Evaluation of different temperature effects to mycelial growth of Ganoderma subresinosum.. Evaluation of different pure culture media effects to mycelial growth of Ganoderma subresinosum
INTRODUCTION
Introduction
Ganoderma, commonly known as reishi mushroom or Ling Zhi, is a valuable medicinal macrofungus that has been cultivated for over 4,000 years, especially in Far East countries such as China, Japan, and Korea This genus belongs to the family Ganodermataceae and is recognized for its health benefits.
Extensive clinical studies, particularly in Japan, have demonstrated the medicinal and therapeutic value of mushrooms, especially Ganoderma species, in preventing and treating various conditions such as cancer, viral diseases, hypercholesterolemia, blood platelet aggregation, and hypertension Key active substances, including polysaccharides like β-glucans, nucleic acid derivatives such as eritadenine, lipids, peptides, proteins, and glycoproteins, have been isolated and identified Notably, some mechanisms of action, such as antiviral activity through the stimulation of interferon production, have been elucidated Additionally, while some medical claims are less well documented, they may still hold validity and warrant further investigation.
Reishi mushrooms, particularly Ganoderma lucidum, have been extensively studied for their numerous benefits; however, research on the black Reishi, or Ganoderma subresinosum, remains limited despite its ease of cultivation and practical applications Vietnam, with its rich natural resources and skilled workforce, presents an ideal environment for mushroom cultivation, making it a valuable contributor to sustainable economic development Therefore, this thesis focuses on Ganoderma subresinosum as the primary subject of research.
To successfully cultivate Ganoderma subresinosum, it is essential to consider environmental conditions alongside the selection of varieties and farming methods The specific location and climate of each country play a crucial role in identifying optimal growing conditions, an area that has not been extensively researched By determining these ideal conditions, we can enhance the production of the best black Reishi mushroom strains, making this a vital step in the isolation and cultivation of black Reishi strains sourced from nature This article focuses on the "Determination of Suitable Cultural Conditions for the Growth of Black Lingzhi Mushroom (Ganoderma subresinosum)."
Objective
Research and determine the most suitable growing conditions for
Ganoderma subresinosum strain, including temperature, pH, agar media and different nutritional supplements
Requirement
Evaluate the growth of Ganoderma subresinosum on different levels of temperature, pH and agar environment to determine the optimal environment for
Ganoderma subresinosum strains Evaluate the growth and development of
Ga21 strain on different sawdust substrates and then select the most potential substrate for production.
LITERATURE REVIEW2
The situation of research in the world and Viet Nam
2.1.1 The situation of research in the world
Nowadays, more than 2000 species of mushrooms have been identified
Approximately 80 edible and medicinal mushroom species, including oyster, straw, wood ear, and shiitake mushrooms, have been successfully cultivated Due to their scarcity in nature, Ganoderma species are artificially cultivated in solid culture to meet the demand for medicinal herbs in international markets In Taiwan, researchers have collected and cultivated over 10 different Ganoderma species, while in Korea, Ganoderma holds a significant market share, particularly the Ancient Lingzhi hay Artists conk species known for its high anti-tumor effects However, China remains the world's largest center for the cultivation and production of Reishi mushrooms, with global output reaching significant levels in the late 1990s.
China's Ganoderma sp production reached 4,300 tons, with an output of 3,000 tons According to Wang X J (1994), the cultivation of Ganoderma sp in China dates back to 1621 The first report of Ganoderma sp in China was made by Teng in 1934, identifying four species.
G lucidum and one variety Later, many researchers extensively studied
Chinese Ganodermataceae and introduced more than 100 species in four genera:
Ganoderma, Amauroderma, Haddowia and Humphreya (Zhao & Zhang 2000,
Ganoderma, commonly known as "Lingzhi," has been recognized as its scientific binomial in various studies on Chinese edible and medicinal mushrooms Recent research, particularly by Cao et al (2012) and Dai et al (2017), has provided robust molecular and morphological evidence supporting this classification Additionally, mycochemical studies have reported varying species counts within the Ganoderma genus.
Ganoderma (Peng et al 2014, Yan et al 2013)[11, 12] However, the majority of Ganoderma species reported from China have not been subjected to systematic studies (Baby et al 2015)[13]
By 1936, Zenzaburo Kasai and Yukio Naoi successfully cultivated
Ganoderma sp cultivation at Tokyo Agricultural University, Japan, has significantly advanced, with production increasing 40 times from 1979 to 1995 Japan leads in the successful cultivation of Ganoderma sp and ranks among the top globally in scientific research and the preparation of medicinal products Additionally, Korea has developed advanced technology for Reishi mushroom production.
2.1.2 The situation of research in the Vietnam
In 1978, the Institute of Medicinal Materials in Hanoi successfully cultivated the Chinese variety of Ganoderma By 1987, scientists from the University of Natural Sciences identified the wild Lingzhi variety in the Lam Dong mountain forest for propagation, leading to successful production at the Ganoderma farm of Central Pharmaceutical Enterprise 24, with notable results achieved in 1988.
The Vietnamese Medicine and Pharmacy, following the legacy of Ly Thoi Tran, recognized the medicinal properties of Lingzhi mushroom Le Quy Don described Lingzhi as "the source of rare products of Dai Nam land" over 200 years ago Additionally, Hai Thuong Lan Ong and Le Huu Trac advocated for the motto "Men pharmacy to cure the Men," incorporating Lingzhi into various remedies (Dinh Xuan Linh et al., 2012).
Research on the taxonomy and distribution of Vietnamese Ganodermataceae has revealed that Vietnam is home to 27 species of the Ganoderma genus Despite this, the classification and distribution of Ganoderma in Vietnam remain subjects of ongoing study, as highlighted in a 2013 report.
Nguyen et al.,[16] 43 species of the Ganoderma genus were identified in the
Highlands of Vietnam Five of these have been successfully cultivated in Vietnam including G lucidum, G applanatum, G australe, G colossum, and
G subresinosum, and one of these the G.lucidum species is most commonly cultivated Today in Vietnam, the research on mushroom species is not very much Mostly done by researchers such as Trinh Tam Kiet et al (2012), Le Xuan Tham (2005, 2009), Ngo Anh (2007), Phan Huy Duc, Ngo Anh (2004), these research focuses on the diversity of the fungal colonies in the northern, central and southern regions For just in the Central Highlands, Le Ba Dung
(2003), Nguyen Phuong Dai Nguyen (2013) has conducted several projects studies on large fungi that including Ganoderma genus
Engineer Nguyen Thanh is recognized as the first cultivator of Ganoderma sp in Vietnam, having introduced the standard variety from China By the end of 1978, with the guidance of Professor Trinh Tam Kiet, the Chinese Hong Chi mushroom successfully fruited for the first time in a laboratory setting.
In 1987, engineer Nguyen Thanh, PhD Nguyen Thien Tich, and Master Co Duc Trong conducted a search for wild Ganoderma in the mountainous region of Lam Dong province to identify seed sources for cultivation research and production.
One of the first studies on the composition and biological activity of
Ganoderma sp in Vietnam was conducted by Nguyen Thi Chinh et al (2005)[18]
Ganoderma sp cultivated in Vietnam is abundant in protein, sugar, lipids, mineral salts, and vitamin C, with a notable 36% content of 1-3-β-D-glucan This mushroom has potential applications in tumor treatment and cancer therapy, as it may aid in the partial repair of damaged genes.
Provincial departments of science and technology have developed various successful mushroom cultivation models, particularly for Ganoderma sp., and are providing guidance to farmers This initiative not only enhances economic efficiency but also addresses the issue of labor surplus.
In 2017, researchers Nguyen Thi Bich Thuy and Nguyen Thi Thu found that various strains of Ganoderma sp., specifically GA1, GA2, GA3, GA4, and GA10, thrive in the climatic conditions of Tam Dao National Park, with an optimal cultivation period from April to September They identified significant levels of triterpenes and polysaccharides in the strains, noting that GA3 exhibited the highest ganoderic acid content at 0.238%, followed closely by GA2 at 0.208% Additionally, strain GA1 cultivated in Tam Dao had the highest polysaccharide content at 0.734%, surpassing the 0.546% found in GA1 grown in Hanoi.
Scientific classification
According to Dinh Xuan Linh et al -2012:[15]
- This mushroom has scientific name is ganoderma subresinosum (Curtis) P Karst
- Common name are: Ling zhi, Reishi
According to Trinh Tam Kiet (2011)[20] Ganoderma sp belongs to: Kingdom : Fungi
Distribution
Ganoderma species thrive in northern Eastern Hemlock forests and are found in both tropical and temperate regions across North and South America, Africa, Europe, and Asia They act as parasites or saprotrophs on a diverse range of trees, primarily targeting living hardwoods These fungi can grow solitarily or in groups, often near the base of trees.
The Ganoderma genus, which encompasses approximately 80 species of mushrooms, is found in tropical, subtropical, and temperate regions globally Historically, these mushrooms have been mined in China, Vietnam, and India Today, their cultivation has expanded significantly due to advanced technology in countries like Japan, Korea, and China, as well as emerging growth in some Southeast Asian nations and South America.
Life cycle of Ganoderma sp
In the summer and autum, the fruiting bodies or mushroom of
Ganoderma sp release millions of spores into the air, which can be carried to new locations If conditions are favorable, these spores germinate and form a primary hypha The spores are released from the underside of the fruiting body, while the tougher topside does not contribute to spore release Within hours, the surrounding micro-environment becomes covered in spores, allowing the life cycle to begin anew.
Fig 2.1 Life cycle of Ganoderma sp
The primary hypha must find a mate to progress in its growth cycle, a process known as plasmogamy, where the cytoplasms fuse without nuclear fusion, resulting in a dikaryon state (n+n) The fungus then produces secondary hyphae that generate hydrolase, a powerful decomposing enzyme that breaks down wood, allowing for prolonged growth as long as there is sufficient wood These hyphae branch out to enhance nutrient absorption, collectively forming a mycelium When environmental conditions become unfavorable, the fungus produces fruiting bodies, or mushrooms, which contain basidia In these structures, nuclear fusion occurs through karyogamy (2n), followed by meiosis, resulting in the formation of four haploid spores (n) that are released into the air, allowing the life cycle to restart.
Biological characteristics of Ganoderma sp
Ganoderma species are characterized by their vivid red, yellow, or brown colors and a varnished appearance Commonly referred to as a "soft, corky, and flat mushroom with a prominent red-varnished, kidney-shaped cap," it holds significant cultural importance, known as Lingzhi (Soul's Herb) in China and Reishi (King of Herbs) in Japan, reflecting its esteemed status in ancient traditions.
The mature Reishi mushroom exhibits various morphological characteristics, primarily consisting of two main parts: the stalk and the cap The stalk can be long, short, or nearly sessile, typically attached to the concave underside of the cap Initially white, the stalk later transitions to yellow or brown, covered by a glossy sheath that resembles the cap in color and texture Its shape is cylindrical, nearly round, or slightly flat, with dimensions ranging from 3 to 20 cm in length and 0.5 to 2 cm in diameter Notably, the tissue of the stalk is similar to that of the cap (Trinh Tam Kiet, 2011).
The kidney-shaped mushroom slide exhibits a nearly round or fan-shaped appearance, often featuring concentric deformations with radioactive trimming Its color ranges from lemon yellow to saffron, yellow-brown, orange-yellow, red-brown, and purple-brown, with a smooth, varnish-like texture As the mushroom ages, the reddish-brown chalky coating on its surface darkens The size of the mushroom ears varies significantly, measuring between 5 to 12 cm in diameter and 0.8 to 3.3 cm in thickness, with attachments that can be either raised or concave, resembling an umbilical cord.
The underside of a mushroom cap is flat and typically white or light yellow, featuring numerous tiny holes where fungal spores are produced and dispersed As the fungus matures, the spore-forming area darkens to a deep brown The spores themselves are egg-shaped or resemble truncated eggs, characterized by a colorless appendage surrounding the germination hole, which is rust-yellow Each spore has a two-layered shell, with a smooth, colorless outer membrane and a rusty brown inner membrane that develops spikes extending towards the outer layer The dimensions of the spores range from 5-6.5 x 8.5-11.5 µm.
Factors effect on Ganoderma sp growth and development
Effect of favourable nutrient sources:
Carbon plays a crucial role in energy synthesis and forms the fundamental physical structure of cells, constituting approximately 50% of the dry weight of mushrooms (Trinh Tam Kiet, 2012) Ganoderma sp., commonly known as Lim mushroom, is a saprophytic fungus that thrives on decaying organic matter, particularly in ironwood forests This fungus possesses the ability to break down lignin, cellulose, and hemicellulose through enzymatic processes, making substrates such as sawdust, straw, waste cotton, bagasse, and corncob suitable for cultivating Lingzhi.
Nitrogen is a crucial element for fungal growth, as it is vital for the formation of amino acids and nucleic acids Mycelium can absorb both organic and inorganic nitrogen sources, including broth, yeast extract, soybean meal, peptone, (NH4)2SO4, asparagine, alanine, and glycine While nitrogen accelerates mycelial growth, excessive amounts can hinder the formation of fruiting bodies Therefore, the optimal carbon to nitrogen (C/N) ratio during the mycelium development phase is 25:1, while it shifts to 30:1 or 40:1 during the fruiting stage.
Minerals make up a small amount in mushrooms but are essential:
+ Source of sulfur: supplied into the environment from a source of sulfate and needed to synthesize some amino acids
+ Sources of phosphate: participate in ATP synthesis, nucleic acid, phospholipid membrane The source of phosphorus is usually phosphate salts
+ Source of potassium: plays a role as a cofactor, providing active enzymes Simultaneously plays a role in balancing the gradient (gradient) inside and outside the cell
+Magnesium: Necessary for the activity of certain enzymes, magnesium source is provided from magnesium sulfate
Organic molecules, while not a primary energy source for cells, play a crucial role in enzyme activity as vitamins Mushrooms, in particular, absorb vitamins from external sources and only need them in trace amounts Two vital vitamins for mushrooms are biotin (vitamin H) and thiamin (vitamin B1) (Le Duy Thang, 2001).
To screen for mineral salts suitable for the mycelial growth of
A minimal media for cultivating Ganoderma sp mushrooms was created using a 0.1% concentration of various acids, including acetic, citric, maleic, lactic, succinic, and fumaric acid The inoculated Petri dishes were incubated in the dark at 25℃ for five days, after which the mycelial growth, density, and color of the colonies were assessed.
The growth of Ganoderma sp is significantly affected by external factors, with key influences on its morphology being humidity, light, and temperature Additionally, while air quality, pH levels, culture conditions, and water availability also play a role in morphological development, their impact is comparatively less critical.
Water is a vital nutrient for all species, including mushrooms The fruit formation stage requires consistent moisture to facilitate fruit dissociation (Wang, 2006; Dinh Xuan Linh et al., 2012) High humidity is crucial for the growth and development of mushrooms, particularly Ganoderma This includes moisture in both the substrate and the surrounding air Optimal humidity levels vary throughout the development stages, with substrate humidity ideally between 60-65% and air humidity ranging from 80-95% (Dinh Xuan Linh et al., 2012).
Light is one of the very important elements of mushroom farming The mycelium needs different levels of light depending on the different growth stages
Temperature plays a crucial role in metabolism and growth by influencing enzyme activity High temperatures can lead to elongated stalks, thin follicles, increased pests and diseases, dry fruit, and premature aging, while low temperatures result in poor fruit development and susceptibility to mold Optimal growth occurs at temperatures between 20-30°C, with the ideal fruiting stage ranging from 22-28°C.
Proper ventilation is crucial during both mycelial growth and the fruiting stage of mushrooms, as it ensures the right balance of carbon dioxide (CO2) and oxygen (O2) Fungi, being filamentous organisms, consume oxygen and emit carbon dioxide For optimal fruiting, CO2 levels should remain below 0.2%; concentrations between 0.4% and 0.6% can completely inhibit fruiting body formation, while levels from 0.2% to 0.4% result in elongated stems and thin caps Adequate ventilation is essential for the successful cultivation of Lingzhi mushrooms (Dinh Xuan Linh et al., 2012).
The growth of fungi is significantly influenced by environmental factors, particularly pH levels, which impact enzyme activity and the permeability of substances through the mycelium cell membrane This, in turn, enhances cellular metabolism, boosts enzyme activity, and promotes ATP formation.
The Lingzhi mushroom influences the metabolic process by releasing organic acids into its growing medium, which consistently shifts the pH towards acidity To enhance this environment, supplements like light powder (CaCO3) and gypsum powder (CaSO4) are incorporated, serving to supply calcium ions (Ca2+) while also regulating humidity and pH levels.
pH significantly affects the permeability of substances through the mycelium membrane, enhances cellular metabolism, boosts enzyme activity, and promotes ATP formation Ganoderma thrives in neutral to slightly acidic conditions, specifically within the pH range of 5.5 to 7 (Dinh Xuan Linh et al., 2012).
Table 2 1.Ecological conditions of Ganoderma sp cultivation
Factor Period Appropriate interval Note
Fruiting body 800 - 1.000 lux Scattered pH Mycelium 5.0 – 6.0
(According to Le Xuan Tham, 1996)
2.6.3 Composition of chemical and biological compounds
The analysis of G-Bing Lin has proved the general pharmacokinetic components of Lingzhi as follows: (Nguyen Huu Dong et al., 2002):[21]
Other Ingredients: K, Zn, Ca, Mn, Na, essential minerals, vitamins, amino acids, enzymes and alkaloids
Since the 1980s, advanced techniques such as UV, IR, mass-gas chromatography (GC-MS), nuclear magnetic resonance, solid magnetic resonance spectroscopy, high-pressure liquid chromatography (HPLC), and plasma spectrometry (ICP) have been employed to accurately identify nearly 100 active ingredients and their derivatives in Lingzhi (Le Xuan Tham, 1996).
2.6.4 Major bioactive components isolated from Ganoderma sp
Mushrooms have been utilized for centuries for their nutritional and sensory benefits, and they are recognized as functional foods due to their bioactive compounds Over 50 mushroom species demonstrate immunological potential and exhibit anticancer activity in vitro and in animal models, with some studies extending to human cancers Given that cancer is a leading cause of death globally, mushrooms offer significant health benefits, including ergothioneine, selenium, fiber, and various vitamins and minerals Their bioactive compounds, such as β-glucans, β-proteoglycans, lectins, triterpenes, ergosterol, glutamine, and arginine, contribute to their antitumor and anticarcinogenic properties This literature review explores the positive effects of mushroom compounds on cancer treatment and their potential compatibility with chemotherapy management.
Medicinal mushrooms are recognized for their therapeutic properties and positive impact on human health Historically, they have served as a vital food source, with their consumption linked to disease prevention, treatment, and increased longevity in various cultures In many Eastern countries, these mushrooms have been integral to traditional medicine for treating various ailments.
Recent scientific and medical research conducted over the past 30 years in Japan, China, Korea, and the USA has validated the unique compounds and properties of mushrooms in the prevention and treatment of cancer, with over 200 species exhibiting medicinal benefits These distinct mushroom species are rich in essential chemical constituents, macro- and micronutrients, and serve as a rare source of ergothioneine, proteins, selenium, essential amino acids, vitamins, and dietary fibers.
MATERIAL AND METHODS
Time and location
All the experiments were conducted during July 2019 - January 2020 in the Mushroom Research and Development Center, Faculty of Biotechnology, Vietnam National University of Agriculture
Research object
Strains Ga21 is maintained and preserved at the laboratory of the Mushroom Research and Development Center, Faculty of Biotechnology, Vietnam National University of Agriculture.
Materials
Materials used for pure culture media preparation:
-Inorganic substances such as NH4Cl , NH4NO3, (NH4)2SO4, HCl, NaOH
Materials used for the Preparation of Grain master spawn of Ganoderma subresinosum
- Paddy grains: We used fresh, unbroken, insecticide or fungicide untreated and insect undamaged
- Sawdust: no oil, no mouldy
Materials used for cultivation substrates of Ganoderma subresinosum
- Sawdust: no oil, no mouldy
Equipment
- Incubation room, Laminar flow cabinet, temperature control cabinet, autoclave (steam sterilization), pH meter
- Petri-dish, test tube, Erlenmeyer flask 500ml, alcohol burner, forceps, glass funnel, bottle, inoculation loop, plastic wrap, scissors, cotton, rubber band
- Thermometer use for measures: pH level
- Room store the strains must have air conditioner
- Net house for growing mycelium must be clean, airy, airtight, low light, humidity from 65 to 80%, temperature 20-30 o C
- The area for composting and mixing material must be large enough, cool, and clean, not close to sources of pollution
- Bag packing: Polypropylene bag with dimension 19x37cm.
Contents of the study
Content 1: The effect of different temperature on mycelial growth of
Content 2: Evaluation of different pH effects to mycelial growth of
Content 3: Evaluation of different pure culture media effects to mycelial growth of Ganoderma subresinosum
Content 4: Evaluation of different paddy grain substrates (grain master or grade 2) effects to mycelial growth of Ganoderma subresinosum
Content 5: Evaluation of different substrates effects on ruiting body formation
Methods
Mycelial characteristics (mycelial morphology, density) were evaluated according to Trinh Tam Kiet (2012) Culture conditions were tested following
Methods of material processing
Potatoes were peeled and cut into moderate pieces, then boiled in 500ml of distilled water until tender The resulting extract was filtered through a steel mesh, and glucose and agar were added and dissolved The mixture was then diluted with water to a total volume of 1000ml and transferred into bottles The media were autoclaved at 121°C for 60 minutes, allowed to cool to 55-60°C, and poured into Petri dishes within a bio-cabinet Finally, the dishes were wrapped in plastic and stored at 25°C until needed.
3.7.2 Preparation of paddy grain substrates
Paddy used fresh, unbroken, insecticide or fungicide untreated and insect undamaged paddy grains for the grain master preparation
Paddy grains were thoroughly washed four times with fresh water and soaked for 12 hours They were then boiled in an equal volume of fresh water until soft Notably, the grains did not split or ooze out starch, after which they were dried.
The mixture consisted of 99% rice grain and 1% CaCO3, which was then combined with sawdust in test tubes Each test tube contained 30g of the mixed medium, which was autoclaved at 121°C for 90 minutes.
3.7.3 Preparation of substrates for cultivation
Process of composting sawdust: Wood sawdust not contain volatile oil, and the poison can be used as a primary agent for the development of Ganoderma subresinosum
- Sawdust mixed with a lime solution (pH: 12) with 4kg of lime / 1000L Water, stir well after incubation for 5-7 days and then continued to incubate for 1-2 more days, the humidity must reach 65%
According to each recipe, sawdust after incubation was mixed with nutritional additives such as wheat bran and CaCO3
The material was placed in 1kg polypropylene bags, which were sealed with heat-resistant PVC pipes After securing the bag necks and inserting cotton plugs, the bags were transported to the sterilization room for autoclaving at 120-125 °C for 120-180 minutes Following sterilization, the bags were cooled and stored in a culture room at 20 °C Host seeds of grade 2 were then inoculated into the sterile bags at a rate of 8-10 g per bag.
After transplanting, the substrate is placed in a culture room until the mycelium fully develops Once the mycelium has completely colonized the substrate, the bags are moved to the harvesting room.
Method of evaluating the effect of substrates on growth and
3.8.1 Experiment 1 Evaluation of different temperature effects to mycelial growth of Ganoderma subresinosum
The study investigated the impact of varying temperatures (20˚C±1, 25˚C±1, 30˚C±1, and 35˚C±1) on mycelial growth in incubators Each temperature treatment was replicated three times, and observations regarding colony diameter, color, and growth patterns were documented after 12 days of incubation.
3.8.2 Experiment 2 Evaluation of different pH effects to mycelial growth of Ganoderma subresinosum
The optimum growth of Ganoderma subresinosum was evaluated across various pH levels ranging from 3 to 10 The pH of the growth medium was adjusted using NaOH or HCl and confirmed post-sterilization Each treatment was replicated three times, and observations regarding colony diameter, color, and growth patterns were documented after 12 days of incubation.
3.8.3 Experiment 3 Evaluation of different pure culture media effects to mycelial growth of Ganoderma subresinosum
Basal medium (200 g infused potato, 15g agar powder, 20g fructose)
Table 3 1 Pure culture media components
1 basal medium + 2g ammonium chloride (NH4Cl)
2 basal medium + 2g ammonium nitrate (NH4NO3)
3 basal medium + 2g ammonium sulfate (NH4)2 SO4
Each treatment was replicated three times, each of 2 Petri-dishes with 9cm of diameter The total number of Petri-dishes: 36 Petri-dishes for 6 formulas
3.8.4 Experiment 4 Evaluation of different paddy grain substrates (grain master or grade 2) effects to mycelial growth of Ganoderma subresinosum
There are 3 formulas for conducted this experiment:
Formula 1: 99% grain of paddy + 1% CaCO3
Formula 2: 69% grain of paddy + 30% sawdust + 1% CaCO3
Formula 3: 39% grain of paddy + 60% sawdust + 1% CaCO3
Each formula was replicated three times, each of 2 test tube (Diameter: 1.8cm x length: 18cm) with 25g of components/ test tube A total number of test tube: 18 test tubes
3.8.5 Experiment 5 Evaluation of different substrates effect to the growth and development of Ganoderma subresinosum
In this experiment, we use sawdust as a basal substrate enriched by wheat bran at different percentages as the following table:
Table 3 2 Different ratio of substrates to cultivated Ganoderma subresinosum
- Each formula was replicated three times
- Each formula was packed 5 bags of sawdust materials with 1kg /bag, so total number of bag is 15 bags/ formula Totally for 6 formulas are 90 bags.
Data collection
For optimization of pH and temperature:
- Mycelial growth rate (mm/day)
- Measure of mycelial growth rate (mm / day) for each formula
- Observation of color, mycelial characteristics
- Mycelial growth rate were calculated by this formula: V = D/T
• V: Mycelial growth rate (mm/day)
• D: The length growth of mycelial
- Period of surface colonization: This period thinks from time we inoculated till mycelial colonized full the bag surface
- Period of bag colonization: Time the mycelium had colonized the bag completely This period thinks from the time we inoculated till mycelium had colonized fully the bag
- The period of antlers appearance (days) thinks from time we inoculated to Germination
- To measures the length and width of fruiting body, the length of stalk (mm)
Data analysis
Microsoft Word and Excel with IRRISTAT 5.0 were employed to analyze experimented data.
RESULT AND DISCUSSION
Effect of temperature on mycelial growth of Ganoderma subresinosum
Temperature is a crucial physical factor influencing mycelial growth and fruiting body formation This study examined the mycelial growth of strain Ga21 on PGA medium at four distinct temperatures: 20˚C±1, 25˚C±1, 30˚C±1, and 35˚C±1, over periods of 2, 4, 6, and 8 days.
The effect of temperature on mycelial growth and morphology of strain Ga21 were provided in Table 1 Specifically, the strain was able to grow at 20-
The optimal temperature for mycelial growth was determined to be 25°C, with a growth measurement of 27.06 mm, while growth at 30°C was slightly lower at 25.69 mm, and 20°C showed minimal growth at 12.34 mm The mycelial density was thickest at 25°C, somewhat thick at both 20°C and 30°C These findings align with the observations made by Song et al.
Table 4 1 Effect of different temperature on mycelial growth of Ganoderma subresinosum
Diameter of mycelial (mm) after days Mycelial characteristics
Mycelia density was somewhat thick and colonized completely light white
25±1 8.86 25.36 35.11 38,89 Mycelial white density was thick and colonized completely cottony
Mycelia density was somewhat and colonized completely lighter white
35±1 0 0 0 0 Mycelia density was can not grow
Fig 4 1 Effect of different temperature on mycelial growth of Ganoderma subresinosum
Diameter of mycelial length (mm)
Fig 4 2 Mycelial growth on different temperature for 4 days after inoculation
Fig 4 3 Mycelial growth on different temperature for 8 days after
Effect of pH on mycelial growth of Ganoderma subresinosum
pH levels significantly influence cell membrane function, nutrient absorption, cell morphology, solubility, ionic states, enzyme activity, and product biosynthesis Mushrooms can thrive across a broad pH spectrum In this study, the growth of strain Ga21 was assessed within a pH range of 3.0 to 10.0, with the results detailed in Table 4.2.
Table 4 2 Effect of different pH on mycelial growth of Ganoderma subresinosum pH Radius growth (mm) after days Mycelial characteristics
Mycelia density was weak and nearly non development light white
Mycelia density was weak , slow development and colonized incompletely light white
Mycelia density was thicker, faster development and colonized completely white cottony
Mycelia density was thick, quite quick development and colonized completely white cottony
Mycelia density was thick, quick development and colonized completely white cottony
Mycelia density was very thick, promptly development and colonized completely white cottony,
Mycelia density was very thick in a flash development and colonized completely white cottony
Mycelia density was thick, quick development and colonized completely white cottony
Ganoderma subresinosum demonstrates optimal growth at a pH range of 8.0 to 9.0, with maximal mycelial growth recorded at pH 8.0 (38.87 mm) and pH 9.0 (39.00 mm) At these pH levels, the mycelium exhibited high density and a white color In contrast, pH levels between 3.0 and 5.0 negatively impacted mycelial growth Therefore, it is recommended to maintain a pH of 8.0 to 9.0 for the cultivation of Ganoderma subresinosum.
Fig 4 4 Effect of different pH on mycelial growth of Ganoderma subresinosum
Fig 4 5 Mycelial growth on different pH for 7 days after inoculation
2 days 4 days 6 days 8 days mycelial diameter(mm) pH levels
Effect of spawn grain substrates (grain master or grade 2) on mycelial
Spawn is essential in mushroom cultivation, with its type influencing both spawn running time and mushroom yield The four main types of spawn include sawdust spawn, grain spawn, liquid spawn, and stick spawn To enhance the cultivation of strain Ga21, we experimented with four distinct formulas that combined rice grain and sawdust as substrates.
Table 4 3 The length of mycelial growth of Ganoderma subresinosumin
Formulas The length of mycelial growth (mm) after days
4 days 8 days 12 days 16 days 20 days
The growth of strain Ga21 mycelium was significantly affected by different spawn grain substrates, with Formula 2 exhibiting the highest growth rate, followed by Formula 3 and Formula 1 Mycelial characteristics revealed that strains grown on Formula 1 and 2 displayed a white color and high density, while Formula 3 resulted in lower density.
Using grain for spawn production can lead to higher manufacturing costs and increased contamination rates To mitigate these issues, we experimented with a mixture of sawdust and rice grain in varying ratios as a spawn substrate, which effectively reduced production costs Consequently, Formula 2 is recommended as the optimal spawn grain substrate for the industrial-scale cultivation of Ganoderma subresinosum.
Table 4.4 Evaluation of different grain substrates for spawn development
Mycelial growth rate(mm/day)
Mycelial characters mycelial density colour
All grains were entirely colonized by the mycelium and tightly held with each other
2 9,78 7,25 all grains were entirely colonized by the mycelium and tightly held with each other
3 10,89 5,77 Mycelial growth was low and thin paler white
Fig 4.6 Effect of different grain substrates on mycelial growth of
Fig 4.7 Mycelial growth for eight days Fig 4 8 Mycelial growth for 14 days
4 DAYS 8 DAYS 12 DAYS 16 DAYS 20 DAYS mycelial diameter(mm)
4.4 Evaluation of different pure culture media effects to mycelial growth of
For level 1 propagation, we can enhance basic agar media by incorporating various inorganic and organic substances To identify the optimal medium for the growth and development of Ga21, while maximizing economic value, we experimented with several inorganic components, including ammonium chloride (NH4Cl), ammonium nitrate (NH4NO3), ammonium sulfate ((NH4)2SO4), peptone, and yeast powder.
Table 4 5 Effect of different pure culture media on mycelial growth of
Media mycelial diameter (mm) after days Mycelial characters
2 4 6 8 10 mycelial density colour NH4Cl
Mycelia density was weak and slow development pale white
Mycelia density was thicker and grows faster pale white (NH4)2SO4
Mycelia density was thicker and grows faster pale white
Mycelium density was thick and grow normally dark white
The mycelium density was very thick and cottony, and grow very quickly dark white
The mycelium density was very thick and grow very quickly pale white
The study evaluated the growth of Ganoderm mycelium on six different media, revealing that it thrived on all tested substrates (Table 4.5) The basic medium yielded the highest growth rate at 38.44, characterized by fast but thin, pale white mycelium This was closely followed by the basic medium supplemented with enamel powder, which exhibited a growth rate of 38.22 and produced dense, fluffy white fibers Other media, including basic medium with ammonium sulfate (30.92), peptone (30.22), ammonium nitrate (29.89), and ammonium chloride (24.39), demonstrated moderate growth rates, resulting in white mycelium with varying fiber densities.
The optimal environment for cultivating Ganoderma subresinosum is a combination of a basic substrate and yeast powder, even though the fiber system may not exhibit the fastest growth.
Fig 4 9 Effect of different pure culture media on mycelial growth of Ganoderma subresinosum
2 days 4 days 6 days 8 days 10 days
Fig 4 10 Mycelial growth on different media for 5 days after inoculation
Fig 4 11 Mycelial growth on different media for 9 days after inoculation
Effect of different substrates on primordia formation and development
Vietnam offers significant opportunities for cultivating Ganoderma due to its abundant agro-industrial waste and human resources This study focuses on using sawdust as the primary substrate for growing Ganoderma subresinosum, despite sawdust's low nutrient content To enhance mushroom cultivation efficiency and shorten the growth period, it is essential to incorporate supplements such as rice bran, wheat bran, and corn bran into the substrate These additions increase the nitrogen content, which is crucial for cellular protein and enzyme synthesis In this experiment, we specifically cultivated Ganoderma subresinosum using sawdust enriched with wheat bran.
Table 4 6 The length of mycelial growth of Ganoderma subresinosumin after 4, 8, 12, 16, 20 days of incubation
Formula The length of mycelial growth (mm) after days
Mycelial growth rate (mm/day)
The study indicates that strain Ga21 successfully grew and formed primordia across all tested formulas Among these, Formula 5 demonstrated the slowest mycelial extension rate, while Formula 1, consisting of 99% sawdust, 0% wheat bran, and 1% CaCO3, had the shortest spawn running period, completing it in just 20 days of incubation Additionally, primordia formation was noted on day 34 for Formula 6 and day 36 for other formulas.
38 (Formula 1, 5), and day 41-42 (Formula 2,3,4) Formula II showed the best yield performance with a BE value of 2,95%, followed by Formula 4 (2,66%) and Formula 3 (2,42%) (Table 4.8 and Figure 4.14)
Table 4 7 Comparison of antlers appearances period and mycelial growth rate
Fig 4.12 Mycelial colonized half the bags
Fig 4.13 Mycelial colonized fully the bags Biological efficiency
Table 4 8 Effect of different formulas on formation fruiting body and
Biological Efficiency of Ganoderma subresinosum
Formulas length of fruiting body(mm) width of fruiting body(mm) length of stalk (mm) fresh weight/bag (gram)
Note: For the result above are the yields of first harvest
Fig 4.14 Comparison of Biological efficiency in different substrates of Ganoderma subresinosum
The ability of mushroom to form and develop fruiting body are an important criterion that has an important role in determining the yield of
Ganoderma cultivation requires careful management of environmental conditions to ensure successful fruiting body development When the mycelium reaches a certain density and nutrients are sufficiently accumulated, white sprouts emerge It is essential to regularly spray water to maintain humidity, control temperature, and manage light exposure to prevent mechanical damage from pests High temperatures, strong light intensity, and poor ventilation can hinder fruiting body formation, so maintaining an appropriate temperature and ensuring a cool, well-ventilated farming environment is crucial for optimal growth.
Biological efficiency in different substrates
Fig 4.15 Ganoderma subresinosum in the button neck
Fig 4.16 Ganoderma subresinosum in the body of the bag
Compare in 2 methods to find the body of black Ganoderma in the button neck and in the body of the bag we see:
- Fruiting bodies are slow-growing at nodules and thick and short fungal legs
- Fruiting bodies by the slitting method are more rapidly growing in body fruiting bodies, longer bodies and thinner caps
Evaluate the results of the first harvest
Fig 4.17 Ganoderma subresinosum is eaten by worms
Fig 4.18 Ganoderma subresinosum is infected with mold.
CONCLUSION AND RECOMMENDATION
Conclusion
-The optimal temperature and pH for mycelial growth of strain Ga21 were at 25 ° C ± 1 and pH = 8.0-9.0, respectively
-Formula 2 (69% of rice grains + 30% sawdust + 1% CaCO3) was considered as the optimal spawn substrate for luxuriant mycelial growth of strain Ga21
- The highest mycelium growth rate was obtained when cultivated on Treatment 1 (sawdust 99% + wheat bran 0% + CaCO3 1%), whereas the best yield performance was found in formula 2 (2, 95%).
Recommendation
Further studies are needed to:
- Evaluate the medicinal value of strain Ga21
- Evaluate the resistant of strain Ga21 to bacteria, virus, and mould
- Identify the taxonomic position of strain Ga21
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4.5: Investigation of the growth and development of Ganoderma subresinosum cultivated on different substrates
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