INTRODUCTION
Introduction
Plant diseases caused by fungi, bacteria, protists, nematodes, and viruses pose significant threats to agriculture and food security These pathogens can infect nearly all plant types, leading to reduced crop yields and substantial economic losses The impact of plant diseases can range from minor damage to complete destruction of crops, resulting in hunger and famine Common ailments include black spots, various leaf spots, powdery mildew, downy mildew, and blight In Vietnam, fungal plant diseases are particularly prevalent, severely affecting both agriculture and the economy.
Fusarium oxysporum, Fusarium solani, and Alternaria alternata
Fusarium oxysporum is a widespread fungus that poses significant challenges to food plant production, particularly through soilborne diseases These diseases occur when pathogens infect plants via their roots, with Fusarium oxysporum being a key representative of such soilborne pathogens This fungus can persist in the soil as chlamydospores for extended periods, infiltrating plant roots and spreading through the tissues It colonizes the xylem vessels, leading to systemic symptoms such as yellowing, wilting, and ultimately plant death (Arie, 2019).
Fusarium oxysporum is a significant pathogen in Vietnam, affecting crops like bananas, cabbage, tomatoes, and peppers, thriving in soil temperatures above 24°C and surviving indefinitely without host plants Infected plants exhibit stunted growth, pale green to golden yellow leaves, and progressive wilting and death, with dark streaks in the xylem and potential root decay Farmers currently depend on pesticides, raising concerns about toxicity to workers, consumers, and the environment An environmentally friendly alternative is the use of beneficial microorganisms for biological control, which this study aims to explore by evaluating bacterial strains with antagonistic activity against Fusarium oxysporum These isolates have shown the ability to inhibit mycelial growth in vitro and reduce disease severity in infected plants Additionally, some bacteria can promote plant growth by synthesizing plant auxins like indole-3-acetic acid (IAA) The promising results from experimental studies highlight the potential of microbial antagonists in managing plant fungal diseases.
This study investigates the potential of native bacterial strains isolated from six soil samples in Northern regions to antagonize Fusarium oxysporum We identified 20 isolates that exhibit strong antagonistic properties against this pathogen while also promoting plant growth These dual characteristics enhance plants' resistance to pathogens, highlighting the biocontrol potential of these antagonistic strains.
Aim and requirements
Isolating and selecting potential bacterial strains with antagonistic activity against fungal pathogens
Isolation of potential bacterial strains can suppress fungal pathogens from six soil samples
Collected bacterial strains with antifungal activity against fungal pathogens
Screening of potential bacterial isolates with antagonistic activity against fungal pathogens with Fusarium oxysporum, Fusarium solani, Alternaria alternata
Selecting potential bacterial strains have antifungal activity against fungal pathogens (Fusarium oxysporum, Fusarium solani, Alternaria alternata)
Characteristic of the potential bacterial strains.
LITERATURE REVIEW
Bacteria with antifungal activity against plant pathogens
Biological control offers an eco-friendly alternative to chemical pesticides, effectively protecting plants from pathogens while minimizing environmental and health risks Soil-borne bacteria that antagonize plant pathogens can significantly help prevent plant diseases, serving as a viable substitute for chemical pesticides in agriculture Numerous biocontrol agents have been identified by screening various soil and plant-associated microorganisms for their antagonistic properties Among these, bacilli and pseudomonads are the most frequently isolated bacterial species from the rhizosphere The Bacillus genus, in particular, is known for its ability to promote plant growth through mechanisms such as enhanced nutrition, systemic resistance, pest toxicity, and pathogen antagonism Many Bacillus isolates exhibit antifungal activity against phytopathogenic fungi, making them promising candidates for biocontrol applications (Mardanova et al., 2017).
Bacillus species exhibit significant antagonistic activity due to their production of antimicrobial peptides, enzymes, proteins, and volatile organic compounds (VOCs), making them promising biocontrol agents against phytopathogenic fungi These gram-positive, rod-shaped, spore-forming bacteria can form endospores and withstand extreme conditions such as varying pH, temperature, and osmotic pressure, providing advantages over other organisms Bacillus sp not only colonizes root surfaces and promotes plant growth but also effectively lyses fungal mycelia, positioning them as safe biological agents with high potential in agriculture Notable strains include Bacillus thuringiensis, Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis.
Bacillus thuringiensis is an aerobic, soil-dwelling bacterium, produce
Bacillus thuringiensis, a gram-positive bacterium, is renowned for its potent insecticidal proteins and is one of the most effective biopesticides against crop pests Recent investigations into the delta-endotoxin of Bacillus thuringiensis subsp thuringiensis revealed its antifungal activity against pathogenic fungi such as Phytophthora and Fusarium, potentially through mechanisms involving the uncoupling of oxidative phosphorylation and respiration The toxin was found to enhance respiratory activity in fungal cultures, similar to effects observed in insect cells exposed to entomopathogenic bacteria The antifungal efficacy of delta-endotoxin was influenced by concentration and exposure duration, particularly against Fusarium oxysporum f sp Lycopersici, a pathogen responsible for wilt and late blight in tomatoes Additionally, delta-endotoxin exhibited cytostatic effects on various economically significant phytopathogenic fungi, including Fusarium, Bipolaris, Phytophthora, Alternaria, and Rhizoctonia Despite its demonstrated effects, the specific mechanisms of delta-endotoxin's antifungal action remain underexplored Further research into the interactions between delta-endotoxins and phytopathogenic fungi is crucial for developing innovative microbial pesticides aimed at controlling plant diseases.
Bacillus subtilis is a nonpathogenic, gram-positive, rod-shaped bacterium known for its heat-resistant spores, commonly found in soil, straw, and grass This bacterium enhances soil quality and promotes plant growth through various mechanisms, including the decomposition of organic matter, nitrogen fixation, and the production of plant growth hormones Additionally, Bacillus subtilis plays a significant role in treating agricultural by-products, producing microbial fertilizers, and providing biocontrol against pathogenic fungi (Hashem et al., 2019).
Bacillus subtilis is known for producing iturin group peptidolipid compounds with antifungal properties, making it effective in the biological control of fungal plant pathogens and peach storage rot Key antifungal compounds in this group include bacillomycin and mycosubtilin, which inhibit spore germination in various fungi A specific strain, Bacillus subtilis ZD01, isolated from potato rhizosphere, has demonstrated significant antifungal effects against Alternaria solani through both in vivo and in vitro experiments This strain produces volatile organic compounds that not only reduce colony size and mycelial penetration but also significantly alter the morphology of the fungus Overall, Bacillus subtilis shows great potential as a biologically synthesized fungicide in agriculture.
Scanning electron microscope (SEM) observation showed that VOCs released by ZD01 could cause more flaccid and gapped hyphae of Alternaria solani
Also, VOCs produced by ZD01 can inhibit the conidia germination and reduce the lesion areas and number of Alternaria solani in vivo significantly (Zhang et al., 2020)
Bacillus amyloliquefaciens is a gram-positive, nonpathogenic, endospore-forming soil bacterium known for its plant growth-promoting abilities and production of antifungal and antibacterial metabolites This beneficial bacterium is extensively utilized in agricultural production to enhance crop health and yield.
+ Increased resistance in adverse environments: cold, drought, especially saline sodic soil
+ Stimulating plant resistance, increasing resistance to pathogens and stimulating plant growth (Chowdhury et al., 2015)
Bacillus amyloliquefaciens is gaining significant research interest due to its wide applications in agriculture, pharmaceuticals, food, and environmental industries This bacterium promotes plant growth through various mechanisms and serves as a biocontrol agent against soil-borne plant diseases It is commonly utilized as a biofertilizer and biopesticide, inhibiting pathogen growth by competing for essential nutrients, producing antibiotics, and inducing systemic acquired resistance For example, volatile organic compounds (VOCs) produced by Bacillus amyloliquefaciens NJN-6 have been shown to impede the growth and spore germination of the pathogenic Fusarium oxysporum f sp cubense, which causes fusarium wilt in bananas.
Bacillus licheniformis is a rod-shaped, gram positive motile bacterium,
Bacillus licheniformis is a facultative anaerobe characterized by colonies that exhibit both round and irregular shapes, featuring undulate and fimbriate margins The surface of these colonies is typically rough and wrinkled, displaying hair-like growths that give them a "licheniform" appearance The color of B licheniformis colonies varies from opaque to white.
Bacillus licheniformis effectively prevents plant diseases and enhances soil quality This beneficial bacterium improves the microbial ecosystem in the soil, leading to increased fertilizer efficiency.
Biological control with antagonists offers a sustainable alternative to pesticides for managing plant diseases Bacillus species are recognized for their ability to produce a range of antibiotics and antifungal compounds that effectively suppress phytopathogenic fungi.
Bacillus licheniformis has been reported to produce different antifungal molecules such as fungimycin M4, which inhibits the growth of fungi such as
Bacillus licheniformis BC98, a bacterial antagonist isolated from soil, demonstrates significant antifungal activity against the rice blast fungus Magnaporthe grisea In addition to inhibiting Magnaporthe grisea, this isolate also effectively suppresses the growth of other phytopathogens, including Curvularia lunata and Rhizoctonia bataticola.
2.1.1.5 The applicability of some strains of Bacillus sp have antagonistic activity against pathogenic fungi and stimulate plant growth
Chemical fungicides are commonly used to manage significant crop diseases caused by pathogens such as Fusarium oxysporum and Rhizoctonia solani However, their long-term use raises concerns regarding exposure risks, health and environmental hazards, and the development of resistance in pathogenic fungi In response, biopesticides have emerged as an eco-friendly alternative for pest control Notably, Bacillus spp has been identified as a beneficial soil bacterium utilized in agriculture Recent advancements have highlighted biocontrol agents like Pseudomonas sp and Bacillus sp for inducing systemic resistance against plant diseases A combination of Bacillus amyloliquefaciens strain IN937a and Bacillus pumilus strain IN937b has demonstrated effective protection against a wide range of pathogens, including cucumber mosaic virus and Ralstonia solanacearum.
Fungal pathogens
Plant fungal pathogens significantly affect the quality, quantity, and profitability of agricultural production, leading to annual losses in the billions of US dollars These persistent phytopathogens evade plant defenses, resulting in widespread diseases To combat these issues, farmers have traditionally relied on fungicides; however, the emergence of resistance and environmental toxicity has prompted researchers and cultivators to seek alternative solutions This study evaluates 20 isolates with antifungal activity using three different fungi.
Fusarium oxysporum, Fusarium solani and Alternaria alternata
Fusarium oxysporum exhibits a variety of mycelial colony colors, including white, pale blue, purple, and light orange when grown on potato dextrose agar The fungus produces unicellular, colorless, oval microconidia on short conidiophores that branch from the mycelium, alongside arched macroconidia Additionally, chlamydospores, which are resilient due to their thickened cell walls, can persist in the soil for several decades after formation.
On PDA Fusarium oxysporum produces:
• A wide range of pigments produced by fungal colonies growing on agar, from colorless to purple to amaranth
• Mycelium is white to purple (Burgess et al., 2009)
Fusarium oxysporum is a cosmopolitan species that are widely spread in all types of soil worldwide Fusarium oxysporum to causing severe vascular wilts and root rot diseases in various crops
• Fusarium oxysporum cultureted in PDA and water agar medium at 30° for 7-10 days
Fusarium solani is characterized by its white, cottony colonies and lateral conidiophore production from aerial hyphae The colonies are low-floccose, loose, slimy, and sporadic, and they develop rapidly on PDA medium This pathogen is responsible for diseases in several economically significant crops, including avocados, citrus, orchids, passion fruit, peas, peppers, potatoes, and squash.
Fusarium solani rots the roots of its host plant
Alternaria alternata is a widespread fungus responsible for leaf spot and various diseases affecting over 380 plant species This opportunistic pathogen leads to leaf spots, rots, and blights, resulting in significant yield losses in agricultural crops It is particularly known for causing black spots on numerous fruits and vegetables globally.
Alternaria alternata thrives in warm, moist environments, commonly found in humid climates or regions with substantial rainfall This fungus resides in seeds and seedlings and is disseminated through spores.
Pathogenicity of Fusarium oxysporum
Fusarium oxysporum strains are major pathogens responsible for Fusarium wilt and other diseases, posing a significant threat to agriculture This fungus is ranked among the top 10 most damaging fungi globally, leading to severe yield losses in affected crops Additionally, certain strains can cause foot- and root-rot, increasing the risk of devastating crop losses.
Fusarium oxysporum pathogens that have evolved (De Lamo & Takken, 2020)
Soilborne plant diseases are those caused by infection of pathogens in soil via the roots Fusarium oxysporum is representative of soilborne pathogens
Fusarium oxysporum survives in the soil as chlamydospores for extended periods, infiltrating plant roots and spreading through tissues This pathogen colonizes xylem vessels, leading to systemic yellowing, wilting, and ultimately plant death The wilt-inducing strains of Fusarium oxysporum inflict significant damage on numerous economically important plant species.
Fusarium oxysporum and its formae speciales lead to several symptoms, including vascular wilt, yellows, corm rot, root rot, and damping-off, with vascular wilt being the most significant.
Fusarium oxysporum is the most important species Strains that are rather poorly specialized may induce yellows, rot, and damping-off, rather than the more severe vascular wilt
As Fusarium wilt is the most important disease caused by Fusarium oxysporum, the focus of this section will be on this symptom The first sign of
Fusarium wilt is characterized by the drooping of older leaves and vein clearing in younger leaves Infected seedlings may wilt and die shortly after symptoms appear, while older plants exhibit vein clearing, leaf epinasty, stunting, and yellowing of lower leaves Additional symptoms include the formation of adventitious roots, wilting of leaves and young stems, defoliation, and marginal necrosis of remaining leaves, ultimately leading to the plant's death Notably, browning of the vascular tissue serves as strong evidence of Fusarium wilt, with symptoms becoming more pronounced during the blossoming and fruit maturation stages.
Control of Fusarium diseases
Current general status of soilborne Fusarium disease control
Controlling soilborne funeral pathogens is challenging once they establish in the field Common methods for managing diseases caused by these pathogens include soil disinfection with fumigants like chloro pickin, hot water, or solarization, as well as the use of resistant cultivars However, the effectiveness of these treatments often falls short of expectations, and there is an increasing need to minimize their use due to environmental concerns.
In order to solve these problems, other techniques for controlling
Fusarium are being developed Here, studies on biological control and inducing disease resistance in plants as alternatives to controlling fusarium diseases are presented
+ Choose disease-free bulbs Seed bulbs need to be selected in the field and need to be stored separately
+ Harvest at the right time When harvesting, it is necessary to collect and separate the wild plants for use first
+ Rotate crops thoroughly from 3-4 years on severely diseased fields with other crops
+ Planting trees on high ground, easy to drain, free to drain
+ Apply lime powder to raise soil pH on acidic soil
+ Fertilize with decaying organic fertilizers
+ Use biological products Bacte Cinsan to spray to prevent diseases at the beginning of the rainy season, kill pathogens or spray treatment when plants are sick…
+ Use some of the following drugs to prevent: Ningnanmycin (Niclosat
2SL); Trichoderma viride (Biobus 1.00 WP); COPPER-B 75WP; Prevent root nematodes (creating a "gateway" for fungal attack) by periodically watering
Trichoderma Bacillus & EM HLC probiotics that treat nematodes from seedling stage to harvest stage to help prevent diseases Root rot, green wilt, yellow wilt, etc
Figure 2.1: Some probiotics are applied from bacillus strains with antifungal
(Source: Vinong.net Vietnamnongnghiepsach.com.vn)
The primary approach to managing Fusarium infections has traditionally relied on chemical fungicides; however, the use of biological control agents offers a more effective, safe, and sustainable alternative Beneficial Bacillus strains are particularly promising due to their spore-forming nature, which facilitates easy formulation and preservation as inoculants These bacteria can produce various metabolites that enhance plant growth and bolster the plant's immune response against pathogens, either by inhibiting fungal growth or stimulating defense mechanisms Additionally, microbial consortiums can effectively reduce plant diseases, enhance crop growth, and promote environmental health, ultimately leading to improved crop production (Khan et al., 2017).
Studies of bacteria with antifungal activity against plant pathogens
The application of Bacillus sp species in combating Fusarium wilt, caused by Fusarium oxysporum, is gaining significant attention among researchers Numerous studies have highlighted their effectiveness in the biocontrol of this plant disease.
A study isolated 57 antagonistic bacterial strains from the rhizospheres of healthy banana plants in a wilt-diseased field, selecting six strains with superior survival abilities for further investigation The W19 strain significantly reduced Fusarium wilt incidence and enhanced banana plant growth when combined with organic fertilizer Identified as Bacillus amyloliquefaciens through various analyses, W19 produced antifungal lipopeptides, including iturin and bacillomycin D, as well as surfactin Additionally, 18 volatile antifungal compounds with strong antagonistic effects against Fusarium oxysporum were identified This research underscores the potential of bioorganic fertilizers containing Bacillus amyloliquefaciens for controlling Fusarium wilt in bananas and explores the mechanisms of biocontrol.
Combinatorial effect of endophytic and plant growth promoting rhizobacteria against wilt disease of Capsicum annum L caused by Fusarium solani Research shows that rhizobacterial strains (Pseudomonas fluorescens;
Pf1) against chilli wilt disease caused by Fusarium solani (Sundaramoorthy et al., 2012).
MATERIALS AND METHODS
Materials
3.1.1 The culture medium was used as follows
To isolate, screen, and evaluate bacteria that are resistant to fungal diseases, we use some medium types: NA, PDA, SDA, and LB
Medium PDA (Potato Dextrose Agar): Potato Dextrose Agar—39 g, Water—1 L, pH = 7
Medium NA (Nutrient agar): Peptone—5 g, Beef Extract—3 g, Sodium Chloride—5 g, Agar—20 g, Water—1 L, pH = 7
Medium LB (Luria Bertani): Tryptone—10 g, Yeast Extract—5 g, Sodium Chloride—10 g, Water—1 L, Agar—15 g pH = 7
Medium SDA (Sabouraud Dextrose Agar): Agar—20 g, Sabourand Dextrose broth—30 g, Water—1 L
Medium water agar: Agar—20 g, Water—1 L
Medium LB + L-trytophan: Tryptone—10 g, Yeast Extract—5 g, Sodium Chloride—10 g, Water—1 L, Agar—15 g, L-Trytophan (100mg/L) pH = 7
To screen for bacteria that are resistant to plant fungal diseases, we utilized three fungal pathogens: Fusarium oxysporum, Fusarium solani, and Alternaria alternata, all sourced from the Faculty of Biotechnology at Vietnam National University of Agriculture These fungal pathogens were cultured on potato dextrose agar (PDA) medium and kept at room temperature in the dark.
Fusarium oxysporum: In the laboratory, we cultured on PDA medium at
Figure 3.1: Fusarium oxysporum (The photo was taken at the laboratory of the Department of Plant
Biotechnology 15/11/2022) Fusarium solani: In the laboratory, we cultured on PDA medium at 30°C in the darkness
Figure 3.2: Fusarium solani (The photo was taken at the laboratory of the Department of Plant
Alternaria alternata: In the laboratory, we cultured on PDA medium at
Figure 3.3 Alternaria alternata (The photo was taken at the laboratory of the Department of Plant
From 6 different soil samples in Northern regions: Ha Tay, Tuyen Quang, Quang Ninh, Hung Yen, Tue Vien, Thai Binh
Figure 3.4: 6 soil samples (The photo was taken at the laboratory of the Department of Plant Biotechnology 15/11/2022)
+ Schott duran, pipette, eppendorf, Micro Centrifuge Tubes, sieve, Petri dish
+ Magnetic Stirrer, centrifugal machine, laminar airflow cabinet, Vertical loading steam sterilizer…
Chemical: Distilled water, Sterile physiological salt solution (NaCl- 0.85%), glycerol, Fuschine, Lugol, Salkowski test, IAA, Gentian violet…
Methods
The Dilution Plate Method involves sifting soil through a 2 mm sieve and weighing one gram of each of six samples in sterile containers Each sample is added to 90 ml of sterile physiological salt solution (NaCl-0.85%) in a 250 ml Erlenmeyer flask and shaken for at least one minute A sterile pipette is then used to transfer 1 ml of this solution into a flask containing 9 ml of NaCl (0.85%) to achieve a dilution of 10^-3 This process is repeated by transferring 1 ml from the 10^-3 solution into another flask with 9 ml of NaCl (0.85%) to create a 10^-4 dilution Subsequently, 0.1 ml from each dilution is pipetted onto two Petri plates containing NA medium, spreading the solution evenly with a sterile glass stirring rod, starting with the 10^-4 dilution The Petri plates are sealed with Parafilm and incubated at 30°C, with observations made after 24 and 48 hours.
Figure 3.5: Soil sample dilution and inoculation
The Gram stain is a crucial staining technique in microbiology, primarily used to differentiate between gram-positive and gram-negative organisms This differential stain relies on the distinct characteristics of the cell walls of these organisms, which influence how they absorb and retain stains.
Gram staining process (applied process of the Faculty of Biotechnology, Vietnam National University of Agriculture):
- Put a drop of distilled water on the microscope slide slide
- Use a toothpick to dip a stain of bacteria to be stained, then dip it into a small water stain on the microscope slide
- Fix the slide over the flame of the alcohol lamp while stirring with a toothpick until the water dries to form a sterile ring on the microscope slide
- Drop Gentian violet for 1 minute and then rinse with distilled water
- Continue to drip Lugol solution for 1 minute and then rinse with distilled water
- Rinse with alcohol 70° for 30 seconds, then rinse with distilled water
- Then drop Fuchsin solution for 30 seconds and then rinse with distilled water
- Finally, the initial slide examination should use the X40 objective to evaluate the smear distribution, and then they should be examined using the X100 oil immersion objective
Monitoring indicators: Gram (+) bacteria will be purple, Gram (-) bacteria will be pink
(Source: theory.labster.com) 3.2.3 Catalase test
The catalase test is a crucial method for distinguishing staphylococci, which are catalase-positive, from streptococci, which are catalase-negative This test identifies the presence of the enzyme catalase, produced by bacteria that utilize oxygen for respiration, thereby protecting them from harmful by-products of oxygen metabolism Catalase-positive bacteria encompass both strict aerobes and facultative anaerobes, while catalase-negative bacteria may be anaerobes or facultative anaerobes that rely solely on fermentation without using oxygen as a terminal electron acceptor.
3.2.4 Determination of indole -3-acetic acid (IAA) synthesis capability
Rhizobacteria from plant rhizospheres produce phytohormones, with indole-3-acetic acid (IAA) being the most prevalent auxin IAA, a derivative of indole, features a carboxymethyl substituent The Salkowski reagent method is commonly employed to detect indole compounds, particularly IAA, from microorganisms This method utilizes a mixture of 0.5M ferric chloride (FeCl3) and 98% sulfuric acid (H2SO4), which reacts with IAA to produce a pink color, indicating the formation of an IAA complex and the reduction of Fe³⁺ The resulting color signifies the presence of various indole compounds derived from tryptophan metabolism.
To cultivate bacterial strains, use liquid LB medium supplemented with L-tryptophan (100 mg/L) and incubate at 200 rpm in the dark for 72 hours to prevent IAA degradation by light After incubation, centrifuge the culture solution at 10,000 rpm for 10 minutes at 4°C, then transfer 1 ml of the supernatant into a test tube with Salkowski reagent Incubate this mixture for 20 minutes; a pink color indicates the presence of IAA The IAA content is quantified using the Salkowski colorimetric method at a wavelength of 530 nm, and a standard curve is constructed to analyze the relationship between the OD530 value and IAA concentration.
Concentration of working standard solution IAA
Volume of IAA stock standard solution
1000àg/mL in each 10 ml volumetric flask
Using 10 test tubes containing Salkowski, 2ml of IAA solution concentration from 0 to 90àg/mL respectively Measure the color of the IAA standard scale on a spectrophotometer, at 530 nm, the standard curve has the form y=ax+b (y is the OD value, x is the amount of IAA obtained)
3.2.5 Screening of potential bacteria isolates with antagonistic activity against fungal pathogens (Rahman MA, 2009)
The antifungal activity of 20 isolates against fungal pathogens was evaluated using Petri dish assays Briefly, 20 bateria isolates was cultured on
Fungal pathogens were cultured on PDA plates at 30°C for 7 days, followed by the use of a 5mm diameter jelly tip puncher to transfer the fungal culture to a new Petri dish containing PDA medium, ensuring a distance of 3 cm from the plate edge Antagonistic bacterial strains were inoculated 3 cm away from the fungal culture, with an inoculation line measuring 4.5 cm Control experiments included plates inoculated with fungal pathogens without bacterial isolates All samples were incubated at 30°C for 7 days, and the experiment was organized using a Completely Randomized Design (CRD), with each treatment repeated three times.
R1: (A control value) represents the radial growth of fungus in control sets (cm) R2: The radial growth of the fungus in sets with bacteria (cm)
PIRG: Percentage of inhibition of radial growth of bacteria against fungal (%)
RESULTS AND DISCUSSION
Results
4.1.1 Experiment 1: Isolating and screening bacteria isolates with antifungal activity
The serial dilution technique was employed to isolate bacteria using aqueous dilutions of 10^-3 and 10^-4 with 0.85% sodium chloride Samples from each dilution were streaked onto Sabouraud Dextrose Agar (SDA) plates, and after 24 hours, isolates with the potential to inhibit fungal diseases were collected Since July 2022, 86 bacterial isolates from six different soil samples, including those from vegetables, jackfruit, ming aralia, and water spinach, were identified as antagonists to selected plant pathogenic fungi We screened 20 of these bacterial strains for antifungal activity against Fusarium oxysporum and cultured the isolates on NA medium at 30°C For long-term preservation, the bacterial strains were stored at -70˚C in NA-broth with glycerol.
Table 4.1: Summarization of potential bacteria isolates with antifungal activity
Host plant The number of isolates screening
The number of potential isolates
1 Ha Tay Vegetables 17 3 HT1, HT2, HT4
Figure 4.1: The number of bacteria selected isolates from 6 soil samples 4.1.2 Experiment 2: Screening of potential bacteria isolates with antagonistic activity against fungal pathogens
In an experiment, mycelial discs were cultured on potato dextrose agar plates at 30℃ for 7 days A disc was placed 4.5 cm from the edge of a fresh plate, while bacterial strains were inoculated 1.5 cm from the opposite edge After a 7-day incubation at 30℃, inhibition zones were measured This dual culture method led to the isolation of 20 antagonistic bacteria from soil, which were subsequently selected for screening against Fusarium oxysporum.
Figure 4.3: Antagonistic activity of screening isolates against Fusarium oxysporum
4.1.3 Experiment 3: Evaluation of 20 potential isolates with antifungal activity against fungal pathogens
Table 4.2: Effects of bacteria strains on the mycelial growth inhibition to
No Isolates Mycelial growth inhibition (%)
After 3 days After 5 days After 7 days
The study revealed that mycelial growth inhibition percentages varied between 14% and 54% (Table 4.2) Notably, the isolates HT1, HT2, TV2.1, TV2.5, TV2.11, and TV2.12 exhibited the most potent antifungal activity.
The HT2 strain of Fusarium oxysporum demonstrated the highest mycelial growth inhibition, exceeding 52% among 20 evaluated isolates After 3 days of dual culture, the HT2 strain achieved a 16% inhibition rate, which increased to 54% after 5-7 days In contrast, the M10 strain exhibited the lowest effect, with less than 15% inhibition of colony growth in two tested fungi.
Research by Rania Aydi Ben Abdallah demonstrated that Alcaligenes faecalis (S18) and Bacillus cereus (S42) effectively inhibit the growth of Fusarium oxysporum, with mycelial growth reductions of 44.1% and 42.1%, respectively.
S42 and S18 isolates, respectively (Abdallah et al., 2016) Thus, HT2, TV2.11 and TV2.12 strains have higher antifungal activity against Fusarium oxysporum than the two strains S18 and S42 (>45%)
Figure 4.4: Ten isolates showed strongest antifungal activity against
Table 4.3: Effects of bacteria strains on the mycelial growth inhibition to
No Isolate Mycelial growth inhibition (%)
After 3 days After 5 days After 7 days
The results showed that the percentage of mycelial growth inhibition ranged from 49% to 67% (Table 4.3) We can see that isolates: HT4, QN1, QN3,
Strains TV2.5, TV2.12, and NA15 exhibited the strongest antifungal activity against Fusarium solani, with inhibition rates ranging from 60.59% to 67.37% Notably, strain TV2.5 demonstrated the highest efficacy, achieving over 67% inhibition of mycelial growth among the 20 isolates tested Additionally, TV2.5 inhibited Fusarium oxysporum by 44% after 3 days of dual culture, with inhibition rates increasing to 67% after 5-7 days In contrast, strain TV2.9 displayed the lowest antifungal effect, with less than 49% inhibition of colony growth in two of the fungi tested Overall, all 20 isolates exhibited significant antifungal activity.
In the research of Nailea Báez-Vallejo, two bacterial strains, Bacillus sp
CCeRi1-002 and Pseudomonas sp CCeRi5-020, significantly inhibited Fusarium solani mycelial growth, by 50.6 % and 64.7 % (Abdallah et al., 2016)
Thus, HT4, QN1, QN3 and TV2.5 strains have higher antifungal activity against
Fusarium solani than the two strains CCeRi1-002 and CCeRi5-020 (>62%)
Figure 4.5: Ten isolates showed strongest antifungal activity against
Table 4.4: Effects of bacteria strains on the mycelial growth inhibition to
No Isolates Mycelial growth inhibition (%)
After 3 days After 5 days After 7 days
The results showed that the percentage of mycelial growth inhibition ranged from 51% to 62.68% (Table 4.4) We can see that isolates: : HT1, HT2,
NA6, DL10, TV2.5, TV2.11 have the strongest antifungal activity against
Alternaria alternata (61.11% - 62.68%) Moreover, the strain TV2.11 presented the highest activity, causing more than 62% inhibition of mycelial growth among the 20 evaluated isolates For TV2.11 strain, the ability to inhibit
After three days of dual culture, Fusarium oxysporum exhibited a 2% inhibition rate, which gradually increased to 62% after 5-7 days In contrast, strain M10 demonstrated the lowest effect, with less than 52% inhibition of colony growth in two tested fungi Notably, all 20 isolates displayed strong antifungal activity against Alternaria alternata, exceeding 51%.
In the research of Nailea Báez-Vallejo, Bacillus siamensis, strain LZ88, exhibited strong antifungal activity with an inhibition rate of 81.96% (Xie et al.,
2021) Thus, isolated strains have lower antifungal activity against Alternaria alternata than strain LZ88 (49%, HT2 >54%, QN3
TV2.11, TV2.12, and HT4 are gram-positive strains with strong antifungal activity against fungal pathogens, exhibiting efficacy rates of over 35%, 45%, and 52%, respectively Additionally, these strains possess a high capability for synthesizing indole-3-acetic acid (IAA) Therefore, they can be effectively utilized as bioinoculants in agriculture for enhancing the growth of important crops.
Discussion
Utilizing beneficial bacteria in agriculture and disrupted ecosystems can effectively safeguard crops from phytopathogens These plant-associated bacteria not only promote growth and development but also inhibit the growth of harmful microorganisms To enhance biological control methods and combat plant diseases, it is essential to isolate new antagonistic bacterial strains Various bacterial strains demonstrate a suppressive effect on specific phytopathogens, making them viable biocontrol agents.
In this study, 20 strains exhibiting strong antagonistic activity against fungal pathogens were isolated from six distinct soil samples Among these, strains TV2.12, HT2, TV2.5, TV2.11, and QN3 demonstrated superior effectiveness in inhibiting the growth of various fungal pathogens Notably, significant alterations in mycelium morphology were observed, with bacteria causing irregular, distorted, and shrunken structures compared to the control Additionally, these top-performing strains exhibited the highest capability for indole-3-acetic acid (IAA) synthesis.
CONCLUSION AND PROPOSAL
Conclusion
Eighty-six bacterial isolates were assessed for their antifungal activity against Fusarium oxysporum, leading to the selection of twenty isolates that exhibited strong antagonism These twenty isolates were further tested for their antifungal efficacy against additional fungi, including Fusarium solani and Alternaria alternata, over periods of 3, 5, and 7 days Among these, the strain HT2 demonstrated the highest antifungal activity against Fusarium oxysporum, achieving over 52% inhibition of mycelial growth.
Fusarium solani, the strain TV2.5 presented the highest activity, causing more than 67% inhibition of mycelial growth among the 20 evaluated isolates For
The strain TV2.11 of Alternaria alternate demonstrated the highest activity, inhibiting over 62% of mycelial growth among the 20 isolates evaluated Additionally, most isolated strains exhibited the ability to produce catalase and synthesize indole-3-acetic acid (IAA).
We have identified twenty novel bacterial strains exhibiting varying levels of antagonistic activity against several phytopathogenic fungi Additionally, these strains possess the ability to promote plant growth through the production of indole-3-acetic acid (IAA) Consequently, these microorganisms hold promise for use as bioinoculants in agriculture, particularly as biopesticides for important crops.
Recommendations
For agricultural applications, the evaluation of their activity in vivo need to study in the future
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