VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY ******* GRADUATION THESIS CHARACTERIZATION AND IDENTIFICATION OF AN ACTINOMYCES VNUA30 STRAIN WITH BIOACTIVITY AGA
INTRODUCTION
Introduction
Bananas are a vital crop in Vietnam's agriculture, widely utilized in the food industry for their nutritional benefits Ripe bananas are not only a nutritious fruit but also serve as a flavoring agent in muffins, cakes, and breads Additionally, bananas possess medicinal properties that can aid in treating skin diseases, alleviating diarrhea, and promoting stomach health The cultivation of bananas in Vietnam meets consumer demand while contributing significantly to rural economic development However, diseases like Panama, caused by fungi, pose serious threats to banana yield and quality.
Fusarium oxysporum is a significant pathogen, and while chemical treatments have been used, their effectiveness is often limited A more optimal approach to prevent and combat this disease is through the use of bioproducts, which can effectively inhibit and eliminate pathogenic fungi.
Actinomycetes are a widely distributed group of microorganisms primarily found in soil, known for their ability to synthesize bioactive secondary metabolites such as enzymes, herbicides, pesticides, and antibiotics The study and application of actinomycetes and their biologically active substances offer an efficient and environmentally friendly method to inhibit pathogenic organisms This article focuses on the characterization and identification of the Actinomyces VNUA30 strain, which exhibits bioactivity against harmful pathogens.
Fusarium oxysporum causing the Panama disease on Banana”.
Purpose and requirements
Characterization and identification of an Actinomyces VNUA30 strain with bioactivity against Fusarium oxysporum causing the Panama disease on
- Test the actinomyces VNUA30 strain with ability of resistant to fungi
Fusarium oxysporum cause diseases in banana
- Study some morphological and biochemical, physiological characteristics of VNUA30 strain
- Identify VNUA30 strain based on 16S rRNA gene sequencing
LITERATURE REVIEW
Overview of banana and diseases on banana
Bananas, belonging to the genus Musa and the family Musaceae, are among the world's most significant fruit crops This elongated, edible fruit is botanically classified as a berry and is produced by various large herbaceous flowering plants within the genus Musa.
Bananas were first domesticated in Southeast Asia, with references to their consumption found in ancient Greek, Latin, and Arab texts Alexander the Great encountered bananas during his expedition to India After the discovery of America, bananas were transported from the Canary Islands to the New World, establishing themselves in Hispaniola and spreading to other regions Their cultivation grew significantly, making bananas a staple food in many areas, and by the 19th century, they began appearing in U.S markets While Cavendish bananas dominate imports in non-tropical countries, plantain varieties represent approximately 85 percent of global banana cultivation.
The banana plant is a gigantic herb that springs from an underground stem, or rhizome, to form a false trunk 3–6 metres (10–20 feet) high This trunk
The banana plant features a false trunk made up of the basal portions of leaf sheaths, topped with a rosette of 10 to 20 oblong to elliptic leaves that can reach lengths of 3–3.5 meters (10–11.5 feet) and widths of 65 cm (26 inches) A large flower spike emerges from the top, bearing numerous yellowish flowers protected by purple-red bracts, which eventually bend downward to form bunches of 50 to 150 bananas, grouped in clusters of 10 to 20 After fruiting, the plant is cut down, as each trunk produces only one bunch of fruit New trunks, or suckers, arise from the rhizome approximately every six months, allowing the life of a single rhizome to continue for many years, with weaker suckers pruned and stronger ones nurtured into fruit-bearing plants.
The banana, primarily cultivated in tropical regions, is cherished globally for its flavor, nutritional benefits, and year-round availability It ranks as the fourth most valuable food after rice, wheat, and milk, and holds a significant position in global exports, being the most important fruit with world trade reaching substantial figures.
$2.5 billion annually Only 10% of the annual global output of 86 million tons enters international commerce
Raw bananas consist of 75% water, 23% carbohydrates, 1% protein, and negligible fat in a 100g serving This portion provides 89 calories and meets 31% of the US recommended Daily Value (DV) for vitamin B6, along with moderate levels of vitamin C, manganese, and dietary fiber, while lacking significant amounts of other micronutrients.
Ripe bananas are a nutritious fruit that can enhance the flavor of muffins, cakes, and breads In contrast, cooking varieties known as plantains are starchy and serve as a staple food source in tropical regions A ripe banana is composed of up to 22 percent carbohydrates and is rich in dietary fiber, potassium, manganese, and vitamins B6 and C.
Bananas are a nutritious fruit rich in vitamins, minerals, fiber, and simple sugars, containing no fat or cholesterol They provide an excellent source of vitamin B6, folate, vitamin C, magnesium, potassium, and carbohydrates A single banana can keep you feeling full for an extended period.
Being rich in magnesium & potassium, banana aids in maintaining optimum blood pressure, keeps the heart protected and promotes bone health
Bananas possess antacid properties that help combat acid buildup and protect the stomach lining from ulcers Additionally, they defend against harmful bacteria that can lead to gastrointestinal issues.
Bananas serve as an effective home remedy for diarrhea, acting as a natural binding agent They help combat dehydration and replenish essential electrolytes lost during episodes of loose stools.
Post-workout nutrition is crucial, particularly after intense aerobic exercises This ideal food offers simple sugars for immediate energy and potassium, essential for muscle recovery following a workout.
The white lining of banana peels is an effective natural remedy for various skin conditions, including psoriasis and acne Additionally, banana peels have been shown to help treat warts and minor burns.
To alleviate a severe hangover after a night of binge drinking, bananas can be quite effective A banana smoothie made with milk or yogurt, combined with bananas and a few strawberries, provides relief and helps restore balance.
The inside of a banana peel offers an effective and affordable method for whitening yellow teeth By gently rubbing the inner side of the peel on your teeth for approximately two minutes, you can achieve a brighter, whiter smile.
Bananas are a vital agricultural crop, recognized for their nutritional and medicinal benefits They play a significant role in enhancing rural economic development due to their high economic value However, the yield and quality of bananas are severely impacted by various diseases caused by bacteria, fungi, and viruses, making them highly susceptible to such threats.
Bacteria Bacterial wilt Pseudomonas solanacearum
Finger tip rot Pseudomonas spp
Fungi Panama disease Fusarium oxysporum f.sp Cubense
Black root rot Rosellinia bunodes
Virus Bract mosaic Banana bract mosaic virus
Bunchy top Banana bunchy top virus
The majority of diseases affecting bananas are caused by bacteria, fungi, and viruses Consequently, researching actinomycetes and their biologically active substances offers an effective and environmentally friendly approach to inhibiting these pathogenic organisms.
Overview of Fusarium oxysporum and Panama disease
Fusarium oxysporum is a significant plant pathogen that inflicts considerable damage on various agricultural crops, affecting them both in the field and during postharvest storage This ascomycete fungus includes all species, varieties, and forms classified by Wollenweber and Reinking within the section Elegans, and it belongs to the Nectriaceae family.
Fusarium taxonomy relies on the morphological characteristics of the anamorph, such as macroconidia size and shape, the presence of microconidia and chlamydospores, colony color, and conidiophore structure The challenge in defining species based on these traits is highlighted by the various classification systems proposed, which recognize between 30 and 101 species Many of these taxonomic frameworks categorize the species into distinct sections.
Based on morphological criteria, it is sometimes difficult to distinguish F oxysporum from several other species belonging to the sections Elegans and
Liseola To further complicate the picture, plant pathogenic, saprophytic and biocontrol strains of F oxysporum are morphologically indistinguishable
2.2.1.3 Diseases are caused by Fusarium oxysporum
Fungal phytopathogens pose significant challenges to global agriculture and the food industry by damaging crops and economically vital plants both in the field and during storage One notable pathogen is Fusarium oxysporum, which is responsible for Fusarium wilt, commonly referred to as Panama disease.
Panama disease, also known as banana wilt, is a severe affliction of bananas caused by the soil-dwelling fungus Fusarium oxysporum This form of fusarium wilt is prevalent in tropical regions and affects susceptible banana varieties Historically challenging to manage, Panama disease led to the destruction of global Gros Michel banana plantations during the 1950s and 1960s, which were once the backbone of the commercial banana industry Today, the modern Cavendish banana, which replaced Gros Michel, is now facing threats from this devastating disease.
9 strain of the disease known as Tropical Race (TR) 4 since the 1990s; in 2019 TR
4 was confirmed in Colombia, marking the first appearance of the strain in the Americas
Two external symptoms help characterize Panama disease of banana:
- Yellow leaf syndrome, the yellowing of the border of the leaves which eventually leads to bending of the petiole
Green leaf syndrome affects specific cultivars and is characterized by the leaves maintaining their green color, accompanied by the bending of the petiole, similar to yellow leaf syndrome.
Figure 2.1 Banana trees afflicted with Panama disease
The Fusarium fungus typically invades young roots or root bases through wounds, leading to infections that can extend into the rhizome and rapidly affect the rootstock and leaf bases The spread of the fungus occurs via vascular bundles, which exhibit discoloration ranging from brown or dark red to purplish or black As the infection progresses, the outer edges of older leaves turn yellow, and within one to two months, most leaves, except the youngest, will yellow, wilt, and collapse, ultimately covering the trunk (pseudostem) with dead brown foliage.
The Fusarium fungus ultimately leads to the death of the plant, although new shoots may emerge from the base However, these shoots eventually wilt, resulting in the plant's demise within a few years The fungus persists in the surrounding soil, hindering the success of future plantings.
Overview of Actinomycetes
2.3.1 Definition, classification and distribution of Actinomycetes
Actinomycetes are unicellular, Gram-positive bacteria that belong to the Order Actinomycetales
Members of this group are widely distributed in nature and can be found in a variety of habitats across the world
The name Actinomycetes is derived from the Greek words "atkis" which means ray and "mykes/mukes" which means fungi
Actinomycetes, belonging to the kingdom Bacteria, are unicellular organisms with a simple cell structure These Gram-positive bacteria possess a peptidoglycan layer in their cell wall and are found in diverse environments globally However, certain species are known to cause diseases in humans.
Actinomycetes, belonging to the Phylum Actinobacteria, are Gram-positive bacteria known for their high G+C content in DNA These versatile organisms thrive in both terrestrial and aquatic environments and are notable for their ability to produce mycelium.
Subclass: Actinobacteridae - The subclass Actinobacteridaeis is diverse and consists of a wide range of organisms that can be found in various habitats
As a subclass under the phylum Actinobacteria, members of this group produce mycelium
Actinomycetales, commonly referred to as Actinomycetes, are a diverse group of Gram-positive bacteria found in both aquatic and terrestrial environments These bacteria exhibit a filamentous growth pattern and are primarily aerobic, although some members can thrive in anaerobic conditions.
Actinomycetes, belonging to the phylum Actinobacteria, are found globally due to their ability to adapt to various environments and their nutritional versatility This adaptability enables them to thrive in diverse regions and effectively compete with other organisms in their ecosystems.
Some of the species are capable of surviving in various extreme environments and can, therefore, be classified based on these habitats:
- Alkalophilic species - Identified in soda lake soil (e.g Bogoriella caseilytica)
- Halophilic species - Survive in areas with high salt concentrations (e.g Saccharomonospora halophila)
- Psychrophilic species - Commonly found in very low temperatures (e.g Modestobacter multistep-tatus)
Depending on the species, Actinomycetes range in size from 0.5um to over 5.0um ( T Roseburry.1994 )
On the solid medium, the actinomycetes forms colonies
The size and appearance of actinomycete colonies are influenced by the cultivation medium and environmental conditions Typically, these colonies exhibit a dry, rugged texture and can take on various forms, including skin-like, limestone, and chalky structures They often display distinctive patterns, such as radioactive or concentric circles, and come in a wide range of colors, including red, yellow, gray, brown, and white Additionally, factors such as species, culture conditions, medium composition, temperature, and pH significantly affect the size and shape of these colonies.
The colony consists of three distinct layers: the outer layer, which features brained fibers; the middle layer, characterized by a honeycomb structure; and the inner layer, known for its relatively porous nature.
Spore formation involves the septation of aerial hyphae at various intervals, where the plasma membrane invaginates and the inner wall breaks Subsequently, the inner hyphal wall thickens to create a robust structure.
During sporulation, the dividing nuclear material is constricted at the septum and ultimately divides as the septation process concludes A thick wall forms around each spore as new material is added at the septa.
Sporulation can occur through hyphal fragmentation or endogenous spore formation, with some spores remaining sheathed after fragmentation Different species produce various types of spores, including aleuriospores (from unsheathed hyphae), arthrospores (from sheathed hyphae), and endospores (formed within vesicles) The size of these spores typically ranges from 0.5 to 1.5 µm, and many exhibit a thicker wall structure along with sharp features.
13 spines originating from the sheath Moreover, they also contain the genetic material and cytoplasm of the parent
In the soil, or in the event of adverse environmental conditions, the spores (dormant) are able to survive for a long period of time
Under optimal environmental conditions, spores germinate and produce germ tubes, which subsequently grow into mycelium composed of branching filaments called hyphae.
Actinomycetes, like many bacteria, possess a mycelial cell wall that encases the organism This cell wall is composed of various components, including amino acids, sugars, and amino sugars, and features a peptidoglycan layer predominantly containing diaminopimelic acid in most species.
The cell wall is composed of three distinct layers: the outermost layer, which is thick and measures approximately 60-120 Å (and can reach up to 150-200 Å in older cells), a solid middle layer with a thickness of about 50 Å, and an inner layer also around 50 Å thick Unlike the cell walls of fungi, actinomycetes lack cellulose and chitin; instead, they contain enzymes that play a crucial role in metabolic processes and material transport across the membrane.
2.3.2.4 Physiological – biochemical characteristics of actinomycetes
Actinomycetes are heterotrophic, predominantly non-motile organisms Research into their physiological and biochemical characteristics focuses on their ability to assimilate carbon and nitrogen sources, oxygen requirements, pH tolerance, optimal temperature, salt tolerance, and their interactions with antibiotics These organisms can produce antibiotics and other specific metabolic products They utilize various organic compounds, including sugars, starches, organic acids, lipids, amino acids, and proteins, as sources of carbon and energy.
14 nitrogen are usually nitrate salts, ammonium sakts, peptones, proteins…The ability to assimilate substances in each actinomycetes strain is different
Actinomycetes are primarily aerobic microorganisms that thrive in moist environments, with some species being thermophilic They typically grow best in media with a neutral to slightly alkaline pH, and the optimal temperature range for their growth is between 25°C and 45°C However, the physiological and biochemical characteristics of actinomycetes can vary significantly among different species, especially those isolated from diverse sources and regions around the world, allowing them to adapt to various living conditions.
Application of actinomycetes
Actinomycetes are a widely distributed group of microorganisms primarily found in soil, known for their ability to synthesize bioactive secondary metabolites, including enzymes, herbicides, pesticides, and antibiotics Key enzymes like amylase, lipase, and cellulases produced by actinomycetes are essential in various industries, including food, fermentation, textiles, and paper Additionally, certain enzymes are utilized as therapeutic agents in treating human cancers, particularly acute lymphoblastic leukemia Actinomycetes also contribute to cancer treatment, bioremediation, and the production of valuable antibiotics such as novobiocin, amphotericin, and vancomycin Furthermore, they serve as plant growth promoters, biocontrol tools, and sources of agroactive compounds, highlighting their significant role in producing antimicrobial agents and other industrially important substances.
Therefore, the study and use of actinomycetes as well as biologically active substance that they generate to inhibit pathogenic organisms is a method efficiently and enviromentally friendly.
Materials
Actinomycetes were stored in laboratory of Department of Microbial Technology- Faculty of Biotechnology- Vietnam National University of Agriculture
Pathogenic fungi are preserved in the laboratory of the Department of Microbial Technology at the Faculty of Biotechnology, Vietnam National University of Agriculture The strain GT 1, identified as Fusarium oxysporum, is characterized by its white filamentous appearance, large colony size, and rapid growth, with the mycelium turning purple as it ages.
Figure 3.1 Morphology of Fusarium oxysporum - GT1 on PDA medium 3.1.2 Chemicals, instruments and equipments
The chemicals used in the study of microorganisms and some other chemicals such as: Soluble starch, Glucose, Fructose, Yeast Extract, Malt Extract, K2HP04 , MgS04, NaCl, KN03, FeS04,…
The instrucments and equipment used in Microbial Technology Laboratory- Faculty of Biotechnology- Vietnam National University of Agriculture: Vortex shaker, optical microscope, shaking machine, incubator, pipettes, refrigerators, pH meter,…
- Gause I medium (g/l): 20g of Soluble Starch, 0.5 g of K2HP04, 0.5g of MgS04.7H20, 0.5g of NaCl, 0.5g of KN03, 0.01g of FeS04.7H20, 20g of Agar, 1 liter of distilled water, pH 7-7.4
- Gause II medium (g/l): 30 ml of Meat Extract Water, 5g of Peptone, 5g of NaCl, 10g of Glucose, 20g of Agar, 1 liter of H20, pH 7-7.2
- PDA medium (g/l): 200g of potato, 20g of glucose, 1 liter of distilled water, pH 6
- ISP1 medium (g/l): 5g of Tryptone, 3g of Yeast Extract, 20g of Agar, 1 liter of distilled water, pH 7- 7.2
- ISP2 medium (g/l): 4g of Yeast Extract, 10g of Malt Extract, 4g of Glucose, 20g of Agar, 1 liter of distilled water, pH 7.3
- ISP3 medium (g/l): 20g of Oatmeal, 20g of Agar, 1.0 ml of Micronutrient Salt Solution, 1 liter of distilled water, pH 7-7.4
- ISP4 medium (g/l): 10g of Starch, 1 g of , 1g of MgS04.7H20, 1g of NaCl, 2g of (NH4)2S04, 2g of CaC03, 1.0 ml of Micronutrient Salt Solution, 1 liter of distilled water, pH 7-7.4
- ISP5 medium (g/l): 1g of L- asparagine, 10g of glycerol, 1 g of K2HP04, 1.0 ml of Micronutrient Salt Solution, 1 liter of distilled water, 20g of Agar, pH 7-7.4
-ISP6 medium (g/l): 10g of peptone, 1g of Yeast Extract, 0.5g of Xitrat, 20g of Agar, 1 liter of distilled water, pH 7-7.2
Micronutrient salt solution of IPS: 0.01g of FeS04.7H20, 0.1g of MnCl2.4
H20, 0.1g of ZnS04 7H20, water to 100ml, pH 7-7.2
The carbon sources: D- Glucose, D- Fructose, Saccharose, D- Xylose, Dextrin, Maltose, D- Sobitol, α- Lactose
B solution: 0.64g of CuS04.5 H20, 0.11g of FeS04.7H20, 0.79g of MnCl2.4 H20, 0.15g of ZnS04 7H20, 100ml of distilled water
- Location: This thesis was performed in the laboratory of Department of Microbial Technology- Faculty of Biotechnology- Vietnam National University of Agriculture
- Time: The thesis was done from 9/2020-2/2021
Research methods
3.2.1 Selection of actinomycetes capable of antagonisms with pathogenic fungi - Actinomycetes culture method
Actinomycetes were cultured on Gause I medium Before testing the antagonistic ability, we conducted activating actinomycetes on Gause I medium,the transplantation was repeated 2 times and then proceeded test
- Test capable of antagonisms with pathogenic fungi by dual culture method on PDA medium (Dhanasekaran et al, 2012)
+Step 1: The actinomycete was placed at one end of the petri dish
+Step 2: After 2 cultured days, actinomycete grew and we put the pathogenic fungi at the center of the petri dish
+Step 3: The plate was incubated at 30°C for 5-7 days and we determined the result
-The actinomycetes were maintained in 30% glycerol solution at -21°C condition
Fungal strains were cultured on PDA medium (200g of potato, 20g of glucose, 1 liter of distilled water, pH 6)
3.2.2 Research on biological characteristics and identified actinomycetes 3.2.2.1 Morphological characteristics
Actinomycetes were cultured in isolated colonies on Gause I medium and observed after 5-7 days at 30°C The resulting colonies exhibited various characteristics, including rough, chalky, opaque shapes with radiating lines, as well as smooth and wrinkled forms.
-Color of arial and substrate mycelium
Actinomycetes were cultured on various media, including Gause I, ISP1, ISP2, ISP3, ISP4, ISP5, and ISP6 After five days, we examined the color of the aerial and substrate mycelium, as well as the pigments secreted into the medium, following the methods outlined by Shirling and Gottlieb (1996) This study also focused on the observation of conidiophores and spores.
Actinomycetes were inoculated in Gause I medium using a zigzag pattern, with the lamina pinned down at a 45° angle along the transplant line The cultures were incubated at 30°C, and observations of spore chains, conidiophores, and spore surfaces were conducted using an optical microscope.
- Testing the effect of pH on the growth of actinomycetes
Actinomycetes were cultured on Gause I medium at pH from 4 to 12 at temparature of 30°C, observing the growth after 5 days
-Testing the effect of temparature on the growth of actinomycetes
Actinomycetes were cultured on Gause I medium at different temparatures: 5-25-30-37-40-50°C and observing the growth after 5 cultured days
Add NaCl with concentration from 1-7% into Gause I medium Actinomycetes were transplanted, cultured at 30°C in incubator Observed the
19 growth of actinomycetes after 5-7 days After that we found the appropriate salt concentration for the development of actinomycetes
-The ability to assimilate carbon sources
Actinomycetes were cultures on ISP-9 medium with 1% added sugar sources such as: Glucose, D- Fructose, Maltose, Saccharose, Dextrin…
Actinomycetes were cultured on a sugar-enriched medium in an incubator at 30°C for 5-7 days to assess their growth This study aimed to evaluate the potential of various carbon sources for the growth of actinomycetes.
3.2.2.3 Identification of the actinomycetes based on 16S rRNA sequence
Total DNA was isolated from VNUA30 strain
Actinomycetes 16S rRNA gene was amplified by PCR reaction Two primers 27F and 1429 R have respective sequences:
Reaction was operated according to the following amplification process: 95°C (5min) following by 30 cycles of 95°C (30 second), 53°C (30 second), and 72°C (1 min) and followed by a final extention step of 7 mins at 72°C
The results of the PCR reaction were visualized through gel electrophoresis The obtained PCR product was purified using the QIAquick gel extraction kit (Qiagen, Germany) The purified PCR product was sequenced in Singapore, allowing for the identification of phylogenetic neighbors and the calculation of pairwise 16S rRNA gene sequence similarities (Chun et al 2007) The sequence obtained was nearly complete.
The sequences of phylogenetic neighbors were aligned using the Clustal W Program, focusing on the 1434 bp of strain 30 Phylogenetic trees were constructed utilizing neighbor joining and maximum-parsimony algorithms, with bootstrap values derived from 1000 replications, employing MEGA 6.0 software (Felsenstein 1985; Tamura et al 2013).
RESULT AND DISCUSSION
Screening actinomycetes capable of antagonism with fungi
Actinomycetes isolated from various sources were preserved in 30% glycerol and subsequently activated on Gause I medium I then screened these actinomycetes for their antifungal properties using the Dual culture method on PDA medium, as described by Dhanasekaran et al (2012).
From 30 actinomycete strains, there is only one actinomycete strain with capable of resistant to fungi cause disease in banana by Dual culture method VNUA30 strain had capable of strong resistance to pathogenic fungi on Banana
Figure 4.1 The results of testing the antifungal activity of actinomyces VNUA30 strain by dual cultured method after 5 cultured days
- The VNUA30 strain was grown on PDA plate for 2 days before inoculating the fungi at the center of petri dish Growth inhibitory activity was observed after 5 cultured days at 30°C
- After 5 cultured days, the VNUA30 strain had capable of strong resistance to F oxysporum - causing the Panama disease on banana
Characterization of VNUA30 strain
On gause I medium, the characteristics of colonies, color and aerial mycelium of VNUA30 strain was observed after 5 cultured days at 30°C
Figure 4.2 Morphology of VNUA30 strain on the Gause I medium after 5 cultured days at 30°C
After 5 cultured day, the VNUA30 strain had off- white colonies, bulging center and globose shape
Microscopes are the traditional instruments used for assessing actinomycete, and they remain as indispensable tools for exploring the morphological, physiological, and genetic diversity present in actinomycetes
The VNUA30 strain was cultured on gause I medium with pined lamellae tilted at 45° to observe the color of aerial mycelium The culture was incubated at 30°C, and daily observations were made to examine the spore stem shape, spore chains, and spores on the lamellae using a microscope at 1000x magnification.
Figure 4.3 Conidiophore morphology of VNUA30 strain under optical microscope at 1000 times magnification after 48 cultured hours
After that, spores released from conidiophore very strong after 60-72 hours
On the different media, color, morphology of actinomycetes were different, the growth and color of VNUA30 strain in some media such as Gause
I, ISP-1, ISP-2, ISP-3, ISP-4, ISP-5 and ISP- 6
Some characteristics of VNUA30 strain after 5 cultured days
Table 4.1 Some characteristics of VNUA30 strain after 5 cultured days at
ISP- 2 +++ Light grey White No
ISP- 3 ++ Grey Dark grey No
ISP- 4 + White Light white No
ISP- 6 ++ Grey Light grey Black
Note: (+): It can grow; (++): Good grow; (+++): Very good growth
VNUA30 strain was cultured in various media having difference in ability to grow, the color of aerial and substrate mycelium (Shirling and Gottlieb,
In 1996, the VNUA30 strain exhibited aerial mycelium primarily in grey and white, while its substrate mycelium transitioned from dark grey to light white This strain demonstrated varying growth capabilities across different media, thriving particularly well on Gause I, ISP-1, and ISP-2 Notably, after five days of culture on ISP-6 medium, the VNUA30 strain was able to produce pigments.
The growth and development of the VNUA30 strain of actinomyces are influenced by salt concentration To investigate this effect, the VNUA30 strain was cultured on Gause I medium with varying NaCl concentrations ranging from 1% to 7%, and the results were observed after five days of incubation.
Figure 4.4 Salt concentration affect the growth and development of actinomyces VNUA30 strain
Results showed that VNUA30 strain can grow on medium with salt concentration from 1%-4%, and grow well in salt concentration is 1% and 2%
Microorganisms can be categorized into three types based on their tolerance to salt concentration: low (2-3%), medium (5-20%), and high (above 20%) Actinomyces cannot survive at salt concentrations of 5% or higher, and they fail to grow if the concentration is below 12% (Larsen, 1986) Notably, the VNUA30 strain thrives in low salt concentrations.
4.2.2.2 Testing the effect of pH on the growth capacity
Hydrogen ion concentration closely related to the growth of microorganism Each microorganism also has a different pH range At the different pH, the ability to grow is different, too.
The microorganisms often live in the soil and water, so the microorganism can grow in wide range of pH Each type of microorganism has
- Lowest pH: the lowest pH at which actinomycetes are still survice and grow
- Appropriate pH: pH at which actinomycetes grow best
- Hight pH: the highest pH at which actinomycetes are still survice and grow
Microorganisms thrive at their optimal growth pH, with growth significantly hindered below the minimum or above the maximum pH levels The growth, development, and compound synthesis of actinomycetes are particularly influenced by environmental pH.
VNUA30 strain was cultured on Gause I medium, in the pH varies from
The study aimed to determine the optimal pH value for the growth of the VNUA30 strain, as well as the pH levels that are detrimental to its survival The findings are illustrated in the figures below.
26 pH 4 pH 5 pH 6 pH 7 pH 8 pH 9
Figure 4.5 Testing the effect of pH
Result showed that VNUA30 strain had capable of growth on the medium with wide pH range They can live and grow at pH fom 4-11 At the pH from 7-
10, VNUA30 strain can grow perfectly, so the pH from 7 to 10 is their optimum growth pH VNUA30 strain can not grow at the pH lower than 4 and higher than
4.2.2.3 Testing the effect of temparature on the growth capacity
Microbial species, particularly actinomycetes, are significantly influenced by varying temperature conditions Frequent temperature fluctuations impact their growth, development, and compound synthesis.
Each microorganism, like VNUA30, has three temperature limits: the lowest, optimal, and highest temperatures for growth To determine the optimal temperature for the VNUA30 strain, I cultured it on Gause I medium at various temperatures: 15°C, 20°C, 30°C, 37°C, 40°C, and 50°C After five days of culturing, I observed the results.
Results are shown in the table below:
Table 4.2 Results of testing the effect of temperature
Note: (-): No growth; (+): Grow; (++): Good growth
Results showed that VNUA30 strain can grow well in the teamperature range from 20°C to 40°C The optimal temperature of VNUA30 strain is 30°C
The temperature less than 15°C and more than 40°C not suitable for strain 30 to survice
4.2.2.4 The ability to assimilate carbon sources
Carbon compounds play a crucial role in the growth and production of antibiotics in actinomycetes While many actinomycetes thrive on starch as a primary carbon source, some strains effectively utilize simple sugars like glucose, fructose, and lactose, as well as disaccharides such as maltose.
To determine the ability to assimilate different carbon sources, we conducted culturing strain 30 on the Gause I medium with addition of various carbon sources at the concentration of 1%
ISP-9 medium added with 1% D-glucose sugar was considered positive control Check the resuls after 7 days of culture
The results are showed in the table:
Table 4.3 The ability to assimilate different carbon sources of VNUA30 strain after 5 cultured days
Carbon sources Ability to assimilate
Note (+): It can assimilate carbon sources; (++): Assimilate strongly sources of carbon; (+++): Assimilate very strongly sources of carbon
The VNUA30 strain demonstrated a strong ability to assimilate various sugar sources, particularly raffinose, starch, glycerol, and D-mannitol However, its growth was limited when utilizing cellulose and D-inositol as carbon sources.
Identification of actinomyces VNUA30 based on 16S rRNA sequence
Total DNA was extracted from VNUA30 strain After extraction, total DNA was loaded in Agarose gel 0.8%
Figure 4.6 The result of total DNA electrophoresis 4.3.2 PCR amplification
Total DNA were enough quality for PCR reaction.We use a pair of primers: 27F and 1249R
Two primers 27F and 1429 R have respective sequences:
Reaction was operated according to the following amplification process: 95°C (5min) following by 30 cycles of 95°C (30 second), 53°C (30 second), and 72°C (1 min) and followed by a final extention step of 7 mins at 72°C
Figure 4.7 The result of PCR reaction marker 1500bp
Figure 4.8 The taxonomic tree based on the sequence of 16S rRNA gene coding
The tree is construted according to the neighbor- joining method The analysis involved 11 nucleotide sequences Evolutionary analyses were conducted in MEGA X
The 16S rRNA gene sequence of the VNUA30 strain shows strong homology with that of the genus Streptomyces neyagawaensis, with a genetic similarity of 76% Additionally, morphological, physiological, and biochemical characteristics of the VNUA30 strain closely resemble those of Streptomyces neyagawaensis Therefore, it is concluded that the VNUA30 strain is classified within the genus Streptomyces and identified as Streptomyces VNUA30.
CONCLUSION – SUGGESTION
Conclusion
We found VNUA30 strain had capable of strong resistance to fungi cause disease in banana
Research morphological and biochemical, physiological characteristics of VNUA30 strain: Optimal temperature, optimal pH, suitable salt concentration for growth of VNUA30 strain
- Morphological: Colonies of VNUA30 strain belong to white group, bulging center and globose shape
- Optimal temperature: VNUA30 strain had temperature range from 20°C -40°C, It can grow well from 30°C-40°C and the optimal teperature of VNUA30 strain is 30°C
- Optimal pH: pH range of VNUA30 strain from 5- 11 VNUA30 strain grow well from 7-10 Therefore, VNUA30 strain belong to alkaline actinomycetes
The VNUA30 strain is capable of surviving in media with sodium chloride (NaCl) concentrations ranging from 0% to 3% However, it cannot survive or grow in higher salt concentrations, categorizing it as a low salt concentration strain.
- The ability to assimilate carbon sources: VNUA30 strain had capable of assimilating different sources from various sugar sources VNUA30 strain had strongly assimilating raffinose, starch, glyxerol and D- Manitol
- VNUA30 strain was identified as Streptomyces VNUA30.
Suggestion
After identified the Streptomycetes VNUA30 I suggest to research the biocontrol and plant growth promoting effect of Streptomyces VNUA30
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Morphological characteristics of VNUA30 strain on some kind of media after 7 cultured days at 30°C
1 Effect of temperature on the growth capacity of VNUA30 strain after 7 cultured days
2 The ability to assimilate carbon sources