Summary of doctoral thesis 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
AND TRAINING

MINISTRY OF AGRICULTURE
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......................................................................


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.

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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

2


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

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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 bioproductsReishi 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 - 250C and the most appropriate
time for fruit bodies to grow is from february to may (Solar calendar)
(Nguyen Thi Chinh , 2011 ) .

4


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 -150C; 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

5


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

6


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

7


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
.

8


I.

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

E1

E2

E3

I

II
I
II
I
Temperature
0
18 ± 1 C
3
162,5
3 166,6 3
22 ± 10C
3
283,3
3 278,4 2,5
26 ± 10C
2,5 312,5 2,5 315,6 2,5
30 ± 10C
1,5 354,1 1,5 361,1 1,5
34 ± 10C
1
273,7
1 270,3 1
CV%
4,1
4,1
LSD0,05
20,8
20,9
*Note:
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)

E4
II

I

148,1 3
263,8 2,5
296,3 2,5
351,8 1,5
266,2 1
3,7
17,9

E5
II

I

E6
II

I

II

157,4 3 159,4 0,5 34,1

268,5 3 269,5 1
45,5
305,5 3 319,4 3 132,5
361,1 1,5 351,9 2,5 145,8
260,7 1 264,3 2 131,6
4,1
3,3
3,4
20,4
16,3
6,1

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

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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

E1
E2
E3
E4
E5
E6
CV%
LSD0,05

34,6
29,4
22,7

23,8
30,9
20,6
4,5
2,2

41,6
39,8
31,4
29,7
23,9
30,4
3,2
1,9

148,8
145,6
126,4
128,8
129,7
96,5
6,5
14,8

1,6
1,4
2,1
2,4
1,6
3,6

5,5
0,2

Average
weight
of fruiting
body (gr)

Yield
/bag
(gr)

141,6
118,2
62,7
63
111,2
40,2
5,6
8,9

197,6
165,4
145,7
149,5
157,9
144,7
4,3
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.

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400
350
300
250

Tốc độ
sinh
trưởng Tra
1 (µm/h)

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
Criteria
Spawn

Time

Tra-1 November - January
February - April
May - July
August - October
CV%
LSD0,05

Tra-2 November - January
February - April
May - July
August - October
CV%

Vertical
size
of
mushroo
m cap
(mm)

Horizontal
No of
size of
Weight of fresh
mushroom
mushroom
mushroom/ bag
cap layers
(gram)
cap
/penicle
(mm)

54,2
16,2
42,8
6,3

4,78

78,4
18,3
56,7
5,7
5,86

6,8
2,6
5,4
7,6
0,75

81,8
42,6
65,4
4,5
5,72

54,6
15,8
42,1
6,7

77,9
18,0
55,8
4,2

6,6
2,5
5,5
4,6

80,9
42,4
65,2
6,3

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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
1.00
1
0.80
1.00
2
0.70
0.79
1.00
3
0.71
0.79
0.85
1.00
4
0.67
0.70
0.68
0.75
1.00
5

0.64
0.67
0.66
0.66
0.68
1.00
6
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

12

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
Criteria Mycelia
Mycelia
Mycelia
Spawn
pellet
pellet
biomass
size(mm)
(gr/1000ml)
Temperature
density
0,44
2
9,7

King oyster
18 ± 1°C
spawn
0,76
3
12,8
22 ± 1°C
E1
1,28
4
18,6
26 ± 1°C
1,65
4
17,9
30 ± 1°C
CV%
3,4
5,2
0,67
1,43
LSD0,05
0,72
1
13,8
Yunzhi
22 ± 1°C
spawn
0,94
2

17,6
26 ± 1°C
Tra-1
1,18
4
21,5
30 ± 1°C
1,26
2
15.6
34 ± 1°C
CV%
2,7
3,8
0,52
1,24
LSD0,05
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.

13

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
Initial pH
Spawns

King
oyster
spawn
E1

Yunzhi
spawn
Tra-1

3
4
5
6
7
8
CV%
LSD0,05
3
4
5
6

7
8
CV%
LSD0,05

pH value
after
sterilization

pH value at
the end of
cultivation
process

Mycelia
pellet
density

2,93
3,95
4,95
5,95
6,96
7,98

2,02
2,84
3,82
4,84
5,74

6,58

1
2
3
4
3
2

2,94
3,93
4,96
5,95
6,96
7,97

1,95
2,74
3,67
4,75
5,71
6,66

1
1
2
4
4
2

Mycelia
biomass
(g/1000ml
)
9,8
14,6
16,1
18,6
16,3
14,1
4,0
1,05
10,3
14,2
17,4
27,6
26,3
17,8
4,8
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)

14


Table 3.11. The affects of liquid medium element to the growth of
King oyster and Yunzhi mushrooms
Spawn Treat
s
ment

Mycelia
pellet size
(mm)

Mycelia
density
2

Mycelia
biomass
(g/1000ml)

CT1
CT2
CT3

0,84

CT4

1,08

1,67
3,4

LSD0.05

0,78

CT1

1,43

CT2

1.32

CT3

1,28

CT4

1.3

CT5

1,46

18,4

CV%

4,5

18,8

0,11

Smooth, liquid
dispersion
Smooth, liquid
suspended linkage form
Slick, liquid suspended
linkage form
Slick, liquid suspended
linkage form
Slick, liquid dispersion

4,8

LSD0.05

Yunzhi
spawn
Tra-1

0,87

CT5
CV%

King
oyster
spawn
E1

1,96

Mycelia pellet
charcteristics

2,12

4

20,4

5

28,5

5

29,2

3

24,8
2,2
0,98

1

15,4

3

22,8

5

35,2

4

30,1

2

Hyaline, fringed mycelia
pelle, scattered density.
Hyaline, fringed mycelia
pelle, medium density.
Hyaline, fringed mycelia
pellet, thick density.
Hyaline, fringed mycelia
pelle, thick density
Hyaline, fringed
mycelia pelle, scattered
density.

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

15


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
Criteria
Mycelia
pellet size
(mm)

Mycelia
pellet
density

Mycelia
biomass
(g/1000ml)

10

2,34

2

24,6

20

1,82

3

28,1

30

1,23

4

32,4

40

0,60

5

28,4

CV%
LSD0.05
10

2,9
0,82
1,55

2

2,3
1,24
22,8

20

1,37

3

28,6

30

1,05

4

34,7

40

0,63

5

29,2

CV%
LSD0.05

3,1
0,67

2

3,5
1,89

Spawn Percentage
of original
spawn (% )

King

oyster
spawn
E1

Yunzhi
spawn
Tra-1

Characteristics

Spheric form mycelium,
some smooth, some have
tassels, scattered
Spheric form mycelium,
some smooth, some have
tassels, scattered
Spheric form mycelium,
smooth, scattered
Small
spheric
form
mycelium, smooth, strong
cohesion

Big
mycelia
pellet,
scattered, tassels
Big mycelia pellet, equal,
scattered, with tassels.

Medium mycelia pellet,
cohesion, with tassels.
Small mycelia pellet, strong
cohesion, with tassels .

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).

16


40

Sinh khối sợi
nấm Sị vua (
g/1000 ml)

30
20
10
0
Thời gian ni (giờ) 60 72 84 96 120
48

Sinh khối sợi
nấm Vân chi (
g/1000 ml)

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
Mycelia
Mycelia Mycelia
pellet size density biomass
(mm/ KLC)
(g/1000ml)

Aeration
supply
mode (V/V/M)
King oyster
0,4
spawn
0,5
E1

0,6
0,7
0,8
CV%
LSD0.05
Yunzhi
0,4
spawn
0,5
Tra-1
0,6
0,7
0,8
CV%
LSD0.05

1,74
1,58
1,30
1,06
0,57
4,3
0,98
1,62
1,47
1,02
0,81
0,54
3,3
0,65

1
2
3
4
5

1
2
4
4
5

19,6
26,2
32,1
34,8
33,6
2,0
1,06
21,8
28,9
35,2
34,6
32,8
3,3
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.

17


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
Criteria Mycelia pellet Mycelia Mycelia
size
density biomass
Spawn
(mm)
(g/1000ml)
Percentage
of spawn (%)
3
1,72
1
19,8
5
1,54
2
28,2
King oyster
7
1,28
4

33,7
spawn
10
0,88
4
32,8
E1
15
0,55
5
30,6
CV%
3,3
3,3
LSD0.05
0,72
1,72
Yunzhi
3
1,58
1
20,6
mushroom
5
1,41
2
26,8
Tra-1
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).

18


Table 3:17: Influence of medium components on the growth of the
commercial King oyster mushroom and Yunzhi mushroom
Varieties

King oyster
spawn
E1

Yunzhi
spawn
Tra-1

Formula
1
2
3
4
CV%
LSD0.05
1
2
3
4
CV%
LSD0.05

48

hours
13,7
14,5
16,8
16,9
3,5
1,1
15,8
16,2
16,4
16,7
3,9
1,3

Mycelia biomass (gr/1000ml)
60
72
84
96
hours hours hours
hours
15,9
19,8
21,2
21,8
18,3
22,6
23,4
23,7
20,5

25,7
27,6
27,9
20,7
23,9
25,6
25,9
3,1
3,7
2,7
1,8
1,2
1,7
1,4
0,9
20,6
24,5
26,4
26,8
21,7
28,8
30,2
30,9
22,7
28,7
30,0
30,8
22,6
28,4
29,8

30,2
1,6
1,6
1,3
4,4
0,7
0,9
0,8
2,6

120
hours
21,9
25,7
27,7
25,8
2,8
1,4
26,7
30,7
30,5
30,2
3,9
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.

19


Table 3.22: The effects of medium mixture formulas on the size of
fruiting bodies and yield of fresh mushroom of King Oyster
Spawn

King
oyster
liquid
spawn
E1

King
oyster

solid
spawn
E1

Treatment
1
2
3
4
5
6
CV%
LSD0.05
1
2
3
4
5
6
CV%
LSD0.05

Mushroom cap
diameter
(mm)
28,9
36,7
42,5
48,8
52,5

52,6
2,8
2,2
27,7
35,2
40,4
46,8
50,4
50,3
2,8
2,1

Stalk
diameter
(mm)
20,7
25,6
29,8
31,6
34,8
34,5
3,2
1,7
20,1
24,3
27,9
30,4
32,6
32,5
6,1

3,1

Stalk length
(mm)

Bio- efficientcy
(%)

98,7
105,8
121,6
134,7
148,4
143,2
3,4
7,6
96,5
102,6
120,1
132,4
146,2
143,4
2,5
5,5

36,8
48,9
52,7
58,5
67,4

67,2
2,3
2,3
33,5
41,7
46,8
51,4
59,4
59,1
2,7
2,3

Table 3.23. The effects of medium mixturebformulas on the size of
fruiting bodies and fresh mushroom yield of Yunzhi
Spawn

Yunzhi
liquid
spawn
Tra-1

Yunzhi
solid
spawn Tra1

Treatment
1
2
3
4

5
6
CV%
LSD0.05
1
2
3
4
5
6
CV%
LSD0.05

Vertical size of
mushroom cap
(mm)
31,5
48,7
60,8
68,2
67,4
65,6
3,4
3,5
30,7
46,8
60,2
67,8
67,5
65,9

3,9
3,96

Horizontal size of
mushroom cap
(mm)
43,8
65,4
72,5
85,4
84,7
81,8
1,9
2,4
43,2
64,8
71,9
85,1
85,3
81,5
3,7
4,8

20

Number of
cap
layers/cluster
3,6
5,7

6,2
7,8
7,8
7,5
5,3
0,6
3,4
5,2
5,9
7,3
7,7
7,6
4,4
0,49

Bio
efficient
(%)
5,7
12,8
19,6
24,5
24,3
24,3
4,9
1,6
5,2
12,3
18,9
24,1

24,0
23,8
3,2
1,03


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

21


Table 3.25. The influence of commercial spawn quantity to the growth
of King oyster and Yunzhi mushrooms on the cultivation materials

Spawn

King oyster
spawn
E1

Yunzhi spawn
Tra-1

Spawn
quantity
10
15
20
25
30

CV%
LSD0,05
10
15
20
25
30
CV%
LSD0,05

9,8
7,6
5,7
5,7
5,8

Time for
mycelium to
cover all
material
(daysy)
31
27
23
23
23

Time for
fruiting
bodies to

appear
(days)
42
40
38
38
38

9,3
6,8
5,2
5,4
5.5

34
29
25
25
25

47
42
39
38
37

Percentage
of disease
infection
(%)

Bio-efficent
(%)
44,3
53,7
66,4
66,5
66,4
4,6
1,25
13,4
19,2
24,6
24,7
24,8
4,3
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.

22


Table 3.26. Economic efficiency of cultivating King Oyster and
Yunzhi mushrooms by liquid spawns
Unit: 1000 VND
Spawn source

King oyster
liquid spawn
King oyster
solid spawn
(the control)
Yunzhi liquid
spawn
Yunzhi solid
spawn
(The control)

YIeld
(kg/ton of
material)

Total
input

Selling
price

Tổng
thu

Net interest
rate
Net
Interes
incomparison
interest t rate
with the
control (%)

Labour
cost

Material
cost

6.000

8.430

14.430

404,4

50

20.220

5.790

0,40

7.000

7.530

14.530

356,4

50

17.820

3.290

0,23

5.000

6.170

11.170

36,75

400

14.700

3.530

0,32

6.000

5.970

11.970

36,15

400

14.460

2.490

0,21

75,99

41,77

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

23