In vitro and In silico investigation deciphering novel antifungal activity of endophyte Bacillus velezensis CBMB205 against Fusarium oxysporum

Abstract

Endophytes from medicinal plants are potential biocontrol agents against Fusarium oxysporum f. sp. cubense (Foc), which is the causative fungus of banana wilt disease. In the present study, the endophytic bacterium was isolated from Globba racemosa and their antagonistic activities against Foc were studied, and the probable molecular mechanism of antagonism was predicted by molecular docking studies. The 16SrRNA sequencing confirmed the endophytic isolate to be Bacillus velezensis CBMB205 (EG2). The antagonistic activities of the isolates by distortion of fungal hyphae were illustrated in SEM. The probable metabolites present in endophytic isolate were identified by FTIR, suggesting the presence of C-H, CH3 and O-H groups. Two major metabolites such as β-amyrin and dihydroxy octadecenoic acid (DA) were confirmed by LC-MS analysis. Molecular docking studies suggested that these metabolites showed potential binding with chitin synthase 1 and fungal 1,3-glucan synthase of pathogenic fungi. The binding energy (BE) of the molecular interaction between β-amyrin and chitin synthase-1 (CS-1), and 1,3-glucan synthase (1,3-GS) were estimated to be -10.17 kcal/mol and − 9.5 kcal/mol, respectively. The BE of the interaction between β-amyrin and CS-1 and 1,3-GS were determined to be -2.43 kcal/mol and 3.4 kcal/mol, respectively. The current study demonstrated the antagonistic activities of EG2 towards Foc and provided a probable molecular mechanism by in silico studies. The study also provides a potential insight into developing endophytic metabolite-based antifungal agents for various agricultural applications.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-77926-1.

Introduction

Bananas are one of the most important tropical fruit crops in the world, ranking fourth in underdeveloped countries as the most widely produced food crop. They serve as both a staple food and a source of income for farmers’ livelihood¹. Since the mid-20th century, the banana industry, particularly Musa acuminata (AAA Group) ‘Gros Michel’ variety cultivar has been severely affected by Fusarium Banana Wilt (FBW), caused by the soil-born fungus- Fusarium oxysporum f. sp. cubense (Foc) race 1. This has been recorded as one of the most destructive plant diseases^2,3. Fusarium oxysporum f. is a fungal pathogen that causes Fusarium wilt, a devastating disease in various plant species⁴. The combined effects of soil nematodes and Fusarium have led to the“Panama Wilt” disease, resulting in a reduction of resistance in banana plants against Foc invasion⁵.

Traditionally, controlling this pathogen has involved the use of chemical fungicides, but concerns are growing about the negative impacts of these chemicals on the environment and human health⁶. Therefore, there is a need for alternative, eco-friendly methods to manage Fusarium wilt. Efforts have been made to develop banana crops with better genetic resistance, in addition to using chemical insecticides and fertilizers to combat pests and diseases. However, the banana industry is currently threatened by the emergence of a new variant of Foc, Topical Race 4 (FocTR4). One of the reasons for the resurgence of new races is the overuse of chemicals and monoculture, which has led to an imbalance in the soil microbiome and increased disease intensity^7–9. The pathogen is characterized by the generation of three types of propagative structure: Microconidia, Macroconidia, and Chlamydospores. The latter are highly resistant double membrane propagules that enable the pathogen to survive for decades in the soil and make it difficult to manage and rendering the soil unusable for planting crops^10,11.

The high demand for bananas has forced the world to produce them in large quantities. It is often argued that using resistant cultivars is the only method for managing this disease¹². However, the pathogen can overcome resistance in just a few years, as seen in the case of the Cavendish cultivar infected with FocTR4. There are no effective control measures other than destroying the infected plant parts to reduce the amount of inoculum in the infected area, thereby maintaining biosecurity by preventing the spread of the fungus. However, the public’s resistance to accepting new plant materials, either due to their organoleptic characteristics or because they are genetically modified, discourages potential solutions^13,14. One of the activities to combat Foc is crop rotation. The rotation of bananas with chillies modifies the physiochemical composition of the soil, enhancing the niches occupied by beneficial soil microbes that might help to prevent F. oxysporum-related wilt disease¹⁵. With suitable soil composition and microbial activity, a pathogen fails to successfully establish itself. The physicochemical changes in the soil are caused by soil suppressors¹⁶. Consequently, the assembly of the soil microbiome, agricultural practices, and the genotype of the plant are critical in protecting plant roots from soil pathogens^17–19. Plant-associated micro-biota thrive in variety of environments such as rhizosphere- the thin layer of soil adhering to the roots, rhizoplane- the root surface, endosphere- the root interior and the inner tissues of the plant²⁰. Each zone is strongly influenced by plant exudates, allowing it to modulate its microbiome even when exposed to stress conditions^14,21–24. It is believed that only a small fraction of the organisms found in the rhizosphere can enter plant tissues and permanently colonize the endosphere²⁵;these micro-organisms are known as endophytes. Continuous research is being conducted to study the potential of endophytes in biological control, as it represents a potential approach to improve the production and sustainability of many crops. The discoveries of microbial potential to promote soil health and crop production offer possible alternatives to cease the spread of infection, which is crucial for food security^26,27.

India, being the country that produces the most variety of bananas worldwide, confirmed the existence of the TR4 race in 2017 and predicted losses of over $7 billion annually^28,29. Africa, the world’s third largest banana producer suffered the infection without spare. Foc TR4 officially reached the continent in 2013; it is reported to have destroyed 15,000 plants every week. The predominance of the microbe was reported to be due to the high costs of tissue culture that lead to disease multiplication^30–32. The discovery of TR4 in Latin American plantations has lately been revealed in La Guajira, Colombia, and the province of Piura, Peru; putting worldwide banana exports at risk, particularly in America^13,33–35. The researchers predict that 1.70 million hectares of banana output will be impacted by 2040 by fusarium invasion³⁶.

Endophytes are microbes that colonize internal plant tissues and coexist symbiotically with their host plant, inflicting no visible harm^37,38. Beneficial plant bacteria that are endophytes or free-living organisms in the soil promote plant growth and shield it from biotic and abiotic threats. This influence is through various mechanisms in exchange for a niche to carry out its life cycle³⁹. Their mutualistic interaction is determined by their site in the plant tissue [ intercellular or intracellular] forming a robust symbiotic relationship that boosts crop yields and provides disease resistance in harsh environments⁴⁰.

Such an association between microorganisms and plants may have existed since plants first arose on earth, allowing these microbes to devise genetic systems and evolve metabolic pathways in plants^41,42. This interaction indirectly stimulates development through mechanisms such as the generation of antibiotics, the reduction of ethylene levels in plants, and the induction of systemic resistance against the attack of pathogens⁴³. Therefore, endophytic bacteria can be considered as plant probiotics⁴⁴. Recent studies have reported the positive role of endophytes of the Bacillus species against phytopathogens of important crops such as rice⁴⁵. Different Bacillus species have confirmed bio control activity against FocTR4 even under field conditions^46–49.

The plants in India have evolved to resist hot temperatures and dry spells because of the country’s hot environment, which averages 32 °C annually. Prior researches indicate that plants grown in these conditions favour for antagonistic effect of plants during pathogen invasion^50,51.

Plants contain endophytic bacteria, which can be recovered from all parts of the plant. These bacteria can create a wide range of secondary metabolites that find application in agriculture, pharmaceuticals, and industrial biotechnology^52,53. The discovery of endophytes associated with medicinal plants offers a great perspective to find new biomolecules and greatly increase the production of secondary metabolites of their host plants⁵⁴, which are essential in the treatment of many human and animal diseases as well as plant pathogens⁵⁵. Simple alkaloids, anthraquinones, phenolics, rotenoids, saponins, and terpenes are among the bioactive molecules responsible for these medicinal properties of plants⁵⁰. These chemicals are either created by the plants themselves or by endophytes. For instance, the medicinal plant – Ficus tikoua endorses a Bacillus endophyte that modulates systemic resistance induced against Rizhoctonia solani in rice plants⁵⁶. The metabolites generated from the isolate of this plant have promising antifungal efficacy against the plant pathogens Rhizoctonia solani and F. oxysporum⁵⁷. The ability of endophytic Bacillus, especially the endophytes from medicinal plants to generate aromatic compounds, lipopeptides, plant hormones, polysaccharides, and various enzymes involved in the metabolism of phenylpropanoids (essential components that provide protection) is commendable. India, as a tropical country with high biodiversity, provides greater opportunities to seek endophytes capable of producing bioactive chemicals that pose favorable effects on plant growth and fitness, as well as crop management tactics^58–61. However, there is diminished effort to understand the action of inhibition by the endophytes. Though the researches are directed toward crop management, target-specific inhibitions are of present focus.

Thus, the current study aims to focus systematic approach of isolation, screening, and characterization of endophytic bacteria from Indian medicinal plants to evince the potential of endophyte as an antagonist towards the fungal pathogen Fusarium oxysporum f. sp cubense (FocTR4). The study also provides a comprehensive overview of the inhibitory action of the isolated endophyte toward FocTR4 by in vitro antagonistic studies and in silico molecular docking studies.

Materials and methods

Fusarium oxysporum

The fungal pathogen Fusarium oxysporum f. sp. cubense (Foc) race 4 was procured from the stock culture of Kuvempu University, Shivamogga Dist., Karnataka, India. Foc was grown on potato dextrose agar (PDA, HIMEDIA) plates and incubated at 27 °C for 4–6 days; this culture plate was treated as stock culture and stored at 4 °C for posterior assays (Fig. 1).

Fig. 1.

Macroscopic (front and reverse) and microscopic characteristics of Foc in PDA plates; (A) Colony of rapid growth, irregularly shaped with white fluffy mycelium which turns pink or purple after a week. (B) Purple central pigmentation with concentric halos turning pink and a white halo on the outside. (C) Septate mycelium and short mono phialides (40X). (D) Macro conidia are slightly moon-shaped cells and microconidia are oval and kidney-shaped (40X).

Isolation of endophyte

Healthy leaves of five medicinal plants were utilized as explants: Pandanus sp., Annona muricata, Nycteanthes arbor-tristis, Ocimum tenuiflorum, and rhizome from Globba racemose (Fig. 2); collected from the surroundings of N.M.A.M. Institute of Technology, State Highway 1, Karkala, Karnataka, India (13°10 × 55.46” N 74°56 × 02.26” E).

Fig. 2.

Medicinal plants as source of endophytes; (1) Pandanus sp. (2) Annona muricata. (3) Nycteanthes arbor-tristis. (4) Globba racemosa. (5) Ocimum tenuiflorum. 

For surface sterilization, the tissue samples were submerged in 70% ethanol for one minute, submerged in 1% sodium hypochlorite for 3 min, and rinsed three times in sterile distilled water⁶². The tissues were macerated in a mortar with saline solution (0.1% NaCl)0.100 µL of the homogenates were serially diluted up to 10⁵ dilutions and spread on a 1%w/v nutrient agar plate (HIMEDIA). This was incubated at 30 °C for 24 h. Distinct colonies were developed after sub-culturing of the plates and each of the bacterial colonies was used for antagonism study in plates and broth (supplementary -Table 1).

In vitro antifungal activity assay

A standard spot inoculation procedure was used to evaluate the capacity of the isolates to inhibit the growth of Foc in vitro⁶³. A phytopathogenic fungal plug (5 mm) was placed gently in the center of the potato dextrose agar (PDA, HIMEDIA) plate. After1.5 days, the isolated bacteria were inoculated at four symmetrical lines about 3 cm apart from the plate center. A PDA plate fortified with a fungal disc was used as a control. After incubation at 28 °C for 5–7 days, the zone of inhibition was recorded by measuring the distance between the fungal mycelium edge and the endophyte. The percentage inhibition of the radial growth (PIRG) was calculated using the following formula:

PIRG = [(C – T)/C] × 100.

where C and T represented the radius of fungal mycelial growth in the control and the treatment groups, respectively⁶⁴.

Following the inhibition activity exhibited against Foc, the colony named- EG2 was selected as a potent strain. The impact of the bacterial antagonism on Foc was examined by microscopic analysis (Olympus CX33, Mag vision) of hyphae morphology in the plates and compared with the control experiments.

Antagonism study in broth

Quantitative evaluation of antagonism by endophytes was measured in potato dextrose broth (PDB) medium. The pure cultures of bacteria and Foc were prepared by inoculating Foc to PDB (48 h) and endophytic strain EG2 in nutrient broth (NB) (24 h) separately. For the antagonism study, 1 mL of each stock culture was inoculated to 50 mL of freshly prepared PDB in conical flasks and grown together at 27 °C on a rotary shaker for 6 days. Broth inoculated with only the fungus served as control. After 6 days of incubation, the differences in biomass between the control and test cultures were determined. Dry weights of biomass were noted⁶⁵.

The percentage reduction in weight in the test culture was calculated using the formula:

(w₁-w₂/w₁) ×100.

Where w₁ represented the biomass in the control culture and w₂ represented the biomass of the test culture. In addition to the dual culture assay, secondary metabolites of the potential endophyte were extracted from liquid media using organic solvents.

Characterization of the isolated endophyte and phylogenetic analysis.

The bacterium with the most effective inhibitory activity (EG2) was identified based on the sequencing of the 16SrRNA gene by amplification using forward primer - ‘AGA GTT TGA TCC TGG CTC AG’ and Reverse primer- ‘CGG TTA CCT TGT TAC GAC TT’ by Sanger di-deoxy Sequencing method. The phylogenetic tree analysis was performed to bracket the ancestral evolution of the isolated bacterium. MEGA 11 was employed for phylogenetic analysis⁶⁶. The inference was drawn by evaluating the sequence by maximum likelihood and neighbor-joining method using the BioNJ algorithm⁶⁷.

Extracellular enzyme assay

The activities of extracellular hydrolytic enzymes produced by the isolate were determined on culture plates by streaking endophyte strain on the medium containing enzyme substrate, incubated at 30 °C. A control plate without the endophyte was compared against the test plate. All experiments were carried out in triplicate.

Protease production assay

Protease production by the isolate was evaluated by using skimmed milk agar. The appearance of a clear zone around the colonies after three days was considered a sign of proteolytic activity⁶⁸.

α-amylase production assay.

α-amylase production was evaluated on starch agar. After 48 h Gram’s iodine solution was applied, exhibiting clear zones around the colony was marked as α-amylase producer⁶⁹.

Lipolytic activity assay

Lipolytic activity was observed on peptone agar medium supplemented with 1.0% Tween. The appearance of opaque halos around the colonies after six days was noted as an indication of lipase production⁷⁰.

Cellulase production assay

Cellulase production was evaluated on carboxymethylcellulose agar (HIMEDIA), After seven days, Gram’s iodine solution was applied, and isolates showing clear halos around the colonies were marked as cellulase positive⁷¹.

Phosphate solubilization assay

Phosphate solubilization was screened on NBRIP agar, strain showing clear halos around the colonies after 14 days was marked as a phosphate solubilizer⁷².

Ammonia production assay

EG2 strain was screened for its ability to produce ammonia by inoculation into tubes containing peptone water. After four days, Nessler’s reagent was added to the tubes and ammonia production was indicated by the development of a yellow to a brown color⁷³.

SEM and FT-IR analysis.

The destruction of fungal hyphae by the compounds released by endophytes was visualized using scanning electron microscopy (SEIZZ). From the fungal culture plate, hyphae of initially grown fungus followed by the introduction of endophytes were sampled for the analysis. The functional group commonalities were investigated amongst the three-broth media (fungus, endophyte, and antagonism) using FT-IR (BRUKER) analysis. The comparison of common functional groups of chemicals released by the three-broth culture revealed the chemical compounds that might have been utilized to prevent the growth of fungus.

Extraction of bioactive compounds for LC-MS analysis.

The endophyte, fungal, and test culture broth were centrifuged at 10,000 rpm at room temperature for 30 min. The supernatant was subjected to acidification at pH-2 and left overnight for precipitation at 4 °C. After centrifugation of the content, the pellet was neutralized with NaOH to pH 7. The treated broth of the endophyte, Foc, and their interaction test culture was homogenized by mixing with 10% methanol. Ethyl acetate and methanol were used as organic solvents in the solvent extraction technique to remove the metabolite. An equal amount of the solvents was added to the filtrate to create two clear, immiscible layers. Using a separating funnel, the Ethyl acetate solvent layer containing the extracted chemicals was separated. The crude metabolite was extracted by evaporating the solvent. This was suspended in methanol and chloroform at a 1:1 ratio⁷⁴. The bioactive sample thus obtained was subjected to LC-MS analysis.

In silico studies

The two major probable molecules identified from the LC-MS analysis were selected and the structure of these compounds was retrieved from the NCBI PubChem databases. The effectual binding of these compounds towards the identified prospective molecular targets of the fungal pathogen was predicted by molecular docking studies to model the molecular mechanism of the probable antagonistic activities.

Preparation of the ligand molecules

The 2D structures of the chemical compounds extracted from the broth cultures were β-amyrin (Fig. 3 (a)) (PubChem CID-73145) and dihydroxyoctadecenoic acid (DA) (PubChem CID-71403936) (Fig. 3 (b)) with the molecular weight of 426.7 g/mol and 314.5 g/mol respectively were retrieved from PubChem database⁷⁵. β-amyrin has a topological polar surface area of 20.2Ų and DA with a topological polar surface area (TPSA) of 77.8Ų. The SMILES of each molecule were utilized as a query by PubChem sketcher V2.4 for drawing the 2D chemical structure of ligand molecules⁷⁶.

Fig. 3.

(a) Structure of β-amyrin (PubChem CID-73145); (b) Structure of dihydroxy octadecenoic acid (PubChem CID-71403936).

Selection of the protein targets

The active chitin synthase-I enzyme from Phytophthora sojae (PsCh1) and fungal 1,3-beta-glucan synthase were chosen as potential targets based on their functional role in molecular docking studies. Fungus is structurally bound by polysaccharides composed of chitin 10—20% of dry weight and glucan which makes up around 50–60% of their dry weight. Additionally, a β-1,3 linkage is present in 65–90% of these glucan polymers⁷⁷. The inhibition of enzymes associated with the synthesis of fungal cell walls is prominent in fungal destruction. As the cell wall is about 40% of the entire fungal structure, the pharmacological target of anti-fungal agents has been the enzymes associated with the cell wall synthesis⁷⁸. The critical steps of chitin biogenesis are catalyzed by chitin synthase enzymes which are membrane-bound glycosyltransferases that translocate GlcNAc units from the UDP-GlcNAc substrate to an extending chitin polymer⁷⁹. Chitin synthase 1 is a dimer of 206.29 kDa with a hydrophobic thickness of 30.2 Å and ΔG transfer of -112.4 kcal/mol⁸⁰. On the other hand, 1,3-beta-glucan synthase is a complex protein with a molecular weight of 224.58 kDa and 1876 residues. A conserved catalytic domain Fks1 that is divided as trans membrane region (TM) responsible for Glucan transportation and a central cytosolic region that adopts a cellulose synthase shape with a cytosolic conserved GT-A–type glycosyltransferase domain⁸¹.

The 3D structure (X-ray crystal structure) of chitin synthase-I (PDBID: 7WJM) and 1,3-beta-glucan synthase (PDBID: 8JZN) were retrieved from the Protein Data Bank database (PDB; http://www.rcsb.org/pdb)⁸². The co-crystalized ligands and water molecules in the structure of the target were removed. The amino acids’ missing bonds were restored using the ‘missing bond’ option in the tools. The binding pockets of these targets were predicted by the CASTp server.

Molecular docking

The effectual binding of the ligand molecules towards the target proteins was modeled by molecular docking studies by AutoDock [82]. The ligands were uploaded in the MGL tools associated with AutoDock tools and ligands were processed for docking studies. The root atoms were identified and the torsion terms were defined. The number of torsions was adjusted and the processed ligands were saved in pdbqt format. The protein targets were also uploaded, polar hydrogen bonds were added, and charges were applied. The prepared targets were saved in pqbqt format. The pdbqt files of the targets and ligands were loaded in the AutoDock tool and the binding sites were set using the Autogrid program. The coordinates files for X, Y, and Z- dimensions were adjusted, and the size of the grid boxes concerning the center was also adjusted. The grid parameter files of the receptor were saved in gpf format. The docking was performed by the Lamarckian genetic algorithm by setting the number of conformations and other default parameters. The output files were saved in dpf format. The Auto grid and AutoDock programs were separately executed and the resultant log files were analyzed and visualized in MGL tools and Discovery studio viewer [83]. Among the many conformations, the best-docked poses were selected based on binding energy (kcal/mol), inhibition constant (nM), ligand efficiency, Intermolecular energy, interacting residues, and number of hydrogen bonds. The interaction patterns were visualized in Discovery Studio visualizer⁸³.

Results and discussion

 In vitro antifungal activity assay

The isolated bacteria from the four medicinal plants were tested for antagonism against Foc. A control plate inoculated with only Foc (Fig. 4, A) was compared to the antagonism plate inoculated with both endophyte and Foc. Proficient inhibition of 40% was observed in a gram-positive bacterium (EG2). Antifungal activity of EG2 against the phytopathogenic fungus on PDA plates (Fig. 4, a) was exhibited as visual growth inhibition after 6 days of incubation. On microscopic visualization at 40X, the foc hyphal morphology appeared intact and normal indicating the uniterrupted growth (Fig. 4, B). Upon observation, the hyphae displayed mycelial deformations with swollen margins loss of cytoplasmic content, and production of intercalary chlamydospores (cl) as a defence mechanism of the fungus (Fig. 4, b). In contrast, the fungal mycelium of the control sample showed normal morphology and was intact without the presence of chlamydospores (Fig. 4, C). Additionally, the fungus produced microconidia that were clearly visible under microscopy (Fig. 4, c).

Fig. 4.

(A) Light microscopy observations of the impact of selected EG2 strain on Foc hyphae morphology (40×): The antagonism study plate inoculated with endophyte and foc (a) Arrows mark the hyphae in the antagonism presented mycelial deformations with swollen margins, loss of cytoplasmic content and, chlamydospore formation (b); fungal mycelium of the control sample showed a normal and intact morphology (B). The endophyte invalidated the production of macroconidia, arrows mark only microconidia (c), while the control maintained abundant macro and microconidia (C).

Fig. 5.

Antagonism in broth: The fungal culture; control (A), inoculated with endophyte (a); Reduction in biomass production by the EG2 endophyte (b), after 6 days compared to the control (B); Flourishing hyphae of the control broth (C), Formation of intercalary double chlamydospores in the presence of the endophyte (c).

The Gram-positive endophyte with antagonistic activity against FocTR4 in vitro, affected fungal morphology and spore formation. Bacillus species have a significant advantage over other beneficial microbes in the realm of biological control due to their ability to generate spores⁸⁴, and they are characterized by rapid growth and the ability to synthesize a large number of secondary metabolites that play a key role in antibiosis against many soil-borne pathogens^85,86.

The bacterial strain EG2 was able to alter the morphology of Foc. Similar findings demonstrate how the genus Bacillus can influence the morphology of phytopathogens fungi in vitro. A Bacillus(V26) employed for in vitro testing resulted in morphological deformations of the fungal hyphae, including vacuolation, protoplast leaking, and mycelia cracking⁸⁷. Similar deformation of the tips of the Foc hyphae was observed in the present study⁶²,wherein it was identified that Bacillus subtilis could deform hyphal tips into spherical structures that were markedly constrained by FocTR4. Besides, the production of chlamydospores is triggered when Foc confronts the endophyte⁸⁸, wherein it has been visualised that the metabolites produced by Bacillus subtilis induce the formation of chlamydospores in Fusarium solani f. sp. radicicola in PDA. This demonstrates that microorganisms in the soil represent a biotic stress for Fusarium species by inducing the formation of survival structures.

Antagonism in broth medium

Endophyte EG2 showed antagonism through pathogen biomass reduction by 95% in the broth (PDB) dual culture assay (Fig. 5A and a) compared to control. The control evidently exhibited higher biomass production after inoculation (Fig. 5B); However, the growth inhibition of Foc by the endophyte was observed on the 6th day of post inoculation (Fig. 5b). The microscopy (Olympus magnus CX-23; 10X) revealed the structure of hyphae growing profusely in control; whereas, the hyphal tips were distorted and observation of chlamydospores were visualized in the presence of endophytes (Fig. 5C and c).

The study of antagonism in broth culture ensued a high percentage of Foc biomass reduction (Biomass obtained: control-0.64 g, antagonism-0.03 g) (Fig. 5B and b), and, as in the solid medium, the production of chlamydospores was triggered. In an antagonism study against Foc, prolonged incubation in PDB containing Fengycin and one of the lipopeptides produced by B. subtilis, it was observed that these intercalary and terminal structures can be released from the hyphae during hyphal lysis, demonstrating that they can survive for decades in soil⁸⁹.

Fig. 6.

Macroscopic and microscopic characteristics of the EG2 strain: (A) Irregularly shaped colony, flat with wavy border and cream color, rough appearance with opaque density and friable consistency. (B) Bacilli Gram positive in 100X. (C) Spore staining; bacterium depicts production of central endospore.

The current antagonism study correlates with previous studies, in which the isolated endophyte from Indian soybean presented an antagonistic capacity towards Foc of 40–54%. Of the six isolated endophytic bacteria, Bacillus amyloliquefaciens (VB7) had the maximum inhibition (70.58%) of the mycelial growth of Foc^90,91. This is noticeable because the endophyte in the present study is closely related to this bacteria phylogenetically. Also, endophytic Bacillus from the Indian medicinal plant Ocimum tenuiflorum L modulates the plant for systemic resistance by induction of resistance against Rizhoctonia solani in rice plants⁹². This study highlights the importance of endophytes to induce defense mechanisms in plants by their influence. The formation of chlamydospore in Foc is one of the indication of inhibition by an external agent (Fig. 5 (c)) reported in a study of Bacillus velezensis EB1 wherein an endophytic bacterium isolated showed significant antagonistic activity against Foc in vitro causing morphological, ultrastructural, and alteration changes in the Foc hyphae with the inhibition rate of 75.43% ⁹³; Whereas, EG2 showed the inhibition up to 95%. A strong antifungal activity against Trichoderma harzianum and Gliocladium roseum wherein chlamydospore production was demonstrated by the bacterium Bacillus subtilis C2 culture filtrate indicates the antifungal potential of the species⁹⁴. The isolated endophyte in this study is phylogenetically in close relation with Bacillus subtilis and exhibited chlamydospore formation from hyphae.

Characterization and identification of endophyte.

Plant sample yielded 14 isolates that were purified and characterized by Gram staining. Isolated endophytes were visualized and a predominance of Gram-positive bacteria (thicker cell wall) was observed. This trend has been studied in sorghum plants; changes were found in the root microbiome under drought conditions²¹ where the Gram-positive bacteria predominated and, on the contrary, under normal irrigation the Gram-negative ones predominated, this could be explained by the fact that Gram-positive bacteria’s much thicker peptidoglycan cell helps them survive under drought stress⁹⁵.

The potential antagonist endophyte EG2 was isolated (Fig. 6) from the rhizome of the medicinal plant Globba racemosa from Indian soil (Fig. 6 (A)). The endophyte was gram-positive (Fig. 6, (B)) and in addition, the EG2 strain could form endospores that enabled it to withstand unfavorable conditions (Fig. 6 (C)). Since the Bacillus species are the most dominant bacterial group isolated in a Foc suppressive soil, they succor in field evaluations⁹⁶. According to recent research, the genus Bacillus has the best performance as possible biological control agents, allowing for more extensive in vivo research and characterization of their interactions with fungal pathogens⁹⁷.

Fig. 8.

a) SEM of Foc after the 5th day of inoculation; (b) fungal hyphae showing the chlamydospore formation after the introduction of endophyte (c) gradual destruction of fungal hyphae due to antagonism (d) the hyphae degraded by the antifungal activity of endophyte

The endophyte EG2 showed the highest similarity with Bacillus velezensis CBMB205n based on nucleotide homology and phylogenetic analysis (Fig. 7). The 16sRNA sequence obtained was submitted to GenBank (Accession number-SUB12387104 SeqID1_EG_2 OP975660).

Fig. 7.

Phylogenetic tree of the isolated endophyte –EG2.

As mentioned in Bacillus velezensis strain CBMB205 showed 99.79% of the homology with the isolate; Bacillus species such as B. amyloliquefaciens, B. vallismortis, B. subtilis are closely related to the endophyte having a similar evolutionary pattern. It is interesting to note that the endophytes belonging to this genus prevail in soil and plant roots and improve plant growth by resisting infections by phytopathogens^46,98–100. The cyclic lipopeptides produced by B. subtilis induce Chlamydospore in Fusarium species¹⁰¹. The secondary metabolites produced from Bacillus amyloliquefaciens such as Bacillomycin and Macrolactin are known to suppress soil-borne pathogens and improve yield by promoting plant growth⁴⁷. Several secondary metabolites released from Bacillus velezensis AK-0 also act as antagonists against apple bitter-rot disease¹⁰². Therefore, all these analyses summarize that the EG2 isolate is closely related to most soilborne microbial bio control agents.

Table 1.

Phylogenetic analysis of EG2 isolate showing closely related species.

Description	Max Score	Total Score	Query Cover	E value	Per. Ident	Accession
Bacillus velezensis strain CBMB205	2580	2580	100%	0.0	99.79%	NR_116240.1
Bacillus velezensis strain FZB42	2573	2573	100%	0.0	99.72%	NR_075005.2
Bacillus amyloliquefaciens strain NBRC 15,535	2573	2573	100%	0.0	99.72%	NR_041455.1
Bacillus vallismortis strain DSM 11,031	2573	2573	100%	0.0	99.72%	NR_024696.1
Bacillus amyloliquefaciens strain MPA 1034	2573	2573	100%	0.0	99.72%	NR_117946.1
Bacillus vallismortis strain NBRC 101,236	2569	2569	100%	0.0	99.64%	NR_113994.1
Bacillus amyloliquefaciens strain NBRC 15,535	2569	2569	100%	0.0	99.64%	NR_112685.1
Bacillus subtilis subsp. subtilis strain 168	2567	2567	100%	0.0	99.64%	NR_102783.2
Bacillus subtilis strain DSM 10	2567	2567	100%	0.0	99.64%	NR_027552.1
Bacillus amyloliquefaciens strain BCRC 11,601	2567	2567	100%	0.0	99.64%	NR_116022.1

SEM analysis:

The Foc and antagonism cultures in a Petri plate (Fig. 4, (A)) were subjected to SEM imaging to confirm the morphological modifications exhibited by Foc against the endophyte. The Foc was allowed to grow profusely for five days, followed by inoculation onto a petri plate. Proliferating fungal hyphae was observed in SEM image at a resolution of 20 μm at a working distance (WD) of 5 mm and a magnification of 1 K X (Fig. 8, a). After the introduction of endophyte to the fungal culture, the hyphae initiated the production of chlamydospore as depicted in Fig. 8 (b) at the resolution of 4KX and WD- 4.8 mm; this observation was also confirmed by the light microscopy (Fig. 5, (c)). The long mesh network of fungus slowly disintegrated due to the release of foreign substances into the media by the endophyte. On observation, the fungal hyphae edge appeared to be dry with degradation (Fig. 8, c). There were distorted and completely damaged structures aiding the inhibitory activity of the endophyte against fungus (Fig. 8, d). The fungal growth was inhibited by the compounds released by the endophyte. The scanning electron microscopy confirmed the destruction of the fungal hyphae by the inhibitory activity of the compounds released by the endophyte.

FT-IR analysis:

The culture broth of Foc, endophyte, and antagonism were subjected to FT-IR analysis respectively.

The presence of intense and broad peaks was observed from 3000 to 3400 cm⁻¹ which was common to the three-culture broth (Fig. 9 (a, b,c)). This corresponds to the presence of O-H and vibrations in the broth that necessarily transuded during solvent extraction of bioactive compounds from the broth¹⁰³. Dominance of endophyte over the Foc proliferation was elucidated explicitly by the peaks at 2947.38 cm⁻¹, 2835.65 cm⁻¹ (Fig. 9, b); 2946.10 cm^−1, and 2833.88 cm⁻¹ (Fig. 9,c). These peaks correspond to the C-H stretching of bioactive compounds in broth¹⁰⁴. Slight drift in the wavenumber was encountered due to the vibration; interestingly, these peaks were unnoticeable in the foc culture medium. Similarly, intense peaks at 1018.62 cm⁻¹, 1015.98 cm^−1, and 1015.50 cm⁻¹ were common to three broth systems. A sharp peak from 1000 to 1600 cm⁻¹ corresponds to the O-C-C stretch present commonly in culture broth¹⁰⁵. Considerably, C = C stretching was found in Foc 1556.42 cm⁻¹ as well as in the antagonism culture broth at 1567.33 cm⁻¹ depicting the similar structure of compounds; further, the alkene stretching was significant in all three broth systems at 1640.99 cm⁻¹, 1656.70 cm⁻1 and 1650.54 cm⁻¹, with shifts in wavenumber as a response to vibration^106,107. From 1400 to 1450 cm⁻¹ CH₂ and CH₃ stretching was observed¹⁰⁸. FT-IR depiction is aided by the fact that endophytes prevailed during antagonism by suppressing the production of fungal compounds that lead to its inhibition.

Fig. 9.

(a) FTIR of Foc culture medium; (b) FTIR of endophyte (EG2) culture broth; (c) FTIR of antagonism culture broth

Enzyme assay

The isolated bacteria EG2 was positive to produce protease (Fig. 10(a)), cellulase, and ammonia (Fig. 10(b)) and negative to produce lipase (Fig. 10(c)) and amylase and phosphate degrading enzymes (Fig. 10(d)). The zone of clearance was evident in the culture plates (Fig. 10). It is interesting to notice that the endophyte was positive for cellulase (Fig. 10(e)) and negative for α-amylase production (Fig. 10(f)) though both the polymers have common monomers. It might be due to the structural difference in the substrate that starch has C₁-C₄ α-glycosidic linkage and cellulose has C₁-C₄ β-glycosidic linkage.

Fig. 14.

LC-MS of DL-2, showing ionization for beta-amyrin

Fig. 10.

Enzyme test results: (a) Protease production; (b) Positive for ammonia degradation; (c) negative for lipase; (d) Negative for phosphate degradation; (e) Cellulase production; (f) negative for Amylase production.

Bacillus is widely used to control plant diseases, especially those diseases caused by soil-borne pathogens. The bacilli can produce protease, amylase, lipase, metabolites and substances such as ammonia, siderophores, hydrogen cyanide, and salicylic acid that degrade the cell wall, leading to altered cell structure and function¹⁰⁹.

Similar to other studies, the production of ammonia has been evidenced by Bacillus spp against Foc, and even Bacillus have been isolated from unusual environments, for example, Bacillus subtilis isolated from the dung microflora produced α-amylases and cellulase inhibiting the growth of Fusarium oxysporum¹¹⁰. On the other hand, a thermo tolerant marine Bacillus, showed ammonia production as well as potent antifungal activity against Foc¹⁴.

The ability of endophytes to produce cellulase and pectinase may provide a host resistance mechanism against pathogen invasion, to obtain nutrition from the host, or to be a latent pathogen¹¹¹. In addition, Bacillus can colonize roots of banana to suppress fusarium wilt under field conditions¹¹², this ability may also be driven by the production of cellulases by the bacteria. A rhizospheric Bacillus showed a high antagonistic activity towards Foc both at the in vitro level (77%) and at the in vivo level with a disease incidence of 10% ¹¹⁴. On the other hand, it was shown that the entry into the plant of the endophytic bacterium Enterobacter JM22 was assisted by the ability of the bacteria to hydrolyze the cellulose attached to the plant cell wall¹¹⁴. Cellulose being the prime constituent of the fungal hyphae, is easily degraded by the bacteria and hence the fungal growth is inhibited. Thus, the ability of endophytic strain EG2, especially of the Bacillus genus, to colonize plant roots and induce resistance to systems such as bio prospecting in the bio control of Fusarium Banana Wilt is demonstrated.

LC-MS Analysis:

The biomolecules present in the broth were determined using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry; Liquid Chromatography (LC) followed by laser desorption ionization and MS (Mass spectroscopy) detection. The compound adducts of the biomolecules [M + H] ²⁺, could also be determined by the generated peaks. Screening of the samples over the entire wavelength range in real time was possible to achieve with DAD.

The chromatogram of liquid chromatography (Figs. 11, 12 and 13) of Foc culture broth (DL-1), Bacteria culture broth (DL-2), and antagonism culture broth (DL-3) depicted the interesting results. DL-2 and DL-3 have three compounds in common as shown in the chromatogram. A common biomolecule at RT-3.91 was produced at a higher amount in DL-3 as compared to DL-1. The compounds with RT-4.12 and 4.23 appear to be common in DL-2&DL-3 but absent in DL-1. This signifies the elimination of the compounds released by the Foc in antagonism culture and proves the bioactives released by the bacteria precede over Foc mycotoxin. According to the DL-1 LC graph, the compound with a retention time of 0.35 covers the major part of the aliquot¹¹⁵.

Fig. 11.

LC-MS of Purified Foc culture broth (DL-1): Liquid chromatogram

Fig. 12.

LC-MS of Purified Endophyte culture broth (DL-2): Liquid chromatogram

Fig. 13.

LC-MS of Purified Antagonism culture broth (DL-3): Liquid chromatogram

The retention values in the graph depict the possible variation in the release of metabolites. By observation, it was determined that few components are commonly present in all three broths such as; the compound at R.T (Retention time) − 3.91 has RI (Relative intensity) of 20–30% in DL-1, whereas, in DL-2, it has a slight shift in R.T of 3.92 RI 30–40% with 1.79% presence; furthermore, in DL-3 the RI increases to 50–60%. A common compound was present at R.T 2.96 which could be the media component used in broth preparation. Another compound at R.T- 4.12 was present in the concentration 3.14% and RI 30% in DL-2; comparatively, in DL-3, 6.69% of the same compound was present at above 50% RI. DL-2 R.T -3.74 nearly 40% DL-3 nearly 60% intensity. From this, it was observed that there is an increase in the amount of the compounds released by the bacteria in the broth during antagonism.

The mycotoxins of Fusarium trichothecenes like T2 toxins, and HT-2 toxins were^116–119. Through LC-MS analysis, the researchers determined that the Foc can produce endotoxins such as fusaric acid (FA) and beauvericin (BEA). 180.13 was determined to be the m/z ratio for FA; 806.47, 801.27, and 784.33 were determined to be the m/z for BEA¹²⁰. It is commonly recognized that the endophyte B. velezensis has antifungal properties. The primary secondary metabolites found as antifungals in B. subtilis are members of the cyclic lipopeptide family, including fengycin and iturin^{5,102,113,121–126}.

Heavy compounds like proteins with high molecular weight posed challenges during interpretation. Owing to the complexity of the compounds from LC-MS, the perceived compounds from the analysis were beta-amyrin (Fig. 14) and dihydroxy octadecenoic acid (Fig. 15) with RT- 2.723 and 2.385 respectively. The fragmentation of the molecule beta-amyrin at 425.3329 m/z and Dihydroxyoctadecenoic acid at 314.1480 m/z indicates their presence in the broth.

Fig. 15.

LC-MS of DL-2, showing ionization for dihydroxy octadecenoic acid

In silico studies.

The molecular basis of the probable antagonistic activities of endophytic isolate by the extracted compounds, probably, beta-amyrin and dihydroxyoctadecenoic acid have been predicted by molecular docking studies. The effectual binding of beta-Amyrin and dihydroxy octadecenoic acid towards the structures of chitin synthase 1 (PDB: 7WJM) and fungal 1,3-beta-glucan synthase (PDB: 8JZN) has been predicted by molecular docking. This interaction model probably provides a structural biology basis for the antagonistic activity of the isolated endophytes towards the fungal pathogens.

The molecular docking studies suggested that the interaction between Beta-Amyrin with chitin synthase 1 from Phytophthora sojae (PDB ID: 7WJM) stabilized with various interactions with a binding energy of -10. 17 kcal/mol. The inhibition constant (nM), ligand efficiency, and intermolecular energy of the interaction were estimated to be 34.98, -0.33, and − 10.47, respectively. The major chains and amino residues that interacted with the ligands were found to be B- Ile 642, B- Ile 643, B - Ile 641, A-ILE 641, B- Gly 646, A-Leu 645, A-Cys 735, A -Ile 731, A-Gly 644, A-Leu 640, A –Ile, 643, A-Met 639, A-Gly 646, B-Leu 645, A - Ile 642, B-Cys735, B-Leu 640, B- Ile 731, B-Met 639, B- Gly 644 and B- Met 639 with the formation of six hydrogen bonds (Table 2). The interaction also stabilized by several weak interactions such as covalent bonds, van der Waals, and alkyl interactions (Fig. 16). The interaction modeling of Beta-Amyrin with fungal 1,3-beta-glucan synthase (PDB ID: 8JZN) is shown in Table 3. The ligand and receptor interaction is stabilized with the binding energy of -9. 5 kcal/mol. The inhibition constant (nM), ligand efficiency, and intermolecular energy were estimated to be 109.25, -0.31, and − 9.8, respectively. The major chains and amino acid residues that stabilized the receptor-ligand complexes were identified to be A-Leu1378, A-Leu1377, A-Val1371, A-Phe1370 A-Gln 1376, A-Val 1375, A-Ile 1373, A-Pro 1372, A-Ala 1386 and A-Phe 1390 with four hydrogen bonds. The 2D interaction diagram and binding pockets are shown in Fig. 17. From the interaction modeling, it is clear that the ligand Beta-Amyrin demonstrated substantial binding with multiple targets of fungal pathogens with stable binding, which might be one of the reasons for the antagonistic activity of the endophytes towards the fungal pathogens.

Table 2.

Interaction of Beta-Amyrin with chitin synthase 1 from Phytophthora sojae (PDB ID: 7WJM).

Ligand	Receptor	Binding energy (kcal/mol)	Inhibition constant (nM)	Ligand efficiency	Inter molecular energy	Interacting residues	Number of hydrogen bonds
Beta-Amyrin	chitin synthase 1	-10.17	34.98	-0.33	-10.47	B- Ile 642, B- Ile 643, B - Ile 641, A-ILE 641, B- Gly 646, A-Leu 645, A-Cys 735, A -Ile 731, A-Gly 644, A-Leu 640, A –Ile, 643, A-Met 639, A-Gly 646, B-Leu 645, A - Ile 642, B-Cys735, B-Leu 640, B- Ile 731, B-Met 639, B- Gly 644, B- Met 639	06

Fig. 16.

(a) The interaction pose of beta-amyrin with showing the interacting residues and the types of bonds involved during docking (b) The 3-D model of the docked chitin synthase 1 molecule with beta-amyrin at the active site.

Table 3.

Interaction of Beta-Amyrin with fungal 1,3-beta-glucan synthase (PDB ID: 8JZN).

Ligand	Receptor	Binding energy (kcal/mol)	Inhibition constant (nM)	Ligand efficiency	Inter molecular energy	Interacting residues	Number of hydrogen bonds
Beta-Amyrin	fungal 1,3-beta-glucan synthase	-9.5	109.25	-0.31	-9.8	A-Leu1378, A-Leu1377, A-Val1371, A-Phe1370 A-Gln 1376, A-Val 1375, A-Ile 1373, A-Pro 1372 A-Ala 1386, A-Phe 1390	04 A-Leu1378, A-Leu1377, A-Val1371, A-Phe1370

Fig. 17.

(a) The interaction pose of beta-amyrin with showing the interacting residues and the types of bonds involved during docking (b) The 3-D model of the docked 1,3-beta-glucan synthase molecule with beta-amyrin at the active site.

The molecular docking studies further illustrated that dihydroxy octadecenoic acid also demonstrated profound effectual binding with both fungal targets. The interaction modeling suggested that dihydroxy octadecenoic acid interacted with chitin synthase 1 and showed an interaction with a binding energy of -2.43 kcal/mol. The inhibition constant (nM), ligand efficiency, and intermolecular energy were estimated to be 16.53, -0.11, and − 7.8, respectively. The chains and major interaction residues were found to be A-Phe 635, A-Leu 634, A-Ala 812, A-Asn 808, A- Pro 589, A- Ala 590, A- Trp 804, A- Ala 805, A- Ile 641, A-Leu 637, A-Leu 809, A-Leu 638, B-Leu 638, B-Leu 634, B-Leu 809 and B- Leu 813 (Table 4). The weak interactions that stabilized in the binding are shown in Fig. 18. Further, dihydroxy octadecenoic acid showed effectual binding with fungal 1,3-beta-glucan synthase with a binding energy of -3.74 kcal/mol. The inhibition constant (nM), ligand efficiency, and inter-molecular energy of the interaction were estimated to be 1.83, -0.17, and − 9.11, respectively. The main interacting residues that are responsible for the binding were found to be Arg1778, Leu1774, Lys1775, Leu1771, Met1770, Phe1489, and Leu1395 with the formation of two hydrogen bonds (Table 5). The best binding pose and other weak interactions responsible for the binding are shown in Fig. 19.

Table 4.

Interaction of dihydroxy octadecenoic acid with chitin synthase 1 from Phytophthora sojae (PDB ID: 7WJM).

Ligand	Receptor	Binding energy (kcal/mol)	Inhibition constant (nM)	Ligand efficiency	Inter molecular energy	Interacting residues	Number of hydrogen bonds
Dihydroxy octadecenoic acid	chitin synthase 1	-2.43	16.53	-0.11	-7.8	A-Phe 635, A-Leu 634, A-Ala 812, A-Asn 808, A- Pro 589, A- Ala 590, A- Trp 804, A- Ala 805, A- Ile 641, A-Leu 637, A-Leu 809, A-Leu 638, B-Leu 638, B-Leu 634, B-Leu 809, B- Leu 813	Nil

Fig. 18.

(a) The interaction pose of Dihydroxyoctadecenoic acid with showing the interacting residues and the types of bonds involved during docking (b) The 3-D model of the docked Chitin synthase-I molecule with Dihydroxyoctadecenoic acid at the active site.

Table 5.

Interaction of dihydroxy octadecenoic acid with fungal 1,3-beta-glucan synthase (PDB ID: 8JZN).

Ligand	Receptor	Binding energy (kcal/mol)	Inhibition constant (nM)	Ligand efficiency	Inter molecular energy	Interacting residues	Number of hydrogen bonds
Dihydroxyoctadecenoic acid	fungal 1,3-beta-glucan synthase	-3.74	1.83	-0.17	-9.11	Arg1778, Leu1774, Lys1775, Leu1771, Met1770, Phe1489, Leu1395	02, Lys1775, Arg1778

Fig. 19.

(a) The interaction pose of beta-amyrin showing the interacting residues and the types of bonds involved during docking (b) The 3-D model of the docked 1,3-beta-glucan synthase molecule with beta-amyrin at the active site

Following the cryo-EM structural snapshots of PsChs1, during the chitin synthesis, there are dimeric and oligomeric CHS complexes being formed⁷⁹. The catalytic domains of the enzyme formed Ile-641, A-Ile-642, B-Ile-642, Leu-645, A-Cys-735, B-Cys-735, and A-Ile-731, B-Ile-731 alkyl interactions and six hydrogen bonds with β-amyrin. The cytosolic domain of the protein seems to have Vander Waals force of attraction with β-amyrin since the binding residues are closely associated with the interaction with Nikkomycin Z at the A-Ile-643, B- Ile-643, A-Met- 639, B-Met-639, A-Leu-640, B-Leu-640 and Gly-646 residues⁸⁰. Since β-amyrin is pentacyclic triterpenoid it offers higher interactions with the ligand with spatial restrictions as well. Interestingly, the binding pattern of dihydroxy octadecenoic acid follows the pattern of captafol (fungicide) in the binding pocket of chitin synthase 1. The residues Ala-805, Trp-804, Ile- 641, Leu-638, Leu-809, Pro-589, Leu- 809, and Leu-638 residues of side chain- A exhibit the alkyl interaction while the similar residues and Asn-808 on B chain are bonded to dihydroxy octadecenoic acid by van der Waals force of attraction¹²⁷.

The enzyme 1,3-beta-glucan synthase has a predominant unit of Fks1 for the synthesis of glucan which is produced during favorable conditions⁸¹. Researchers have determined the mutant active site of Fks1 and molecular architecture to enable the novel binding sites¹²⁸. β-amyrin forms hydrogen bond at A-Leu-1378, A-Glu-1377, A-Val-1371 and A-phe-1370 whereas dihydroxy octadecenoic acid forms hydrogen bonds at A-Arg-1778 and A-Lys-1775. The vander waals interactions exist at the catalytic region with residues at A-Gln-1376, A-Pro-1372, A-Ile-1373, A-Phe-1390, and A-Val-1375 on interaction with β-amyrin. On the other hand, two such bonds are formed by dihydroxy octadecenoic acid at A-Phe-1772, A-Pro-1491, and A-Met-1770. The alkyl interaction of residue A-Ala-1386 was observed for β-amyrin interaction and A-Leu-1771, A-Phe-1489, A-Leu-1395, and A-Leu-1774 were identified in the case of dihydroxy octadecenoic acid (Fig. 17 (a&b), Fig. 19(a&b)). The inhibition constants were 109.25nM and 1.83 nM which suggest the fact that the ligand- protein complex of beta-amyrin dissociates faster than that of dihydroxyoctadecenoic acid.

Previous studies suggested that the interaction models of Kaurane-16,18-diol-acetate and chitin synthase-1 stabilized with a binding energy of -7.0 kcal/mol¹²⁷; Wherein the interaction models of β-amyrin and chitin synthase-1 showed binding energy of -10.17 kcal/mol and the theoretical models of β-amyrin and 1,3-beta glucan synthase showed a BE of -9.5 kcal/mol. Change in the conformation of the catalytic domain residues of cell wall proteins essentially disorients the biosynthesis of cell wall polymers. Thus, there is a crisis in the cell wall production process and hence the fungal hyphae are distorted from production.

From the present theoretical modeling by Insilco studies, it is clear that Beta-Amyrin demonstrated better interaction (BE -10.17 kcal/mol) towards chitin synthase 1 from Phytophthora sojae and 1,3-beta-glucan synthase (BE -9.5 kcal/mol) when compared with the interaction of Beta-Amyrin and chitin synthase 1 (BE -2.43 kcal/mol) and 1,3-beta-glucan synthase (BE -3.74 kcal/mol). Thus, among the two ligands, Beta-Amyrin is probably the potential inhibitor towards chitin synthase 1 and 1,3-beta-glucan synthase acid compared with dihydroxy octadecenoic acid. The more chemical bonds that are engaged in the interaction, the more stable the complex of ligands and receptors. Thus, hydrogen bonds and alkyl bonds formed between beta-amyrin and chitin synthase 1 and 1,3-beta-glucan synthase with several amino acids probably provided better interactions. Nevertheless, the interaction modeling suggested that the presence of these molecules in the endophytes probably showed a substantial role in the antagonistic activity exhibited by the endophytes towards the fungal pathogens. The present study surely offers profound insight into understanding the molecular mechanism of the antagonistic activities exhibited by the isolated endophyte toward the fungal pathogen and the present computational model provides a prototype in further analysis with the aids of molecular dynamic simulation studies and free energy calculation and possible future experimental studies and validation.

The present study revealed that the endophyte EG2 not only releases cellulase but is also involved in the production of secondary metabolite in plants that antagonize fungal invasions. Further, there is evidence that the endophytes from Ocimum sanctum and Aloe vera showed antifungal activity against Rhizoctonia solani and Fusarium oxysporum. Previous studies have evaluated the potential of Indian medicinal plant extracts against Foc and showed that those extracts prepared with solvents exhibited an antagonistic effect^129,130. These studies also showed the potential of endophytic microorganisms to produce various secondary metabolites for crop protection to reduce chemically synthesized fungicides. There have been increasing concerns about the risks associated with using chemical fungicides and hence research is in progress to screen and identify alternative natural products. The present study certainly provides insights for screening natural inhibitory agents by the aid of endophytic organisms against various fungal pathogens.

Conclusion

In the present study, the endophytic strain EG2, phylogenetically identified as Bacillus velezensis CBMB205, a Gram-positive bacteria, isolated from Globba racemosa. It has shown potential as an antagonist against Fusarium oxysporum Foc Race-4 by producing cellulase and secondary metabolites that destruct the cell wall and induce the formation of chlamydospores in the pathogen. This is distinctly revealed by SEM, FT-IR and LC-MS analysis. Two major compounds namely beta-amyrin and dihydroxy octadecenoic acid, were identified from the endophytes and were found to bind effectively to chitin synthase 1 and beta 1,3-glucan synthase of fungal pathogens through molecular docking. The hypothetical model proposed the potential molecular basis of the antagonistic activities of the isolated endophytes. These computational models provide substantial insight into understanding the structural biology mechanism of the antagonism and could serve as a prototype for future experimentation and the development of novel antifungal metabolites of endophytic origin.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Author contributions

Author’s contributions: Ujwal P: Research design, validation of the research data, reviewed the manuscript and editing; Vibha R: Investigation, Study design, Data curation, writing the main manuscript and editing; Daniela Loaiza Granada: Investigation, writing the main manuscript; Sinosh Skariyachan: Molecular docking, data curation, writing and editing the manuscript; Sandesh K: Reviewing and editing the manuscript.

Data availability

The 16SrRNA sequence of the Endophyte obtained was submitted to GenBank (Accession number-SUB12387104 SeqID1_EG_2 OP975660).

Declarations

Competing interests

The authors declare no competing interests.

Conflict of interest

The authors declare no conflict of interest regarding publication of the manuscript.

Ethical approval

The present work does not require ethical clearance.