexisting in the ecosystem; necessary nutrients. 1.3. Factors affecting the growth of King oyster and Yunzhi mushrooms During the processes of growth, development, King oyster and Yunzhi mushrooms are influenced by many factors. Agents that affect the 6 formation of fruiting bodies of the fungi can be divided into 3 types: external factors such as temperature, moisture, humidity, light, pH, CO2 concentration and wind speed as well as the role played by other microorganisms coexisting in the ecosystem; necessary nutrients. 1.4. The role of King oyster and Yunzhi mushrooms King Oyster and Yunzhi are 2 types of mushrooms that have high medicinal and nutritional values. Especially Yunzhi, a kind of mushroom that is studied deeply on its medicinal properties by a lot of scientists around the world. There have been many published articles that announced the uses of Yunzhi such as for cancer treatment, anti HIV virus, immune enhancing effects ... Besides, the cultivation of King oyster and Yunzhi mushrooms have contributed to the use of all by-products from agricultural waste, contributing to environmental protection. Chapter 2 RESEARCH MATERIALS AND METHODOLOGY 2.1 . Research Materials 2.1.1 . Mushroom spawn : Including 6 spawns of King oyster mushroom and 2 Yunzhi mushroom spawns of different origins, currently stored at the Center for Plant Biotechnology, Agricultural Genetics Institute. 2.1.2. Materials and chemicals * Materials used in DNA extraction - 23 RAPD primers were used in the study of biodiversity of King oyster mushrooms, 10 RAPD primer used for Yunzhi mushroom * The chemicals used in propagation experiments of spawns level 1 and intermediate level + Glucose, yeast extract, peptone, MgSO4.7H2O, KH2PO4, thiamine, ... originating from Merck Company. * Materials used in commercial spawn propagation experiments and spawn cultivation + Glucose, yeast extract, peptone, MgSO4.7H2O, KH2PO4, thiamine, ... originating from China. + Some other materials, such as ordinary paddy rice, rice bran, straw, sawdust. 2.1.3 . The conditions and equipments used in the experiment 2.2. Research Contents 2.2.1. Some biological characteristics of King oyster and Yunzhi mushrooms 2.2.2. Evaluation of the genetic differences of King oyster and Yunzhi mushrooms 7 2.2.3. Propagation of liquid spawns of King oyster and Yunzhi mushrooms 2.2.4. Using liquid spawns to cultivate King oyster and Yunzhi mushrooms 2.3. Research methodology 2.3.1. Research some biological characteristics of King oyster and Yunzhi mushrooms Experiment 1: The growth and development of King oyster and Yunzhi mushrooms in pure culture Experiment 2 : Evaluation of the growth and development of King oyster mushrooms and Yunzhi on cultivation materials 2.3.2 . Assessment of genetic differentiation of King oyster and Yunzhi mushrooms 2.3.2.1 . DNA extraction method 2.3.2.2 . PCR method 2.3.2.3 . Methods of data analysis 2.3.3 . The research to propagate liquid spawns of King oyster and Yunzhi mushrooms 2.3.3.1 . Experiments to propagate the first grade liquid spawns of King oyster and Yunzhi mushrooms Experiment 3 : Effect of temperature to the growth of King oyster and Yunzhi Mushroom in the liquid medium. Experiment 4 : Effect of pH to the growth of King Oyster and Yunzhi mushrooms in the liquid medium. Experiment 5 : Effect of the liquid medium components to the growth of the first grade liquid spawns of King oyster and Yunzhi mushrooms. Experiment 6 : Effect of shaking mode to the growth of King oyster and Yunzhi mushrooms in the liquid medium. Experiment 7 : Effect of base rate of original spawns to the growth of King oyster and Yuanzhi mushrooms in the liquid medium. Experiment 8 : Effect of incubation time to the growth of King oyster and Yuanzhi mushrooms - liquid spawn types. 2.3.3.2. Research to propagate King oyster and Yunzhi mushroom Liquid spawns at intermediate Grade. Experiment 9: Effect of aeration mode to the growth of King oyster and Yunzhi mushrooms of intermediate grade. Experiment 10: Effect of the spawn implantation rate to the growth of King oyster and Yunzhi mushrooms of intermediate grade. 2.3.3.3. Research to propagate commercial liquid spawn of King oyster and Yunzhi mushrooms 8 Experiment 11: Effect of liquid environmental components to the growth of King oyster commercial mushrooms. 2.3.4. The experiment using liquid spawns to the cultivate King oyster and Yunzhi mushrooms. Experiment 12: Evaluation of the impact of the original source of spawns and cultivating materials to the growth of King oyster and Yunzhi mushrooms. Experiment 13: Effect of age of liquid spawns to the growth of King oyster and Yunzhi mushrooms on farming materials. Experiment 14: Effect of quantity of commercial spawns to the growth and development of King oyster and Yunzhi mushrooms on farming materials. 2.4. Methods of environment preparation 2.5. Experimental designing method - The experiment was repeated 3 times arranged and carried out as follows: + For the first grade spawns: 5 flasks of 500ml /treatment, each flask contains 200ml liquid nutrient medium. A total of 15 flasks /1 treatments. + For the spawns of intermediate grade: 5 Duran flasks of 5000ml/treatment, each Duran flask containing 3000 ml liquid nutrient medium. A total of 15 Duran flasks/1 treatment. + For commercial spawn: 30 liters liquid nutrient medium / 1 bioreator / treatment. + In the breeding period: each treatment, 100 bags/ experimental layout. A total of 300 bags of materials / treatments; tracking targets growth of 50 bags / treatments, taking samples at 5 diagonal points, 10 bags /each point. * Observation criteria - Monitoring the growth characteristics in pure culture - Monitoring the growth characteristics in the liquid spawn period + Mycelia pellet size (KLC) (mm) + Mycelia biomass (gr/1000ml) + Mycelia pellet density * King Oyster Mushrooms (1) Number KLC from 10-40 KLC/1ml (2) Number KLC from 41-80 KLC/1ml (3) Number KLC from 81-120 KLC/1ml (4) Number KLC from 121-150 KLC/1ml (5) Number KLC above150 KLC/1ml * Yanzhi mushroom (1) Number KLC from 10-30 KLC/1ml (2) Number KLC from 31-60 KLC/1ml (3) Number KLC from 61-90 KLC/1ml (4) Number KLC from 91-120 KLC/1ml (5) Number KLC above 120 KLC/1ml - Monitoring the growth characteristics on their cultivation phase . 9 2.6. Methods of data processing The study results are processed by biostatistics methods by Excel and software of IRRISTAT 4.0. 2.7. Location and time of the experiments - The experiment was conducted at the Center for Plant Biotechnology; Division of Genetic Technique - Agricultural Genetics Institute; Institute of Microbiology and Biotechnology - Hanoi National University; Van Giang Mushroom Research Center, Van Giang - Hung Yen - Duration of experiment: from 4/2008 - 4/2013. CHAPTER 3 RESULTS AND DISCUSSION 3.1. Study on some biological characteristics of King oyster and Yunzhi mushrooms 3.1.1. The growth and formation of fruiting bodies of King oyster mushrooms in pure culture In this study, six strains of King oyster mushroom are incubated in pure culture in different temperature ranges. Table 3.1. Effect of temperature on the growth of King oyster mushroom mycelia system in pure culture Spawn Temperature E1 E2 E3 E4 E5 E6 I II I II I II I II I II I II 18 ± 10C 3 162,5 3 166,6 3 148,1 3 157,4 3 159,4 0,5 34,1 22 ± 10C 3 283,3 3 278,4 2,5 263,8 2,5 268,5 3 269,5 1 45,5 26 ± 10C 2,5 312,5 2,5 315,6 2,5 296,3 2,5 305,5 3 319,4 3 132,5 30 ± 10C 1,5 354,1 1,5 361,1 1,5 351,8 1,5 361,1 1,5 351,9 2,5 145,8 34 ± 10C 1 273,7 1 270,3 1 266,2 1 260,7 1 264,3 2 131,6 CV% 4,1 4,1 3,7 4,1 3,3 3,4 LSD0,05 20,8 20,9 17,9 20,4 16,3 6,1 *Note: I. Thickness of mycelia system (unit) 3 unit thickness: very thick mycelia system 2.5 units thickness : thick mycelia system Growth rate of the mycelia system (µm/h) 2 unit thickness : average mycelia system 1.5 units thickness : thin mycelia system 1 unit thickness: very thin mycelia system The results showed that 5 varieties of King oyster mushrooms from E1 - E5 have many similar characteristics when cultured on agar in the studied temperature range. The growth rate of mycelia system of E1 strain is the fastest. The features of strain E6 are clearly different with the above 10 5 strains on the growth duration and characteristics of fiber systems, the formation of fruiting bodies. 3.1.2. The growth and formation of fruiting bodies of King Oyster mushroom on cultivating materials Table 3.3 . Some growth characteristics of the King oyster mushroom strains Strains Stalk diameter (mm) Peripileic diameter (mm) Length of stalk (mm) No of fruiting bodies / bag Average weight of fruiting body (gr) Yield /bag (gr) E1 34,6 41,6 148,8 1,6 141,6 197,6 E2 29,4 39,8 145,6 1,4 118,2 165,4 E3 22,7 31,4 126,4 2,1 62,7 145,7 E4 23,8 29,7 128,8 2,4 63 149,5 E5 30,9 23,9 129,7 1,6 111,2 157,9 E6 20,6 30,4 96,5 3,6 40,2 144,7 CV% 4,5 3,2 6,5 5,5 5,6 4,3 LSD0,05 2,2 1,9 14,8 0,2 8,9 12,3 On the culture material of King oyster mushroom strains, there are very clear rules of growth as compared with that in pure culture. Of all 6 strains of King Oyster mushroom in the study, the strain E1 gave the highest weight of fruiting body/bag (197.6 gr/bag). Other strains in the study all gave lower fruiting body weight/bag; strain studied marketing them for their weight E1/ bag highest (197.6 gr / bag ). All other strains in the study gave lower weight/bag, the fruiting body of strain E6 collected gave lower weight/bag (144.7 g / bag). 3.1.3. Characteristics of King oyster mushroom spores Spores of the fungus species were observed on a scanning electron microscope with magnification of 500-15,000 times. In 7,500 times magnification, King oyster mushroom spores have ovoid shape put closely together attached to tapered basidium. 3.1.4. The growth and formation of fruit bodies of Yunzhi mushroom in pure culture The growth of the Yunzhi mushroom mycelium in pure culture were recorded in Figure 3.32. 11 Figure 3:32: growth rate of Yunzhi mushroom mycelia system in different temperature conditions In different temperature range of the culture, the growth rate of Yunzhi mushroom mycelia system differs, characteristics of mycelia system are also different. During the whole process of rooting and the after the mycelium covered the medium surfaces, at any temperature, lighting conditions, there are not signs of formation of fruiting bodies in pure culture of the two Yunzhi mushroom strains. 3.1.5. Studies on the growth and fruitingbody formation of Yunzhi on farming materials The results show that: from February to April: The mycelia systems and fruiting bodies of 2 Yunzhi mushroom strains have good growth, less affected by diseases. Two Yunzhi strains Tra-1 and Tra-2 have a lot of similiar characteristics: mycelia sprouting time, fruiting body formation time, fruiting body size, color, yields of fresh fruiting bodies of 2 strains in different times of the year of the study…. Table 3.6: Affects of cultivating time to fruiting body size and yield of Yunzhi mushroom Spawn Criteria Time Vertical size of mushroo m cap (mm) Horizontal size of mushroom cap (mm) No of mushroom cap layers /penicle Weight of fresh mushroom/ bag (gram) Tra-1 November - January February - April 54,2 78,4 6,8 81,8 May - July 16,2 18,3 2,6 42,6 August - October 42,8 56,7 5,4 65,4 CV% 6,3 5,7 7,6 4,5 LSD0,05 4,78 5,86 0,75 5,72 Tra-2 November - January February - April 54,6 77,9 6,6 80,9 May - July 15,8 18,0 2,5 42,4 August - October 42,1 55,8 5,5 65,2 CV% 6,7 4,2 4,6 6,3 250 300 350 400 Tốc độ sinh trưởng Tra 1 (µm/h) 12 LSD0,05 5,05 4,25 0,45 7,92 3.1.6. Characteristics of microscopic Yunzhi mushroom spores On scanning electron microscope with magnification of 2,000 times, Yanzhi mushroom spores are oligosporous at 15,000 times magnification of the two strains of Tra-1 and Tra-2, there are only a few basidium that formed 4 sterigma on which, there fixed 4 typical basidiums. 3.2. Assessment of genetic differences of strains of King oyster and Yunzhi mushrooms 3.2.1. The genetic differences of King oyster mushroom strains Analysis of genetic diversity Table 3.8. Genetic similarity coefficiences of the fungi samples in the study Sample 1 2 3 4 5 6 1 1.00 2 0.80 1.00 3 0.70 0.79 1.00 4 0.71 0.79 0.85 1.00 5 0.67 0.70 0.68 0.75 1.00 6 0.64 0.67 0.66 0.66 0.68 1.00 Through the analysis of genetic relationship, a relationship diagram of the fungal genetics of the samples in the research have been established. Genetic similarity coefficient with lowest value of 0.64 is between sample 1 and 6. Highest similarity coefficient is between two samples number 3 and number 4 with a value of 0.85. The pair samples of No.1 and No.2 has rather high genetic similarity coefficient of 0.80. 3.2.2. The genetic differences of Yunzhi strains The results of electrophoresis of PCR products of 2 samples with 10 primers, each primer obtained similar number of DNA bands and band size. This result is entirely consistent with the results of the study of biological characteristics of 2 Yunzhi mushroom strains as these 2 strains have similar growth characteristics of the mycelia system as well as the fruiting bodies. 3.3. Results on the propagation of King oyster and Yunzhi spawns From the results of the evaluation of biological characteristics and genetic differences of the mushroom strains in the study, King oyster mushroom strain E1 and Yunzhi strain Tra-1 were selected, these are 2 promising strains in Vietnam. To promote production and sustainable development of the selected fungus, the research will continue to conduct studies on propagation technologies of liquid spawns and use these spawns for cultivation in 13 order to reduce growth duration, increase seed vigor and yield of commercial mushroom. 3.3.1. Result on the propagation of King oyster and Yunzhi liquid spawns, Grade 1 3.3.1.1. Effect of temperature on growth of King oyster and Yunzhi mushrooms in liquid medium The results obtained in studies on the effect of temperature on the growth of King oyster E1 and Yunzhi Tra-1 in liquid medium, we found that, in the temperature range of 26 ± 1°C, King oyster mushroom farming in liquid medium; Yunzhi farming in the temperature range of 30 ± 1°C are the most appropriate, the results are consistent with studies on the growth of mycelia system of these 2 strains in pure culture. Table 3.9. Effect of temperature on growth of King oyster and Yunzhi mushrooms in liquid medium Spawn Criteria Temperature Mycelia pellet size(mm) Mycelia pellet density Mycelia biomass (gr/1000ml) King oyster spawn E1 18 ± 1°C 0,44 2 9,7 22 ± 1°C 0,76 3 12,8 26 ± 1°C 1,28 4 18,6 30 ± 1°C 1,65 4 17,9 CV% 3,4 5,2 LSD0,05 0,67 1,43 Yunzhi spawn Tra-1 22 ± 1°C 0,72 1 13,8 26 ± 1°C 0,94 2 17,6 30 ± 1°C 1,18 4 21,5 34 ± 1°C 1,26 2 15.6 CV% 2,7 3,8 LSD0,05 0,52 1,24 3.3.1.2 . Effect of pH on the growth of King oyster and Yunzhi mushrooms in liquid medium The results showed that in the initial pH range from 5-6, mycelia biomass increases. When the initial medium pH = 6, the density of mycelia pellet and mecelia biomass of King oyster are the largest (18.6 g/1000ml). At the pH = 7, the mycelia biomass begin to show decrease. At a pH value of 8, mycelia rates drops sharply (14.1 gr/1000ml ). So, the mycelia biomass of King oyster is the largest when the initial medium pH value = 6. 14 Yunzhi mycelium biomass reached the highest (27.6 g/1000ml liquid) when cultured under conditions of pH= 6. There is not much difference between the medium pH = 6 and pH = 7. When the pH = 8, the mycelia pellet and biomass density decreased remarkedly (17.8 gr/1000ml). Table 3.10 . Effect of initial pH on the growth of King oyster and Yunzhi mushrooms in liquid medium Spawns Initial pH pH value after sterilization pH value at the end of cultivation process Mycelia pellet density Mycelia biomass (g/1000ml ) King oyster spawn E1 3 2,93 2,02 1 9,8 4 3,95 2,84 2 14,6 5 4,95 3,82 3 16,1 6 5,95 4,84 4 18,6 7 6,96 5,74 3 16,3 8 7,98 6,58 2 14,1 CV% 4,0 LSD0,05 1,05 Yunzhi spawn Tra-1 3 2,94 1,95 1 10,3 4 3,93 2,74 1 14,2 5 4,96 3,67 2 17,4 6 5,95 4,75 4 27,6 7 6,96 5,71 4 26,3 8 7,97 6,66 2 17,8 CV% 4,8 LSD0,05 1,57 3.3.1.3. The effects of nutritionnal elements on the growth of King oyster and Yunzhi mushrooms in liquid medium. The data in Table 3.11 shows that: King oyster mushroom grows best in medium mixture 4 (2.5 g yeast extract + 2.,5 gr pepton e+ 0.5 g MgSO4.7H2O + 1 g KH2PO4 + 100 gr of fresh mushrooms + 15gr glucose); Yunzhi mushroom grows best in medium mixture 3 (2 gr yeast extract + 2 gr pepton e+ 0.2 gr MgSO4.7H2O + 1 gr KH2PO4 + 15 gr glucose + 1.5mg thiamin) 15 Table 3.11. The affects of liquid medium element to the growth of King oyster and Yunzhi mushrooms Spawn s Treat ment Mycelia pellet size (mm) Mycelia density Mycelia biomass (g/1000ml) Mycelia pellet charcteristics King oyster spawn E1 CT1 1,96 2 18,8 Smooth, liquid dispersion CT2 0,87 4 20,4 Smooth, liquid suspended linkage form CT3 0,84 5 28,5 Slick, liquid suspended linkage form CT4 1,08 5 29,2 Slick, liquid suspended linkage form CT5 1,67 3 24,8 Slick, liquid dispersion CV% 3,4 2,2 LSD0.05 0,78 0,98 Yunzhi spawn Tra-1 CT1 1,43 1 15,4 Hyaline, fringed mycelia pelle, scattered density. CT2 1.32 3 22,8 Hyaline, fringed mycelia pelle, medium density. CT3 1,28 5 35,2 Hyaline, fringed mycelia pellet, thick density. CT4 1.3 4 30,1 Hyaline, fringed mycelia pelle, thick density CT5 1,46 2 18,4 Hyaline, fringed mycelia pelle, scattered density. CV% 4,5 4,8 LSD0.05 0,11 2,12 3.3.1.4. Affect of shaking mode to the growth and development of King oyster and Yunzhi mushrooms. The result in Table 3.12 showed that King oyster mushroom gave highest mycelium biomass (26.9gram/1000ml) at shaking mode of 160 rounds/minute; Yunzhi mushroom offered the highest mycelium biomass (36.2 gram/1000ml) at shaking mode of 140 rounds/ minute. 3.3.1.5. Affects of inoculating spawns rate to the growth of mycelia system of King oyster and Yunzhi mushrooms in liquid medium The result showed that the rate of incubated spawns affected much to 16 mycelia pellet density, size and biomass of both King oyster and Yunzhi mushrooms. When using mother spawns with the rate of 30% original spawn, we could collect spawn of high mycelia pellet density, high mycelia pellet size (1.23 -1.05 mm), highest mycelia biomass as compared to other rate of spawn incubation forms (32.4 - 34.7 gr/1000ml). Table 3. 13. Affects of inoculating spawns rate to the growth of King oyster and Yunzhi mushrooms Spawn Criteria Percentage of original spawn (% ) Mycelia pellet size (mm) Mycelia pellet density Mycelia biomass (g/1000ml) Characteristics King oyster spawn E1 10 2,34 2 24,6 Spheric form mycelium, some smooth, some have tassels, scattered 20 1,82 3 28,1 Spheric form mycelium, some smooth, some have tassels, scattered 30 1,23 4 32,4 Spheric form mycelium, smooth, scattered 40 0,60 5 28,4 Small spheric form mycelium, smooth, strong cohesion CV% 2,9 2,3 LSD0.05 0,82 1,24 Yunzhi spawn Tra-1 10 1,55 2 22,8 Big mycelia pellet, scattered, tassels 20 1,37 3 28,6 Big mycelia pellet, equal, scattered, with tassels. 30 1,05 4 34,7 Medium mycelia pellet, cohesion, with tassels. 40 0,63 5 29,2 Small mycelia pellet, strong cohesion, with tassels . CV% 3,1 3,5 LSD0.05 0,67 2 1,89 3.3.1.6. Effect of inoculation time on the growth of King oyster and Yunzhi in liquid medium Mycelium biomass showed clear growth in time range of 48 hours to 72 hours (King oyster Mushroom reached 21.2 -28.2 gram/1000ml; Yunzhi mushroom only reach 18.4 -32.8 gram/1000ml), after 72 hours, mycelia biomass of both King oyster mushrooms and Yunzhi mushroom are increasing but only negligible, Mycelia biomass peaked in 96 hours (King oyster mushroom reached 28.9 gram/1000ml, Yunzhi mushroom reached 33.7 gr/1000 ml). 17 Figure 3:59. The growth curves of King oyster mushroom spawn E1 and Yunzhi Tra-1, liquid forms 3.3.2. Research results on propagating of intermediate liquid spawns of King oyster and Yunzhi mushrooms 3.3.2.1. Effect of aeration mode on the growth of intermediate spawns of King oyster and Yunzhi mushrooms Table 3:15. Effect of aeration intensity on the growth of intermediate spawns of King oyster and Yunzhi mushrooms Spawns Criteria Aeration supply mode (V/V/M) Mycelia pellet size (mm/ KLC) Mycelia density Mycelia biomass (g/1000ml) King oyster spawn E1 0,4 1,74 1 19,6 0,5 1,58 2 26,2 0,6 1,30 3 32,1 0,7 1,06 4 34,8 0,8 0,57 5 33,6 CV% 4,3 2,0 LSD0.05 0,98 1,06 Yunzhi spawn Tra-1 0,4 1,62 1 21,8 0,5 1,47 2 28,9 0,6 1,02 4 35,2 0,7 0,81 4 34,6 0,8 0,54 5 32,8 CV% 3,3 3,3 LSD0.05 0,65 1,84 For King oyster mushroom, the mycelia biomass reached the highest mycelia biomass of 34.8 ml gram/1000 at the air supply of 0.7 V/ V/M. 0 10 20 30 40 Thời gian nuôi (giờ) 48 60 72 84 96 120 Sinh khối sợi nấm Sò vua ( g/1000 ml) Sinh khối sợi nấm Vân chi ( g/1000 ml) 18 For Yunzhi, in the aeration mode of 0.6 V/V/M, mycelia biomass reached 35.2 gram/1000 ml of the spawn liquid. 3.3.2.2. Effect of spawn rate on the growth of King oyster Yunzhi mushrooms of intermediate grade Table 3:16. Effect of spawn rate on the growth of intermediate grade of King oyster and Yunzhi mushrooms Spawn Criteria Percentage of spawn (%) Mycelia pellet size (mm) Mycelia density Mycelia biomass (g/1000ml) King oyster spawn E1 3 1,72 1 19,8 5 1,54 2 28,2 7 1,28 4 33,7 10 0,88 4 32,8 15 0,55 5 30,6 CV% 3,3 3,3 LSD0.05 0,72 1,72 Yunzhi mushroom Tra-1 3 1,58 1 20,6 5 1,41 2 26,8 7 1,08 3 32,4 10 0,86 4 35,6 15 0,52 5 32,5 CV% 4,7 2,7 LSD0.05 0,93 1,47 For King oyster mushroom implanted with 7% spawn will give highest mycelia biomass (33.7 gram/1000ml), uniformed mycelia pellet, the liquid is not agglomerate. For Yunzhi mushroom implanted with 10% spawn will give the highest mycelia biomass (35.6 gram/1000ml). 3.3.3. Research results on propagation of commercial mushroom (King oyster and Yunzhi mushrooms,) liquid types King Oyster mushroom grow best in Formula 3 of medium: 2 g yeast extract + 7 g peptone + 0,5 g MgSO4.7H2O + 1 g KH2PO4 + 15 glucose, mycelia biomass peaked at time from 72 -84 hours (27.6 to 27.9 gam/1000ml); Yunzhi mushroom grows best in medium formula II: 1.5 gr yeast extract + 6 gr peptone + 1 gr KH2PO4 + 0.2 gr MgSO4.7H2O + 15 gr glucose + 1 mg thiamin, mycelia biomass peaked in time of 72-84 hours (30.2 to 30.9 gram/1000ml). 19 Table 3:17: Influence of medium components on the growth of the commercial King oyster mushroom and Yunzhi mushroom Varieties Formula Mycelia biomass (gr/1000ml) 48 hours 60 hours 72 hours 84 hours 96 hours 120 hours King oyster spawn E1 1 13,7 15,9 19,8 21,2 21,8 21,9 2 14,5 18,3 22,6 23,4 23,7 25,7 3 16,8 20,5 25,7 27,6 27,9 27,7 4 16,9 20,7 23,9 25,6 25,9 25,8 CV% 3,5 3,1 3,7 2,7 1,8 2,8 LSD0.05 1,1 1,2 1,7 1,4 0,9 1,4 Yunzhi spawn Tra-1 1 15,8 20,6 24,5 26,4 26,8 26,7 2 16,2 21,7 28,8 30,2 30,9 30,7 3 16,4 22,7 28,7 30,0 30,8 30,5 4 16,7 22,6 28,4 29,8 30,2 30,2 CV% 3,9 1,6 1,6 1,3 4,4 3,9 LSD0.05 1,3 0,7 0,9 0,8 2,6 2,3 3.4. Application of liquid spawn for cultivation of King oyster and Yunzhi mushrooms 3.4.1. The effects of spawn source and cultivation materials on the growth of King oyster and Yunzhi mushrooms. 3.4.2. The effects of cultivation materials on the formation and development of fruiting bodies of King oyster and Yunzhi mushrooms. 3.4.3. The influence of mixing formula on the size of fruiting bodies and yields of King oyster and Yunzhi mushrooms The study results on the influences of material kinds on the formation, development of fruiting bodies and yields of King oyster and Yunzhi mushrooms are shown in Table 3.22 and Table 3.23 The results collected during the observation on the growth, development of King oyster mushroom in different raising formulas with 2 sources of liquid spwn and traditional spawn showed that: the supplement of nutrition during rearing process of King oyster mushroom had clear effects on the growth, development and yields of mushrooms; with the supplement of 20% rice bran, the size of fruiting bodies is the biggest, and highest yield of mushroom. Using liquid spawn will shorten from 12 -14 days of rearing time in all formulas. 20 Table 3.22: The effects of medium mixture formulas on the size of fruiting bodies and yield of fresh mushroom of King Oyster Spawn Treatment Mushroom cap diameter (mm) Stalk diameter (mm) Stalk length (mm) Bio- efficientcy (%) King oyster liquid spawn E1 1 28,9 20,7 98,7 36,8 2 36,7 25,6 105,8 48,9 3 42,5 29,8 121,6 52,7 4 48,8 31,6 134,7 58,5 5 52,5 34,8 148,4 67,4 6 52,6 34,5 143,2 67,2 CV% 2,8 3,2 3,4 2,3 LSD0.05 2,2 1,7 7,6 2,3 King oyster solid spawn E1 1 27,7 20,1 96,5 33,5 2 35,2 24,3 102,6 41,7 3 40,4 27,9 120,1 46,8 4 46,8 30,4 132,4 51,4 5 50,4 32,6 146,2 59,4 6 50,3 32,5 143,4 59,1 CV% 2,8 6,1 2,5 2,7 LSD0.05 2,1 3,1 5,5 2,3 Table 3.23. The effects of medium mixturebformulas on the size of fruiting bodies and fresh mushroom yield of Yunzhi Spawn Treatment Vertical size of mushroom cap (mm) Horizontal size of mushroom cap (mm) Number of cap layers/cluster Bio efficient (%) Yunzhi liquid spawn Tra-1 1 31,5 43,8 3,6 5,7 2 48,7 65,4 5,7 12,8 3 60,8 72,5 6,2 19,6 4 68,2 85,4 7,8 24,5 5 67,4 84,7 7,8 24,3 6 65,6 81,8 7,5 24,3 CV% 3,4 1,9 5,3 4,9 LSD0.05 3,5 2,4 0,6 1,6 Yunzhi solid spawn Tra- 1 1 30,7 43,2 3,4 5,2 2 46,8 64,8 5,2 12,3 3 60,2 71,9 5,9 18,9 4 67,8 85,1 7,3 24,1 5 67,5 85,3 7,7 24,0 6 65,9 81,5 7,6 23,8 CV% 3,9 3,7 4,4 3,2 LSD0.05 3,96 4,8 0,49 1,03 21 Cultivating Yunzhi mushroom on formula 4 (65% sawdust, 24% cotton by product, 10% rice bran, 1% CaCO3) achieved the highest yield. Depending on the concrete conditions, the application of spawn sources, either solid or liquid spawn will be chosen, as for Yanzhi mushroom, the bio- efficient is not much different when using these spawns, there is only difference in growth duration. 3.4.4. The influence of liquid spawn age to growth of King oyster and Yunzhi mushrooms on cultivating materials Different with other researches, the main aim is to get mycelia, so these researches stop at the observation of mycelium. In our research, the final aim is to get mushroom fruiting bodies, as mycelia biomass can not help to evaluate whether the spawn is good or bad. The next steps in our research, solid spawn is used as the control, using liquid spawn for cultivation in the time from 48 -120 hours as trial experiment. The research result showed that : using liquid spawn can help to reduce time for mycelium to cover all cultivating material and earlier time for the start of bearing fruiting bodies, higher yield in comparison with those of solid spawn . When using spawn replacing for cultivating King oyster mushroom, the time for mycelium to cover all material bag is 35 days; time to begin collecting fruiting bodies is 54 days; bio- efficient reached 58,4%. Whereas, using liquid spawn source of 72 - 96h of age for cultivating King oyster mushroom, time for mycelium to cover all material bag decreases down to 24 days, time for collecting fruiting bodies is 38 - 39 days, bio efficiency reached 65.8 - 67.3%. Similarly, when using liquid spawn source of Yunzhi mushroom of 72h- 96h of age, time for mycelium to start growing is only 25 days in comparison with 31 days of solid spawn; using liquid spawn source of ; using liquid spawn 72 -84 hours of age of Yunzhi mushroom, the bio-efficiency reached 25,1%, while using solid spawn of Yunzhi mushroom, bio-efficiency reached 24.4%. 3.4.5. The influence of commercial spawn quantity to the growth of King oyster and Yunzhi mushrooms on the cultivation materials 22 Table 3.25. The influence of commercial spawn quantity to the growth of King oyster and Yunzhi mushrooms on the cultivation materials Spawn Spawn quantity Percentage of disease infection (%) Time for mycelium to cover all material (daysy) Time for fruiting bodies to appear (days) Bio-efficent (%) King oyster spawn E1 10 9,8 31 42 44,3 15 7,6 27 40 53,7 20 5,7 23 38 66,4 25 5,7 23 38 66,5 30 5,8 23 38 66,4 CV% 4,6 LSD0,05 1,25 Yunzhi spawn Tra-1 10 9,3 34 47 13,4 15 6,8 29 42 19,2 20 5,2 25 39 24,6 25 5,4 25 38 24,7 30 5.5 25 37 24,8 CV% 4,3 LSD0,05 1,89 To achieve high economic efficiency, we chose quantity of spawn liquid to implant into cultivating material as 20 ml liquid/bag for each spawn kind. 3.4.6 . The economic efficiency of using the liquid spawns for raising King oyster and Yunzhi mushrooms The economic efficiency of liquid spawns of King oyster and Yunzhi mushrooms for cultivation for 1 ton of dry material is presented in Table 3:26. Using the liquid spawn source for cultivating can not only shorten the starting time for harvesting the fruit bodies of King oyster mushroom from 58 days to 45 days; start time for harvesting the fruit bodies of bodies of Yunzhi mushroom from 56 days to 45 days, but also bring about high net interest. Using liquid spawn for King oyster mushroom cultivation give high net interest (5.790 million VND / ton of material); interest rate as compared to the capital reached 0.4 ; Net profit increased compared to the control using solid spawn is 75.99 % . For Yunzhi using liquid spawn source for cultivation bring about rather high net interest (3.530 millions VND / ton of material ); interest rate of 0.32 comparing to the capital; Net profit increased to 41.77 % comparing to the control using solid spawns. 23 Table 3.26. Economic efficiency of cultivating King Oyster and Yunzhi mushrooms by liquid spawns Unit: 1000 VND Spawn source Labour cost Material cost Total input YIeld (kg/ton of material) Selling price Tổng thu Net interest Interes t rate Net interest rate incomparison with the control (%) King oyster liquid spawn 6.000 8.430 14.430 404,4 50 20.220 5.790 0,40 75,99 King oyster solid spawn (the control) 7.000 7.530 14.530 356,4 50 17.820 3.290 0,23 Yunzhi liquid spawn 5.000 6.170 11.170 36,75 400 14.700 3.530 0,32 41,77 Yunzhi solid spawn (The control) 6.000 5.970 11.970 36,15 400 14.460 2.490 0,21 In summary, using the liquid spawns of King oyster mushroom and Yunzhi mushroom help to shorten the farming cycle than by traditional methods from 11-13 days; with lower investment costs for growing mushrooms by liquid spawns than using solid spawns but higher productivity, this method should be invested and applied to production in industrial scale. CONCLUSION 4.1. Conclusion 1. From the assessment results on indicators of growth and development of 6 spawns of King Oyster mushrooms (Pleurotus eryngii) and 2 spawns of Yunzhi mushroom in pure culture stages and rearing period, King oyster mushroom strain E1 and Yunzhi mushroom strain Tra-1, adaptable to the ecological conditions of Vietnam, have been selected. 2. Optimal conditions for propagating E1 liquid spawn of King oyster mushrooms at the stage of spawn grade 1: temperature for rearing mycelium is 26 ± 1ºC; pH6; medium formula 5; shakeing mode 160 r /min; original spawn seed rate for inoculation is 30% of original spawns/200ml of liquid medium; innoculating time is 96 hours. In the stage of intermediate spawns: aeration rate of 0.7 liters of air / medium 24 liter / min; rate of spawn gradel 1 for seedlings of intermediate spawns is 7% compared to the medium. For the stage of commercial spawns, suitable medium formula for breeding commercial King oyster mushroom E1 is formula 3. Optimal conditions for propagation of Yunzhi Tra-1 (Trametes versicolor) liquid spawn level 1: temperature: 30 ± 1 º C; pH6; Medium Formula 3; shaking mode 140 r/min, the rate of original spawns used for innocubating 30% /200ml of medium; incubation time is 84 hours. In the stage of intermediate spawns: aeration rate of 0.6 l air /1 liter of medium / min; ratio of original spawn of Grade 1 for mediated inoculation is 10% comparing to the medium; suitable medium formula for breeding commercial Yanzhi mushroom Tra-1 is formula 2. 3. The farming of E1 King oyster mushroom achieve the highest efficiency on the formula 5; optimal incubating time is from 84h - 96h of age; appropriate spawn quantity is 20ml liquid spawn/ bag of material. Using liquid spawns for cultivation of King oyster mushroom can shorten 13 days / cultivating cycle. Cultivating Yunzhi mushrooms Tra-1 achieved the highest efficiency by formula 4; optimal incubating age is from 72h - 84h of age; appropriate seed for implantation is 20ml of liquid spawn/ bag of material. Using liquid spawn for mushroom cultivation can reduce 11 days /cultivating cycle. 4.2. Recommendation From the research results obtained, we respectfully request to continue researching to improve the technology; Applicating liquid spawn propagating technologies to production to develop King Oyster mushroom E1 and Yunzhi mushroom Tra-1 in industrial scale, in order to aim at higher efficient and sustainability for mushroom production. PUBLISHED DOCCUMENTS RELATED TO THE THESIS 1. Nguyen Thị Bich Thuy, Trịnh Tam Kiet (2010), Study some biocharacteristics and technique for cultivation of Yunzhi mushroom (Trametes versicolor), Genetics and Application – Special issue of Bio-technology, Volume No 6: page 55-58. 2. Nguyen Thi Bich Thuy, Co Thi Thuy Van, Trinh Tam Kiet (2012), Studying the propagation technology for King Oyster mushroom (Pleurotus eryngii) liquid form Genetics and Application – Special issue of Bio -technology, Volume No 8: page 88-95. 3. Nguyen Thi Bich Thuy, Co Thi Thuy Van, Đinh Xuan Linh, Trinh Tam Kiet, Nguyen Trung Thanh (2013), “Study to multiply Yunzhi mushroom (Trametes vesicolor) liquid form”, Journal of Vietnam Agricultural Science and Technology, No 41, page: 67-73. 4. Nguyen Thị Bich Thuy, Khuat Huu Trung, Ngo Xuan Nghien, Co Thi Thuy Van, Trinh Tam Kiet (2013), “Studying bio-characteristics and genetic diversity of some strains of King Oyster mushroom (Pleurotuseryngii)”, Journal of Vietnam Agricultural Science and Technology, No 41, page: 73-80.