INTRODUCTION
Introduction
Colletotrichum is a significant genus comprising numerous species that are among the most prevalent fungal pathogens affecting various fruits and crops Most crops globally are vulnerable to one or more species of Colletotrichum, making it a critical concern in agriculture According to Dean (2012), this genus ranks as the eighth most important group of phytopathogenic fungi worldwide.
Colletotrichum gloeosporioides and the anthracnose disease cause mounting threat to global agriculture This fungus causes bitter rot in variety of crops worldwide, from monocotyledons to higher dicothyledons (Bailey, 1992)
Colletotrichum species are responsible for over 50% losses in the productivity of fresh fruits and vegetables, affecting key host plants such as citrus, yam, papaya, avocado, coffee, eggplant, sweet pepper, and tomato This fungus causes significant pre- and post-harvest losses globally, acting as a secondary invader of damaged tissue or surviving as a saprophyte Colletotrichum gloeosporioides manifests various symptoms based on the host species and infected tissue, including black or brown lesions on fruits, blight and necrosis in inflorescences, and abnormal colors with dark, necrotic areas on leaves Additionally, stem infections lead to dieback, discoloration, and symptoms of gummosis and resinosis.
Effective plant protection strategies are essential to mitigate the damage caused by fungal diseases While chemical solutions are commonly used, they present significant drawbacks, including the development of drug resistance in pathogens, heightened environmental pollution, disruption of ecological balance, and the presence of harmful chemical residues in the soil, which pose risks to human health.
Biological-origin fungicides, particularly those derived from Actinomycetes, offer a promising alternative to chemical measures for protecting plants from harmful fungi Actinomycetes are known for their production of secondary metabolites with antibacterial and antifungal properties, as well as their ability to generate extracellular hydrolytic enzymes that aid in the decomposition of organic matter in soil This group of microorganisms plays a crucial role in soil biodegradation and humus formation by recycling nutrients from complex polymers like chitin and lignocelluloses Notably, approximately 23,000 bioactive secondary metabolites have been identified, with Actinomycetes contributing around 10,000 of these, accounting for 45% of all known bioactive microbial metabolites Among them, Streptomyces species alone produce about 7,600 compounds Consequently, there has been an increase in research efforts focused on screening for novel active compounds, characterized through various laboratory tests based on morphological and physiological criteria.
"Characterization and identification of Actinomycetes capable of antagonism with fungus Colletotrichum gloeosporioides cause Anthracnose disease in plants"
Purposes and requirements
- Characterization and identification of Actinomycetes capable of antagonism with fungi Colletotrichum gloeosporioides cause Anthracnose disease in plants
- Screen Actinomycetes strain with ability of resistant to fungus
Colletotrichum gloeosporioides cause anthracnose disease in plants
- Study morphological and biochemical characteristics of selected Actinomycetes train
- Classify and identify Actinomycetes strain based on 16S rRNA sequences
LITERATURE REVIEW
Overview of Actinomycetes
2.1.1 Introduction and distribution of Actinomycetes
Streptothrix foersteri, identified by Ferdinand Cohn in 1875, and Actinomyces bovis, discovered by Carl Otto Harz in 1877, are among the earliest described Actinobacteria Actinomyces bovis is known to cause significant infections in livestock.
'Lumpy jaw' disease in cattle is associated with bacteria known as Actinomyces, which have thin filaments ending in club-shaped structures that resemble fungi Despite this resemblance, the 'gonidia' contain actual cells, making the comparison to fungi misleading Other bacteria, including those causing leprosy and tuberculosis, share similar features and were initially thought to belong to the same group It wasn't until the 1960s that their classification as true bacteria was confirmed.
Actinomycetales in 1916, and it became clear that they formed a big diverse group with a wide range of physiological and biochemical features The
Actinobacteria are today regarded one of the bacterial kingdom's largest phyla
Certain corynebacteria exhibit a GC content of 54%, whereas Streptomycetes possess a significantly higher GC content exceeding 70% Notably, Tropheryma whipplei, the pathogen responsible for Whipple's disease, has a GC content of 46.3%, marking it as the lowest known among Actinobacteria (Ul-Hassan, 2009).
Actinomycetes are a distinct group of bacteria characterized by high GC content and diverse habitats, predominantly found in soil, particularly in alkaline and organic-rich environments They play a significant role in the microbial community and are more prevalent in soil compared to other ecosystems, such as marine and freshwater systems While many Actinomycetes contribute positively to their environments, some species are notable human and animal pathogens.
Actinomycetes play a significant role in plant infections, with their population density varying based on habitat and climate In just 1 gram of soil, there can be between \$10^6\$ and \$10^9\$ cells, predominantly consisting of streptomycetes, which make up over 95% of this population.
Actinomycetes, primarily found in soil, play a vital role as saprophytes by decomposing various plant and animal waste Notably, genera like Streptomyces and Micromonospora are prolific producers of antibiotics, enzymes, enzyme inhibitors, signaling molecules, and immunomodulators.
Actinomycetes, like other soil bacteria, are mostly thermophilic, with a growth temperature of 25-30°C Actinomycetes that grow at high temperatures (50-60°C) and Actinomycetes that grow at low temperatures, such as
Arthrobacter ardleyensis, discovered in Antarctic lake sediments at 0°C, represents two types of Actinomycetes (Chen et al., 2008) These microorganisms typically flourish in low-humidity soil, but their growth is limited or halted in dry conditions or overly moist environments Most Actinobacteria prefer neutral soil, thriving optimally within a pH range of 6 to 9 Interestingly, some Streptomyces strains have been found to adapt to acidic soils with a pH as low as 3.5.
Actinomycetes are characterized through a polyphasic approach that integrates phenotypic, chemotaxonomic, and genotypic data to formally describe new taxa According to Tindall (2010), key elements essential for prokaryote characterization studies include the assessment of phenotypic traits, which serve as the foundation for taxonomic classification.
Most actinobacteria are classified and identified primarily through their morphological characteristics, which remain a crucial factor in acquiring detailed information about a taxon.
Actinobacteria is a highly diverse phylum within the bacterial kingdom, comprising five subclasses, six orders, and 14 suborders (Ludwig et al., 2012) This phylum exhibits significant variation in appearance, physiology, and metabolism, and its name reflects its branching position in the taxonomic tree based on 16S rRNA sequences The recent surge in genomic sequencing has provided valuable insights into the evolution of the Actinobacteria genus, which is categorized into six classes: Actinobacteria, Acidimicrobiia, Coriobacteriia, Nitriliruptoria, Rubrobacteria, and Thermoleophilia.
Radial mycelium is highly developed in Actinomycetes, which can be categorized into substrate and aerial mycelium based on their morphological and functional characteristics These microorganisms are capable of producing various reproductive structures, including spores, spore chains, sporangia, and sporangiospores Key morphological features for classifying Actinomycetes include the location and quantity of spores, the surface properties of the spores, the shape of the sporangia, and the presence of flagella on sporangiospores.
Substrate mycelium plays a crucial role in nutrient absorption for Actinomycetes, developing on or within culture media Under microscopic examination, these mycelia appear thin, translucent, and phase-dark, exhibiting more branching than aerial hyphae The individual hyphae measure approximately 0.4 to 1.2 µm in thickness, lack diaphragms, and are capable of branching.
White, yellow, orange, red, green, blue, purple, brown, black, and other hues are seen in substrate mycelia, and certain hyphae can create water- or fat-
Water-soluble pigments can diffuse into culture media, creating a color-matched environment, while colonies exhibiting the same color are formed by non-water-soluble (or fat-soluble) pigments The coloration of substrate mycelia and the presence of soluble pigments are essential indicators for the identification of new species.
Aerial mycelium refers to the hyphae that develop from substrate mycelium and grow into the air Distinguishing between aerial hyphae and substrate mycelia can be challenging To differentiate them, a cover slip is used to observe the samples in a dry environment under a light microscope Substrate hyphae appear thin, transparent, and phase-dark, while aerial hyphae are characterized as coarse, refractive, and phase-bright.
Pseudonocardia and Amycolata exhibit aerial mycelium with a fibrous coating composed of fibrillar components and short rodlets, creating a unique microscopic pattern This fibrous sheath is also found on sporulating aerial hyphae, contributing to the diverse surface ornamentations of the spores.
Overview of Colletotrichum gloeosporioides
Colletotrichum gloeosporioides, initially named Vermicularia gloeosporioides by Penzig in 1882, was first identified from a specimen collected from Citrus in Italy This name has been widely referenced in various literary works to describe this particular fungus.
C gloeosporioides, the asexual stage of Glomerella cingulata, is associated with various diseases in citrus and has over 600 synonyms, reflecting its morphological and physiological diversity Early research highlighted the morphological similarities among different Colletotrichum species based on host preference and questioned their distinctiveness through inoculation tests The fungus is characterized by irregularly shaped spores that appear as brown to black specks, and under humid conditions, mature acervuli vary widely in size and form, releasing pink masses of conidia.
Colletotrichum gloeosporioides thrives optimally at temperatures between 25-28°C and a pH of 5.8-6.5, remaining dormant during dry seasons until favorable conditions arise This pathogen exhibits a hemibiotrophic infection, featuring both biotrophic and necrotrophic phases simultaneously Various media, including potato dextrose agar and malt extract agar, are utilized for its growth and sporulation The fungus produces hyaline, one-celled conidia that are ovoid to oblong and slightly curved, carried on well-developed hyaline conidiophores measuring 12.5–14.8μm x 4.1–4.7μm Abundant subepidermal acervuli form on diseased plant parts, measuring 80–250μm and producing pink or crimson conidial masses under damp conditions when mature.
C gloeosporioides exhibits two germination types: pathogenic and saprophytic Pathogenic germination occurs on plants or hydrophobic surfaces, characterized by rapid mitosis and the formation of a single germ tube leading to appressoria In contrast, saprophytic germination takes place in rich media, is slower, and results in two germ tubes without appressoria formation, meaning these spores do not infect plants Distinct signaling pathways regulate these mechanisms, with saprophytic germination utilizing cAMP pathways This pathogen affects a wide variety of fruits, including almonds, avocados, and mangoes, causing anthracnose disease and impacting crops like cereals and legumes Additionally, Colletotrichum spp can be found on decaying wild fruits Elevated CO2 levels increase spore production per lesion, potentially exacerbating disease severity and spread.
2.2.2 Biology of Colletotrichum gloeosporioides infection process
Anthracnose is the name given to disease symptoms induced by
Colletotrichum species initiate their life cycle with spore germination on the plant surface, leading to the development of melanized infection structures called appressoria that penetrate host tissue During the initial infection, thick infection hyphae are produced in what is known as the biotrophic stage Subsequently, the fungus transitions into the necrotrophic phase, marked by the formation of thin hyphae.
Secondary hyphae emerge from the original hyphae, colonizing nearby cells and causing visible lesions at the infection site Spores then form on the affected tissue and are spread by insects, air currents, and water splashes, initiating a new cycle of infection (Münch et al., 2008).
Anthracnose infections in papaya usually begin during the early stages of fruit development, with the fungus remaining dormant as an appressorium or subcuticular hyphae until the fruit reaches climacteric maturity The first symptoms appear as small, distinct dry pink spots on the fruit's surface, which later progress to dark brown to black deep lesions.
Glomerella cingulata, with its anamorph C gloeosporioides, is responsible for mango anthracnose, leading to issues such as blossom blight, leaf blight, and tree dieback (Ploetz et al., 1996) Symptoms on the fruit surface appear as spherical brown to black lesions with indistinct boundaries In larger fruits, lesions rarely develop initially, as the fungus remains dormant after colonization until the fruit matures As ripening occurs, dark depressed circular lesions emerge, rapidly increasing in size and potentially covering the entire fruit in severe cases These lesions can vary in size and may extend from the base to the distal end of the fruit In advanced stages, the fungus can penetrate the pulp, producing abundant orange to pink conidia masses (Arauz, 2000).
Anthracnose lesions develop on young avocado (Persea americana) fruits due to insect damage Additionally, dead leaves trapped in the tree serve as a significant source of inoculum, leading to considerable fruit losses in affected trees.
Germinated spores develop appressoria that penetrate the cuticle, leading to dormant subcuticular hyphae until the fruit is harvested and ripened This process results in the formation of spherical black sunken lesions on the fruit's surface, which rapidly spread and subsequently infect the pulp, causing rot.
2.2.3 Damage of Anthracnose disease caused by Colletotrichum gloeosporioides in agriculture
C gloeosporioides is a pathogen that causes anthracnose in a wide range of crops all over the world Avocado, mango, beans, cashews, cassava, citrus plant, cotton, cow-pea, cucumber, eggplant, green gram, mango, onion, pepper, pumpkin, papaya, sorghum, soybean, tomato, watermelon, wheat, yam, zucchini, cucurbit, cereals, legumes, and spinach are among the crops affected Wet, humid, warm circumstances encourage anthracnose, which is transmitted by infected seeds, rain splash, and moist breezes
Anthracnose caused by C gloeosporioides was reported from several parts of the world Bitter rot apples (Malus sylvestris Mill) caused by
Glomerella cingulata and C gloeosporioides have been identified in North Carolina orchards, with the disease first noted at the end of June, potentially leading to 100% fruit rot by mid-August (Shane & Sutton, 1981) Additionally, C gloeosporioides and C acutatum are responsible for fruit rot in apples and pears across the Southern, Central, and Mid-Atlantic regions of the United States, as well as in many countries where these fruits are cultivated (Sutton et al., 2014).
C gloeosporioides was reported to cause both pre and post-harvest anthracnose on avocado in several countries including Australia (Fitzell, 1987), Israel (Binyamini & Schiffmann-Nadel, 1972), South Africa (Darvas & Kotze,
1987) and Sri Lanka (Sivanathan & Adikaram, 1989) The pathogen causes severe yield losses The site of infection in avocado is primarily the fruits, but
Infections can manifest on the leaves and stems of avocado plants, but they do not affect the flowers (Nelson, 2008) Anthracnose, caused by C gloeosporioides, has been documented in avocados in Australia and South Africa (Giblin & Coates, 2007), as well as in bananas (Jeger et al., 1995).
The edible seeds and therapeutic roots of Trichosanthes kirilowii Maxim, a gourd family plant, are grown in China Fruit rot induced by C gloeosporioides caused a large loss in 2000 (Li & Zhang, 2007)
C gloeosporioides has also been linked to mango post-harvest disease
(Ploetz et al , 1996) It was first discovered in Puerto Rico (Collins, 1903) and then spread to Hawaii, Florida, Cuba, the Philippines, Columbia, South Africa, Brazil, the United States, and Pakistan
The disease incidence has been reported to be 32% in South Africa (Sanders et al., 2000), 64.6% in Costa Rica in 1990 (Arauz-Cavallini et al.,
Anthracnose, caused by C gloeosporioides, poses a significant threat to mango production in Southeast Asia, leading to substantial yield losses In Himachal Pradesh, India, post-harvest deterioration due to this disease reached 29.6% during the years 1990-92 Additionally, nearly 100% of fruit produced in wet or very humid conditions is affected by this issue.
In Vietnam, an infection affecting oil coffee berries was identified as caused by the pathogen C gloeosporioides through both morphological and molecular methods (Nguyen et al., 2009) This pathogen was previously found on olive trees in Montenegro in September 1995 (Latinovic & Vucinic, 2000) Additionally, C gloeosporioides has been linked to onion anthracnose in Brazil, a leading onion producer globally In Hawaii, this pathogen has also led to anthracnose disease in 17% of papaya fruits, characterized by rounded, water-soaked, and sunken lesions.
25 lesions on the matured fruits' bodies "Chocolate spots" are the name given to these lesions (Dickman & Alvarez, 1983)
MATERIALS AND METHODS
Materials
The Actinomycetes strains were conserved in laboratory of Department of Microbial Technology, Faculty of Biotechnology, Vietnam National University of Agriculture
Pathogenic fungus Colletotrichum gloeosporioides was conserved in laboratory of Department of Microbial Technology, Faculty of Biotechnology, Vietnam National University of Agriculture
- Gause I medium (g/L): 20g of Soluble Starch, 0.5 g of K2HPO4, 0.5g of MgSO4.7H2O, 0.5g of NaCl, 0.5g of KNO3, 0.01g of FeSO4.7H2O, 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 distilled water, pH 7-7.2
- PDA medium (g/L): 200g of potato are boiled, unpeeled in 1 liter distilled water and filtered to take potato extract water, mixed with 20g of glucose and 20g of Agar, pH 5.6-5.8
- 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
The ISP4 medium is prepared by dissolving 10 g of starch, 1 g of K₂HPO₄, 1 g of MgSO₄·7H₂O, 1 g of NaCl, 2 g of (NH₄)₂SO₄, and 2 g of CaCO₃ in 1 liter of distilled water, adjusting the pH to 7-7.4 Additionally, 1.0 ml of micronutrient salt solution is added The mixture is then autoclaved for 30 minutes at 121°C and stored in a refrigerator for future use.
- ISP5 medium (g/L): 1g of L- asparagine, 10g of glycerol, 1 g of
K 2 HPO 4 , 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
- ISP9 medium (g/L): 2.38g of KH 2 PO 4 , 5.65g of K 2 HPO 4 3H 2 O, 2.64g of (NH4)2SO4, 1g of MgSO4.7H2O, 1 ml of B solution, 20g of Agar, 1 liter of distilled water, pH 6.8- 7.0
- Micronutrient salt solution of IPS: 0.01g of FeSO4.7H2O, 0.1g of MnCl2 4H2O, 0.1g of ZnSO4.7H2O, water to 100ml, pH 7-7.2
- B solution: 0.64g of CuSO 4 5 H20, 0.11g of FeSO 4 7H 2 O, 0.79g of
MnCl2 4H2O, 0.15g of ZnSO4.7H2O, 100ml of distilled water
Carbon sources: Maltose, D-Sorbitol, Starch, Lactose, Fructose, D- xylose
- Glass slides and cover slips
- Location: In laboratories of Department of Microbial Technology, Faculty of Biotechnology, Vietnam National University of Agriculture
Methods
3.2.1 Activate Actinomycetes strains preserved in glycerol
To activate Actinomycetes strains preserved in 3% glycerol at -80℃, we incubated them in a water bath at 137℃ for 2 minutes Subsequently, we transferred pieces of agar to three distinct locations on ISP2 medium agar The samples were then incubated at 30℃, and growth was monitored over a period of 3 to 5 days The strains are deemed active if they exhibit robust growth, stability on ISP2 medium, and show no signs of contamination.
3.2.2 Screening of Actinomycetes with antifungal activity
Co-culture method was used to test activity against the fungus
Inoculate a petri dish containing PDA medium with a 4-5 mm piece of Actinomycetes agar, positioning it 20 mm from the edge of the dish, as demonstrated by Rahman et al (2009).
Inoculate a loop of rod with the control fungus or a 4-5 mm piece of agar containing the fungus at the edge of the plate, positioned parallel to the examined Actinomycetes strain Use a PDA dish with only the inoculated fungal pathogen as a control.
After 6 days of inoculation at 25°C, the radii of fungal dispersion (in mm) were measured in both experimental and control dishes The radius of fungal dispersion adjacent to Actinomycetes (d1) was recorded on the experimental plate, while the control plate's radius was noted as (d0) Each experiment was conducted three times to ensure accuracy.
The antagonistic activity was calculated as: d = d0 – d1
Percent inhibition (PIRG) was determined according to the following equation (Rahman et al , 2009)
The morphological characteristics of Actinomycetes strains were assessed by examining culture traits such as the color of aerial and substrate mycelium, as well as soluble pigments, following the methods outlined by Tresner et al (1968) on ISP media (ISP1, ISP2, ISP3, ISP4, ISP5) as described by Shirling & Gottlieb (1966) The inoculated Actinomycetes were incubated at 30℃ and observed after a 7-day incubation period.
Spore chain morphology and spore surface:
Actinomycetes were cultured on Gause I medium inserted cover slips at
After incubating at 30ºC for 24 hours at a 45º angle, the aerial mycelium of actinomycetes was examined using an optical microscope at 1000x magnification If spores were not produced, the observation should be repeated after another 24 hours During sporogenesis, it is essential to monitor the substrate mycelium for fragmentation, the presence of sclerotia, and the morphology of spore chains, as noted by Nguyen Xuan Canh et al (2016).
3.2.4 Ability to assimilate carbon sources
Actinomycetes were tested for their capacity to utilize various carbon sources on ISP-9 medium, which was enriched with 1% of D-sorbitol, D-xylose, fructose, lactose, maltose, and starch A negative control without any carbon source and D-glucose as a positive control were also included in the study (Pridham & Gottlieb, 1948).
Actinomycetes were cultured on sugar-enriched media and incubated at 30℃ for 5-7 days to assess their growth This evaluation aimed to determine the carbon source utilization capabilities of the studied Actinomycetes strain, with the experiment conducted in triplicate for accuracy.
3.2.5 Ability to produce extracellular enzymes
Actinomycetes were inoculated on mineral salt agar supplemented with various substrates, including CMC, starch, xylan, pectin, chitin, and gelatin, following the method outlined by Gulve & Deshmukh (2011) After a 7-day incubation period, colonies on the petri dishes were stained with 1% Lugol dye to assess amylase, cellulase, xylanase, pectinase, and chitinase activities, while protease activity was evaluated using 0.1% amido black 10B The presence of extracellular enzyme activity was indicated by a light ring surrounding the colonies.
To assess catalase activity, we transfer several colonies onto a clean microscope slide and add 30% hydrogen peroxide (H2O2) The presence of air bubbles indicates a positive result, while their absence signifies a negative result Each experiment is conducted in triplicate to ensure accuracy.
3.2.6 Effects of temperature, pH and NaCl concentrations on the growth of Actinomycetes
Effect of temperature on the growth of Actinomycetes
Studying the influence of temperature on the growth of Actinomycetes on Gause I medium, including different temperature conditions: 25, 30, 35, 40, 45, 50ºC After 5-7 days, observe the growth Nguyen Xuan Canh et al (2016)
Effect of pH on the growth of Actinomycetes
Studying the influence of temperature on the growth of Actinomycetes on Gause I medium, including different pH conditions: 5, 6, 7, 8, 9, 10, 11, and 12 After 5-7 days, observe the growth Nguyen Xuan Canh et al (2016)
Effect of NaCl concentrations on the growth of Actinomycetes
To assess the salt tolerance of Actinomycetes, cultures were grown on Gause I agar enriched with varying concentrations of NaCl (1% to 7%) After a period of 5 to 7 days, the growth was evaluated This study was conducted by Nguyen Xuan Canh et al (2016).
Prepare Simmon citrate agar by combining 5.0 g/L NaCl, 2.0 g/L sodium citrate, 1.0 g/L ammonium dihydrogen phosphate, 1.0 g/L dipotassium phosphate, 0.2 g/L magnesium sulfate, 0.08 g/L bromothymol blue, and 15.0 g/L agar, adjusting the pH to 6.9 ± 0.2 Autoclave 5 ml of the medium at 121ºC for 15 minutes in test tubes After autoclaving, tilt the test tube to create a distinct slant and butt, then inoculate the slant with the provided organism sample.
Incubate the labeled tubes containing sterile wire at 37°C for 5 days A positive citrate test is indicated by a color change in the medium to blue, while a negative result shows no change in color (Rao et al.).
2012) The experiment is repeated three times
The hydrolysis of gelatin was assessed using gelatin agar composed of peptone (5.0 g/L), beef extract (3.0 g/L), and gelatin (120.0 g/L) at a pH of 7 (Dela Cruz & Torres, 2012) The media components were dissolved in hot water, and 8 ml of the medium was poured into test tubes before autoclaving Actinomycetes were then cultured in the medium tubes, with a control sample consisting of a culture tube without Actinomycetes The cultured media were incubated at 30°C for 7-10 days before further analysis.
In a controlled experiment, samples were incubated at 5 °C for 1-2 hours to assess the hydrolysis activity of Actinomycetes The control sample, which had high gelatin content, solidified upon cooling, while the tubes containing liquid remained unfrozen, indicating a positive reaction for hydrolysis activity Conversely, test tubes that solidified like the control sample exhibited a negative reaction This experiment was conducted three times to ensure reliability of the results.
RESULTS AND DISCUSSION
Screening and selection of fungal antagonist Actinomycetes
Thirty Actinomycetes strains, isolated from various sources and preserved in 30% glycerol, were activated on ISP2 medium Their antagonistic potential against the fungus Colletotrichum gloeosporioides was assessed through co-culturing on PDA medium at pH 7, as reported by Yang et al (2019).
Figure 4 1 Screening fungal antagonist Actinomycetes: Control fungus (A),
Dual cultured Actinomycetes (B) after 7 days of culture
From 30 Actinomycetes strains, there was only one strain that was able to be resistant to Colletotrichum gloeosporioides is VNUA48 strain Antagonistic
Actinomycetes demonstrate the ability to inhibit the growth of pathogenic fungi, as evidenced by the formation of an inhibitory zone around the control fungal strain after 7 days of incubation at 30℃ In contrast to the rapid mycelial growth observed in the control dish, co-culture dishes showed a noticeable shrinkage of mycelia on the side opposite the antagonistic Actinomycetes The inhibition rate of mycelial growth was approximately 51.11%, indicating that strain VNUA48 exhibits a strong antagonistic capability against Colletotrichum gloeosporioides, aligning with previous findings by Intra (2011), which reported inhibition rates ranging from 13.8% to 70.4%.
The study revealed that Actinomycetes strain VNUA48 did not directly contact the fungus, indicating that its inhibition of C gloeosporioides is likely due to an antifungal metabolite diffusing in the agar Mycelial growth inhibition was first observed after 2 days, with stronger antagonistic effects noted at 4 days, peaking after 7 days of fungal inoculation.
Actinomycetes strain VNUA48 was selected and tested for further studies.
Morphological characteristics
Colony morphology is a fundamental aspect of Actinomycetes taxonomy The Actinomycetes strain VNUA48 was cultured on various media, including ISP1, ISP2, ISP3, ISP4, and ISP5, to assess its colony characteristics The findings revealed distinct differences in the colony morphology of strain VNUA48 across the different culture media.
Figure 4 2 Colony morphological characteristics of Actinomycetes
VNUA48 in ISP field after 7 days of culture
The morphological characteristics of Actinomycetes strain VNUA48 were visually assessed after 7 days of culture On ISP1 medium, the colonies exhibited a crateriform shape with serrate margins, displaying opalescent aerial mycelium and dark yellow substrate mycelium, along with opaque yellow diffusible pigments Observations on ISP2 medium further contributed to the understanding of the strain's colony morphology.
The Actinomycetes strain VNUA48 exhibited distinct colony morphologies across various media On draughtsman colony surface, it displayed a curled margin, white aerial mycelium, white substrate mycelium, and dark yellow diffusible pigment In contrast, on ISP3 medium, the colonies were characterized by a crateriform surface, serrate margin, opalescent aerial mycelium, yellowish-white substrate mycelium, and no soluble color When observed on ISP4 medium, the strain showed a crateriform surface with an undulate margin, milky white aerial and substrate mycelium, and no diffusible pigment Finally, on ISP5 medium, the colony morphology was umbonate with a scalloped margin, yellowish-white aerial mycelium, milky white substrate hyphae, and no diffusible pigmentation.
Table 4 1 Colony morphology of Actinomycetes strain VNUA48 in ISP field
ISP1 Crateriform Serrate Opalescent Dark yellow Opaque yellow
ISP2 Draughtsman Curled White White Dark yellow
ISP3 Crateriform Serrate Opalescent Milky white No
ISP4 Crateriform Undulate Milky white Milky white No
ISP5 Umbonate Scalloped Yellowish white Milky white No
Spore chains and spores morphology
Figure 4 3 Mycelia and spore chains morphology after 50 hours of culture (A), Spore chain and spore morphology after 72 hours of culture (B)
One of the first criteria to study biological characteristics and taxonomy of Actinomycetes is based on morphological characteristics (Miyadoh et al.,
Strain VNUA48 exhibited both aerial and substrate mycelia, with the aerial mycelia characterized by extensively branched fibers thriving on nutrient medium Optical microscopy revealed that after approximately 50 hours of culture, Actinomycetes strain VNUA48 developed spore production chains at the filament tips, which were flexible By 72 hours of culture, the spores began to detach from the chains and disperse into the medium.
Actinomycetes strain VNUA48 was cultured on ISP 6 medium to assess its melanin pigment production capabilities The experiments demonstrated that this isolate can produce melanin, resulting in a brown-black coloration of the surrounding environment.
Figure 4 4 Actinomycetes strain VNUA48 on ISP 6 medium after 7 days of culture
The study of melanin pigments is significant due to their resemblance to humic compounds found in soil While melanin is not crucial for the growth and development of organisms, it enhances their survival and competitiveness Actinomycetes are particularly advantageous for pigment production, as they thrive on economically viable media and exhibit rapid growth This area of research is emerging and holds various industrial applications.
Ability to assimilate carbon sources
Carbon sources are crucial for antibiotic production, with glucose being a significant carbon source for Actinomycetes and other microorganisms However, excessive glucose can disrupt the synthesis of various antibiotics, illustrating the concept that "too much of a good thing can be bad" (Sanchez & Demain, 2002).
For most Actinomycetes, the commonly used carbon source is starch However, Actinomycetes can make good use of different sugar sources, especially monosaccharides that are easy to use
Based on the evaluation of growth ability of Actinomycetes according to the Internal Streptomyces Project (Shirling & Gottlieb, 1966) With two
The growth of Actinomycetes strains on various carbon sources is assessed through a series of controls Strong positive utilization (++), is indicated when growth in basal media matches or exceeds that in glucose-containing media Positive utilization (+) is observed when growth on the tested carbon source significantly surpasses that on a control medium without carbon, yet remains lower than that on glucose If growth on the tested carbon is only slightly better than on the basal medium without carbon and considerably less than on glucose, it is classified as positive doubtful (±) Lastly, negative utilization (-) occurs when growth is comparable to or less than that on a medium lacking carbon.
Figure 4 5 Growth of Actinomycetes strain VNUA48 on: positive control (A), negative control (B), and basal medium containing starch (C)
Strain VNUA48 demonstrates robust growth on basal media containing lactose and starch, while also effectively utilizing fructose and maltose However, its growth on media with D-xylose and D-sorbitol is limited These findings align with the research conducted by Ahmad et al (2017).
Table 4 2 Assimilation of different carbon sources of Actinomycetes strain
Note: "-" negative utilization; "±" positive doubtful utilization; "+" positive utilization; "++" strongly positive utilization
Ability to produce extracellular enzymes
Plate tests are effective for assessing whether a microorganism produces specific digestive extracellular enzymes In this process, Actinomycetes are streaked on agar and incubated, allowing the release of enzymes that break down macromolecules on the plate A lack of reaction indicates the microorganism does not produce an exoenzyme, while a positive reaction confirms its presence, with the type of enzyme identified based on the hydrolyzed macromolecule These extracellular enzymes play a significant role in biodegradation.
Figure 4 6 Ability to produce Amylase, Cellulase, Chitinase, Pectinase, Protease, Catalase of strain VNUA48 after 7 days of culture
Strain VNUA48 exhibits notable extracellular enzyme production after 7 days of culturing, as indicated by the light rings surrounding its colonies While it can produce various enzymes such as amylase, cellulase, chitinase, pectinase, protease, and catalase, the amylase production is relatively weak, evidenced by the small light ring In contrast, significant amounts of other enzymes are released, as shown by the larger rings around the colonies Additionally, the presence of air bubbles on the surface of a microscope slide confirms that strain VNUA48 can produce catalase.
44 the fungal cell wall is chitin, the ability to produce chitinase may be one of the antifungal mechanisms of this Actinomycetes strain
The results closely align with Al-Dhabi's 2019 study on the production of extracellular enzymes by Actinomycetes species isolated from the root-associated soil of tomato plants.
Effects of temperature, pH and NaCl concentrations on the growth of
Different microorganisms adapt to various environmental conditions, influencing their metabolism and growth This study investigates the environmental factors affecting the growth and development of Actinomycetes strain VNUA48 to provide insights into optimal culture conditions for future research Strain VNUA48 was cultivated on Gause I medium under varying temperatures, pH levels, and salt concentrations The growth and development of strain VNUA48 after five days of culture were evaluated, with results summarized in Table 4.3.
Table 4 3 Effects of environmental conditions on the growth of
Condition Optimal condition range Tolerable condition range
Effect of temperature on the growth of strain VNUA48
Temperature is an important factor affecting the living activities of organisms, especially the ability to biosynthesize biologically active compounds
Research indicates that the Actinomycetes strain VNUA48 thrives in temperatures between 20℃ and 40℃, with an optimal growth temperature of 25℃ to 40℃ These findings align with previous studies by Shekhar et al (2014) and Singh et al (2019).
Effect of pH on the growth of strain VNUA48
Soil and water are strongly modified environments, therefore, these microorganisms are affected by environmental influences, especially Actinomycetes One of the important factors affecting the growth of Actinomycetes is pH
The study revealed that the VNUA48 antagonistic Actinomycetes exhibits a remarkable growth ability across a broad pH range, with optimal growth occurring between pH 5 and 8 It thrives in culture media with pH levels ranging from 4 to 11, although its growth is significantly hindered in strongly alkaline conditions at pH 12 These findings highlight the impressive pH tolerance of the VNUA48 strain, aligning with similar optimal pH results reported by Singh et al.
2019) but these reported Actinomycetes cannot survive in the range pH of greater than 8 Therefore, VNUA48 is superior to these Actinomycetes strains
Effect of NaCl concentrations on the growth of strain VNUA48
Soil salinity significantly impacts the growth and development of soil microorganisms, making it essential to assess the salt tolerance of Actinomycetes strains VNUA48 Research findings indicate that this particular Actinomycetes strain can thrive in culture media with salt concentrations as high as 4% Furthermore, the strain exhibits optimal growth under these conditions.
46 range of salt concentrations from 0 to 2% (Appendix 5) There were similar results between my research and Arasu' research (Arasu et al., 2009).
Ability to utilize citrate
Simmons' citrate agar is a selective medium designed to differentiate gram-negative bacteria based on their ability to utilize citrate as a primary carbon and energy source This agar is essential for identifying organisms that can metabolize citrate, aiding in the classification of various bacterial species.
Citrate usage is a metabolic test that assesses an organism's ability to utilize citrate as its sole carbon source The test employs Simmons citrate agar, which contains citrate, ammonium ions, and the pH indicator bromthymol blue This indicator appears green at pH levels below 7.6 and blue at pH levels above 7.6 In this study, a microorganism-free test tube serves as a negative control.
Figure 4 7 Citrate utilization test of strain VNUA48: Negative control tube
(A), test tube containing strain VNUA48 (B)
The study demonstrated that strain VNUA48 can utilize citrate as its primary carbon source, as indicated by the change in color of citrate agar This ability is characteristic of specific microorganisms that can metabolize citrate rather than relying on fermentable substrates.
The citrate utilization test is selective for certain microorganisms, and while Singh et al (2019) reported that Actinomycetes strains can inhibit the growth of the fungus C gloeosporioides, these strains were not found to utilize citrate In contrast, Actinomycetes strain VNUA48 shows promising potential in this regard.
Ability to hydrolyze gelatin
The gelatin hydrolysis test identifies an organism's ability to produce gelatinase, an enzyme that breaks down gelatin through two sequential reactions Initially, gelatinase degrades gelatin into polypeptides, which are subsequently converted into amino acids These amino acids can then be absorbed by the microbial cells for their metabolic processes The presence of gelatinase is determined using a nutrient gelatin medium.
Figure 4 8 Gelatin hydrolysis test of Actinomycetes strain VNUA48: Negative control tube (A), test tube containing Actinomycetes strain
In this research, a negative control tube without Actinomycetes was used The result showed that VNUA48 had the ability to hydrolyze gelatin because the
The 48 gelatin tube failed to gel and remained liquid in the refrigerator, while the negative control tube solidified This indicates that the nutrient gelatin tube was inoculated with a gelatinase-positive microorganism, which secreted gelatinases that hydrolyzed the gelatin, leading to liquefaction In contrast, a nutrient gelatin medium inoculated with a gelatinase-negative bacterium remained solid after cold treatment Singh et al (2019) reported similar findings, demonstrating that Actinomycetes strains RCS252 and RCS260 possess the ability to hydrolyze gelatin.
Ability to decompose urea
Urease is an enzyme that facilitates the hydrolysis of urea in various organisms, including plants, algae, fungi, and bacteria, leading to its widespread presence in soil This enzyme has been immobilized for use as a biosensor in flow cells, enabling continuous urea measurement in dynamic systems Additionally, urease is utilized alongside urea fertilizers to enhance the hydrolysis of ammonium in the soil.
The urease test identifies an organism's ability to hydrolyze urea through urease enzyme activity During the incubation period, urea is transformed into ammonia, leading to an alkaline environment that causes the phenol red indicator to change to a pink-red hue For accurate results, a negative control should be conducted using a medium devoid of Actinomycetes In contrast, organisms that do not produce urease will show no color change or may turn yellow due to acid production.
Figure 4 9 Urea decomposition test: Negative control tube (A), test tube containing Actinomycetes strain VNUA48 (B)
After 7 days of incubation, the color in Christensen urea agar tubes inoculated Actinomycetes strain VNUA48 was pink Ammonia is produced when urea is degraded The alkalinity caused by the visual color change from orange to pink is detected by the phenol red indicator There are differences between my thesis and (Singh et al , 2019) thesis in which the Actinomycetes strains cannot generate urease.
Classification of the Actinomycetes based on 16S rRNA sequences
4.9.1 Total DNA extraction and concentration
Following the total DNA extraction, the concentration and purification of the DNA were assessed With an A260/A280 ratio of 1.86, the DNA is deemed pure The concentration of DNA in the extracted product is 496 ng/µl.
The total DNA product obtained from extraction was analyzed for size using gel electrophoresis on a 1% agarose gel The results were then documented with a gel scanner and photographed, as shown in Figure 4.10.
50 size of product is more than 10 Kb, with a clear thick band
Figure 4 10 Gel electrophoresis of total DNA extraction of Actinomycetes strain VNUA48 4.9.2 Amplification of 16S rRNA sequences
The amplification of 16S rRNA sequences was achieved through polymerase chain reaction (PCR) using primers 27F and 1492R, with an annealing temperature set at 53℃ Gel electrophoresis on a 1.5% agarose gel confirmed the successful amplification of the 16S rRNA gene sequence region from the VNUA48 Actinomycetes isolate, resulting in a clear band approximately 1500 bp in size (Figure 4.11).
Figure 4 11 Gel electrophoresis of PCR product of Actinomycetes strain
4.9.3 Sequencing PCR products and building a phylogenetic tree
The PCR product was purified and sequenced in Singapore Following the sequencing, the obtained sequence was compared with other sequences in the gene bank using the Blast tool A classification tree for strain VNUA48 was constructed using MEGA11 software, with the results illustrated in Figure 4.12.
Figure 4 12 Phylogenetic tree of Actinomycetes strain VNUA48
Actinomycetes strain VNUA48 is classified within the same clade as Streptomyces sp strain PSKA49, supported by a bootstrap value of 82 Additionally, nucleotide sequencing results indicate a high similarity in the 16S rRNA between Actinomycetes strain VNUA48 and related strains.
Streptomyces sp strain PSKA49 was 99.31% In terms of reliability and similarity, these two strains are similar
In addition to the morphological, physiological and biochemical characteristics studied, I found that the studied Actinomycetes strain VNUA48 has many similarities with Streptomyces sp on the gene bank
Therefore, combining biological characteristics and molecular methods, I conclude that strain VNUA48 is closely related to Streptomyces sp strain PSKA49 and I named this strain as Streptomyces sp strain VNUA48
Streptomyces sp strains have demonstrated effectiveness in inhibiting the mycelium growth of pathogenic fungi, including Colletotrichum gloeosporioids and Curvularia eragrostides, which are known to cause diseases in yam (Soares et al., 2006) Additionally, these strains exhibit antagonistic properties against other harmful fungi, such as Sclerotinia sclerotiorum FW43.
Rhizoctonia solani FW408, Fusarium oxysporum f.sp lactucae L74, Pythium ultimum FW407, Thielaviopsis basicola FW406 and Phytophthora sp FW409