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Article

Eco-Friendly Biomass Production and Identification of Active Compounds of Paenibacillus polymyxa EB.KN35 with Potent Anti-Fusarium oxysporum Effect

by
Van Anh Ngo
1,†,
Anh Dzung Nguyen
1,†,
San-Lang Wang
2,*,
Tu Quy Phan
3,
Thi Ha Trang Tran
1,
Dinh Sy Nguyen
3 and
Van Bon Nguyen
1,*
1
Institute of Biotechnology and Environment, Tay Nguyen University, Buon Ma Thuot 630000, Vietnam
2
Department of Chemistry, Tamkang University, New Taipei City 25137, Taiwan
3
Department of Science and Technology, Tay Nguyen University, Buon Ma Thuot 630000, Vietnam
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Microorganisms 2025, 13(4), 800; https://doi.org/10.3390/microorganisms13040800
Submission received: 12 February 2025 / Revised: 16 March 2025 / Accepted: 28 March 2025 / Published: 31 March 2025
(This article belongs to the Special Issue Microorganisms: A Way Forward for Sustainable Development?)
Browse Figures
Versions Notes

Abstract

:
Fusarium oxysporum is a fungal plant pathogen for over 100 agricultural crop species. There are strategies for managing Fusarium wilt, including antagonistic bacteria that offer a promising and sustainable effect. In this work, among the various endophytic bacterial strains, Paenibacillus polymyxa EB.KN35 was selected as the best antifungal strain against F. oxysporum. For eco-friendly biomass production of this bacterium, some agricultural byproducts were tested for cultivation, and a soybean processing byproduct (SPBP) was found to be a suitable C/N source for P. polymyxa EB.KN35 fermentation. The utilization of a 14 L bioreactor system for P. polymyxa EB.KN35 fermentation achieved a high biomass productivity (3.46 × 1011 CFU/mL) in a short time (8 h). In bioactive compound analysis, EB.KN35 was found to be secreting several plant growth-promoting compounds such as GA3, IAA, kinetin, and zeatin (via HPLC) and eleven volatile compounds (via GC–MS). The docking study indicated that some volatile compounds (1, 2, 4, and 9) may play a significant role in inhibiting F. oxysporum. The study results highlight the potential for reusing a soybean processing byproduct as a C/N source for the bioproduction of P. polymyxa EB.KN35 with potential use as a biocontrol agent and biofertilizer.

Graphical Abstract

1. Introduction

Fusarium oxysporum is a soil-borne ascomycete fungus ranked fifth among the most destructive plant pathogenic fungi worldwide and seriously harms more than 100 major agricultural crop species [1,2,3]. F. oxysporum produces chlamydospores that can survive in infected soil for decades [4]. Current strategies for managing Fusarium wilt include cultural practices, chemical treatments, and biological control. Among these, microbial biocontrol agents, such as antagonistic bacteria, offer a promising and sustainable alternative [5,6,7,8,9,10]. Beneficial microbes have various defense mechanisms against pathogens, including direct antagonism, indirect inhibition of pathogens through secondary compounds, antibiotics, or cell wall-degrading enzymes, as well as competition with pathogens [11,12]. In addition, beneficial bacteria also help stimulate plant growth and enhance the sustainability of agroecosystems [13,14].
Paenibacillus polymyxa is an effective biocontrol agent for sustainable crop production. P. polymyxa has been proven to be an effective fungicidal bioagent against various pathogens such as Verticillium longisporum, Fusarium sp., Ralstonia solanacearum, etc. [15,16,17,18,19,20,21,22,23,24]. Notably, P. polymyxa has strong potential against F. oxysporum, causing diseases in watermelon, cucumber, rape, Cryptomeria fortunei, and Urtica dioica [17,18,19,20,21,22,23,24]. However, its potential against F. oxysporum disease in durian plants has not been recorded. P. polymyxa has gained interest in mass production via fermentation for application. Almost all the previous studies used commercial media for cultivation. For cost-effective production, some byproducts have been used in P. polymyxa fermentation, such as sweet potato starch wastewater, molasses, pear bagasse, wheat bran, etc. [25,26,27,28,29,30,31,32,33,34,35]. In recent years, soybean flour has been used in P. polymyxa fermentation. However, the reuse potential of its byproduct has not been considered [33]. In particular, assessing some common byproducts such as soybean processing byproducts, rice bran processing byproducts, and cassava processing byproducts has not been reported. In addition, almost all the previous works [25,26,27,28,29,30,31,32,33,34,35] reported biomass production on a small scale using flasks. The bioactive compounds produced by this bacterial strain and its mechanism of interaction with F. oxysporum trypsin are also not well-studied.
Protein trypsin-like proteases (1TRY) targeting F. oxysporum, also known as triosephosphate isomerase, are crucial metabolic enzymes involved in glycolysis and gluconeogenesis. In Fusarium oxysporum, 1TRY is an important enzyme with multiple functions. It is a key metabolic enzyme, and it also contributes to fungal virulence. In fungal pathogenicity, 1TRY plays a role in immune evasion, oxidative stress resistance, host cell interaction, and host adaptation. These abilities make 1TRY a promising target for antifungal drug development [36,37,38].
In this study, among the various tested endophytic bacterial strains, P. polymyxa EB.KN35 was screened as the most promising strain for the biocontrol of F. oxysporum. Regarding the eco-friendly production of this potential strain, various agricultural processing byproducts were used as C/N sources for fermentation. P. polymyxa EB.KN35 was further tested for fermentation on a larger scale using a 14 L bioreactor system. In addition, the bioactive compounds produced by P. polymyxa EB.KN35 were identified using GC–MS and HPLC. The molecular docking study and DPT calculation were also conducted to study the interaction mechanism of bioactive compounds with protein 1TRY, targeting F. oxysporum.

2. Materials and Methods

2.1. Materials

The pathogenic fungus Fusarium oxysporum and 110 endophytic bacterial strains were stored at the Institute of Biotechnology and Environment—Tay Nguyen University, Vietnam. PDA medium was used for fungal activation, and LB medium was used for bacterial activation at 28 °C. Soybean processing byproducts (SPBPs), rice bran processing byproducts (RBPBPs), and cassava processing byproducts (CPBPs) were collected in Buon Ma Thuot, Dak Lak, Vietnam.

2.2. Methods

2.2.1. Endophytic Bacterial Strains Screening

The antifungal activity of all the endophytic bacterial strains was evaluated according to the method of Ngo et al. [39]. According to this method, the pathogenic fungus F. oxysporum was placed in the middle of a Petri dish, the antagonistic endophytic bacteria were inoculated 3 cm away from the pathogenic fungus with a 1 cm bacterial inoculation line, the activity evaluation medium was PDA medium, and each experiment was repeated 3 times. The experimental plates were incubated at 30 °C until the front edge of the F. oxysporum fungus in the control group reached the edge of the plate. For the control group, only the pathogenic fungus was inoculated on the plate.
The growth inhibition activity was calculated as follows:
The inhibition rate of mycelial growth (%) = [(D1 − D2)] / D1] × 100, where D1 = radius of the mycelium growing on the control plate (cm) and D2 = radius of the mycelium growing on the plate cocultured with bacteria (cm). Figure 1 illustrates the measuring radius of the mycelium.

2.2.2. Method for Assessing Bacterial Density After Culture

Microbial density was assessed using the colony counting method on agar plates [40].

2.2.3. Fermentation Optimization

To achieve high biomass production of P. polymyxa EB.KN35, C/N sources, types of mineral salts, and some parameters were examined.
  • ♦ The effect of C/N sources on the growth of P. polymyxa EB.KN35
Various agricultural processing byproducts, including 1.5% SPBP, 1.5% RBPBP, and 1.5% CPBP, were supplemented into the medium (30 mL) containing 0.5% NaCl, with the initial pH of 7, a shaking speed of 150 rpm for 24 h (*), and the control medium LB (1% peptone, 0.5% yeast extract, and 0.5% NaCl). The fermentation condition was symbolized as (*) and was further used for some experiments. Then, the bacterial density after culture was determined according to the method of Mai et al. [40] to find the most promising substrate.
  • ♦ The effect of agricultural processing byproducts (SPBPs) and LB ratio on the growth of P. polymyxa EB.KN35
SPBP and LB were mixed at various ratios of 0/10, 3/7, 5/5, 7/3, and 10/0 with pH = 7 and used as a C/N source for P. polymyxa EB.KN35 fermentation. The ratios were tested as follows:
+Test 1: 1.5% LB
+Test 2: 0.45% SPBP+1.05% LB
+Test 3: 0.75% SPBP+0.75% LB
+Test 4: 1.05% SPBP+0.45% LB
+Test 5: 1.5% SPBP+0.5% NaCl
P. polymyxa EB.KN35 was cultivated under the conditions mentioned above (*). The bacterial density after culture was determined according to Section 2.2.2 to identify the most suitable substrate ratio.
  • ♦ The effect of C/N source concentration on the growth of P. polymyxa EB.KN35
Several concentrations of the 7SPBP/3LB (1%, 1.25%, 1.5%, 1.75%, 2%, 2.25%) were added to the liquid medium containing 0.5% NaCl under the abovementioned conditions (*). From the density results after culture, the optimal substrate concentration was identified for the next experiments.
  • ♦ The effect of mineral salts on the growth of P. polymyxa EB.KN35
The influence of some types of mineral salts on the growth of P. polymyxa EB.KN35 was determined in the previous studies [25,31,35]. In this study, some other sources of sulfate and phosphate salts were investigated:
The influence of some types of sulfate salts was investigated: MgSO4, CaSO4, MnSO4, ZnSO4, and FeSO4. The culture medium (30 mL) contained 1.75% C/N source, 0.05% sulfate salt, and the control medium (with 0.5% NaCl instead of 0.05% sulfate salt), with an initial pH = 7 and 150 rpm shaking speed for 24 h. Then, the suitable sulfate salts were determined according to Section 2.2.2 and selected for further tests.
The influence of some types of phosphate salts was investigated at 0.1% (K2HPO4, KH2PO4, Na2HPO4, Ca3(PO4)2), as well as 0.05% FeSO4. The culture medium (30 mL) contained 1.75% C/N source, 0.05% sulfate salt, and the control medium (with 0.5% NaCl), with an initial pH = 7 and 150 rpm shaking speed for 24 h. This allowed for the determination of the most suitable phosphate salt according to Section 2.2.2.
  • ♦ The effect of some fermentation condition parameters on the growth of P. polymyxa EB.KN35
Some other parameters that affect the growth of P. polymyxa EB.KN35 during fermentation was also studied according to the previous reports [25,26,27,28,29,30,31,32,33,34,35], including the initial pH of the culture medium (6.25, 6.5, 6.75, 7.0, 7.25, and 7.5), temperature (25 °C, 28 °C, 30 °C, 32 °C, and 34 °C), and culture time (16, 18, 20, 22, 24, and 26 h). The culture medium (30 mL) contained 1.75% C/N source, 0.1% KH2PO4, and 0.05% FeSO4, with a 150 rpm shaking speed for 24 h, and bacterial density was determined after culture according to Section 2.2.2. The culture time experiment involved 30 mL of a medium containing 1.75% C/N source, 0.1% KH2PO4, and 0.05% FeSO4, with a 150 rpm shaking speed, and bacterial density was determined after 16 h, 18 h, 20 h, 22 h, 24 h, and 26 h (method from Section 2.2.2.).
  • ♦ Scaling up the biomass production of P. polymyxa EB.KN35 using a 14 L bioreactor system
A 14 L bioreactor system (BioFlo/CelliGen 115, New Brunswick, NJ, USA) contained 7.2 L of a medium including 1.75% C/N source, 0.1% KH2PO4, 0.05% FeSO4, with the parameters of the system adjusted to match the results of experiment 3.2 (30 °C, dissolved oxygen at 15 vvm, stirring speed at 300 rpm). After that, 0.8 L of bacterial culture were added to the bioreactor, and the microbial density was monitored at 2 h, 4 h, 6 h, 8 h, and 10 h after culture (CFU/mL).

2.2.4. Detection and Identification of Bioactive Compounds Biosynthesized by P. polymyxa EB.KN35

HPLC was used to determine the growth-promoting compounds, including IAA, GA3, kinetin, and zeatin according to the method presented by Nguyen et al. [41]. Besides, the volatile compounds of this strain were identified through the GC/MS system (including ITQ 900 Mass Spec-Thermo and GC-Thermo Trace GC Ultra, Thermo Fisher Scientific, headquartered in Waltham, MA, USA). The TG-SQC capillary column (30 m × 0.25 mm × 0.25 μm) was filled with carrier gas helium (99.99%) at a constant flow rate of 1 mL/min. The bacterially cultivated broth was dissolved in methanol. One μL of the sample solution was injected with a 10:1 split ratio at a 250 °C injector temperature and constant ion source temperature of 230 °C. Initially, the column was kept at 50 °C for 2 min, then increased to 250 °C at a speed of 10 °C/min, and further increased to 280 °C for 10 min. The MS data were collected at the conditions of 70 eV of electron energy, 0.5 s of scanning interval time, and the range of fragments from 35 to 650 Da and were then compared to the standard compounds in the Mass Spectra Library (NIST 17 and Wiley) for identification.

2.2.5. Molecular Docking and DFT Calculation

Molecular docking was performed following the protocols presented in the previous report [42]. The structure of the protein 1TRY targeting F. oxysporum was obtained from the RCSB Protein Data Bank, and its 3D structure was optimized (at a virtual pH of 7) using the MOE-2015.10 software. The most active binding sites on the protein 1TRY were searched using the site-finding function of the MOE-2015.10 software. The structure of the ligands (volatile compounds produced by P. polymyxa EB.KN35) was also prepared using the MOE software with the following optimization parameters: force field MMFF94x; R-field 1: 80; cutoff, rigid water molecules, space group p1, cell size (10, 10, 10); cell shape (90, 90, 90); and gradient 0.01 RMS kcal·mol−1A−2. All the ligands were docked into the binding sites of the protein 1TRY, and the data were obtained from the docking performance at the most active binding site. Some output data, including root mean square deviation (RMSD), docking score (DS), linkage type, distances between linkages, and amino acid compositions, were obtained for analysis. The highest-energy molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of these ligands were examined using density functional theory (DFT) at the B3LYP/6-31G theoretical level.

2.2.6. Statistical Analysis

The results of the experiments in Section 3.1 and Section 3.3 were processed using the Microsoft Office Excel software, and the experiment in Section 3.2 employed ANOVA using the SPSS 22.0 software. All the experiments were randomly designed. The data represent the average value of 3 repetitions ± standard deviation with a significance level of p < 0.05.

3. Results and Discussion

3.1. Screening of Endophytic Bacterial Strains with Potent Fungicidal Efficacy Against Fusarium oxysporum

A total of 110 endophytic bacterial strains were evaluated for their antifungal effect against F. oxysporum. As shown in Table A1, fifty-two strains showed mycelial growth inhibition, with inhibition values in the range of 11.79–79.58%, and five strains were found to show significant mycelial growth inhibition (≥50.83%). Of these, the endophytic strains Bacillus amyloliquefaciens EB.CK9, Bacillus amyloliquefaciens EB.EH18, Bacillus methylotrophicus EB.EH34, and Bacillus siamensis EB.KN10 showed moderate inhibition values of 50.83%, 56.67%, 55.83%, and 51.25%, respectively. Paenibacillus polymyxa EB.KN35 was demonstrated to be the most effective antifungal strain with the highest inhibition value of 79.58%. The inhibition images of these five strains against F. oxysporum on Petri dishes are illustrated in Figure 2.
Paenibacillus polymyxa is an effective biological control agent against various pathogens such as bacteria, root-knot nematodes, and some fungi and oomycetes (Verticillium longisporum, Fusarium sp., Ralstonia solanacearum) through various mechanisms [15,16,17,18,19,20,21,22,23,24]. P. polymyxa is also recognized as a potential biocontrol agent against F. oxysporum [17,18,19,20,21,22,23,24]. Some endophytic bacteria, such as P. polymyxa, isolated from healthy plants, have been shown to control Fusarium wilt on various crops. In addition, they have beneficial effects on promoting plant growth under field conditions [15,16]. Similarly, P. polymyxa PJH16 isolated from cucumber soil and P. polymyxa NSY50 isolated from vinegar waste compost have been identified as biocontrol agents effective in controlling Fusarium wilt, which is a major soil-borne disease in cucumber [20,24]. In this study, we noted that the durian endophytic P. polymyxa EB.KN35 exhibited a strong antagonistic potential against F. oxysporum, a pathogenic durian isolate from the Central Highlands of Vietnam. For the first time, durian endophytic bacterium P. polymyxa has been recorded as effective against F. oxysporum (79.58%). The potential biocontrol of P. polymyxa against F. oxysporum in various crops from the literature review is summarized in Table 1. P. polymyxa showed mycelial growth inhibition against F. oxysporum, with inhibition values in the range of 36–88.36%. Thus, the anti-F. oxysporum activity of P. polymyxa EB.KN35 recorded in this work is comparable to that of the previous studies.

3.2. The Effects of the Substrate, Salt, and Some Fermentation Parameters on the Growth of P. polymyxa EB.KN35

Currently, P. polymyxa is being widely studied, mainly for fermentation in commercial media. There have been some reports on cultivating P. polymyxa using byproducts (wastewater from sweet potato starch, molasses, pear pulp, wheat bran, etc.) to reduce costs and create environmentally friendly products [25,26,27,28,29,30,31,32,33,34,35]. Consequently, this study investigates the possibility of using agricultural byproducts for P. polymyxa EB.KN35 fermentation to achieve high productivity in its biomass and bioactive compounds.

3.2.1. The Effect of the Substrate Source on the Growth of P. polymyxa EB.KN35

P. polymyxa EB.KN35 strain was fermented using several agricultural processing byproducts (RBPBPs, SPBPs, and CPBPs) as C/N sources for cultivation. As shown in Figure 3A, the density of P. polymyxa EB.KN35 grows strongly in SPBPs, reaching 7.8 × 108 CFU/mL. The result of this study is different from the previous reports. Kim et al. [26] used TSB in the cultivation of P. polymyxa T5 and gained 106 CFU/mL. Meanwhile, P. polymyxa BY-28 reuses wastewater from sweet potato starch, reaching 9.7 × 109 CFU/mL; P. polymyxa GBR-1 uses commercial medium BHI, reaching 4.6 × 109 CFU/mL; and P. polymyxa D1 reuses pear pulp and wheat bran, reaching 4.52 × 109 CFU/mL [29,30,31]. In addition, skimmed milk and molasses were used as C/N sources in the fermentation of P. polymyxa Kp10 and P. polymyxa IN937a, reaching a density of 108 CFU/mL and 6.606 × 109 CFU/mL, respectively [33,35]. Soybean processing byproducts (SPBPs) have high nutritional content, including about 50% crude oil, 25% proteins, 10% lipids, and other nutrients [43]. Therefore, SPBPs are a suitable substrate for the fermentation of the endophytic bacterium P. polymyxa EB.KN35, and it was selected for further research. Notably, this is the first record of the ability to reuse these three agricultural processing byproducts for the P. polymyxa strain cultivation.
The different ratios of SPBPs and LB had a clear impact on the fermentation of P. polymyxa EB.KN35. The results in Figure 3B indicate the best growth of P. polymyxa EB.KN35 was recorded at the SPBPs/LB ratio of 7/3, with bacterial densities reaching a high of 9.78 × 108 CFU/mL. Thus, this ratio was chosen for subsequent experiments to use soybean processing byproducts. The C/N source (SPBPs/LB) concentration was also assessed for the effect on bacterial biomass production. The experimental results illustrated in Figure 3C show that the different concentrations of SPBPs/LB (0.5–2.5%) were directly proportional to the growth of P. polymyxa EB.KN35, with SPBPs/LB concentrations of 1.75–2.5% ( 7:3 ratio) showing the highest growth. Therefore, we used the SPBPs/LB concentration of 1.75% ( 7:3 ratio) for the following experiments to save production materials.

3.2.2. The Effect of Mineral Salts and Some Fermentation Parameters

Sulfate and phosphate salts have been reported to enhance bacterial fermentation by achieving higher productivity of biomass and bioactive compounds in numerous works [44,45]. Thus, in this study, the effect of sulfate and phosphate salts on bacterial growth was tested, and the results are presented in Figure 4A and Figure 4B, respectively. As shown in Figure 4A, all sulfate salts were found to promote bacterial biomass growth more than the original environment. Among them, P. polymyxa EB.KN35 showed the highest growth in the medium supplemented with FeSO4, reaching a cell density of 4.12 × 109 CFU/mL. Meanwhile, three out of the four added phosphate salts (K2HPO4, Na2HPO4, and Ca3(PO4)2) were ineffective for the growth of strain EB.KN35 with the bacterial density equal to or even lower than in the original environment (Figure 4B). Notably, supplementation with KH2PO4 strongly promoted the development of P. polymyxa EB.KN35, with a significantly high bacterial density of 5.06 × 109 CFU/mL.
Some culture conditions, such as the initial pH of the culture medium, cultivation temperature, and cultivation time, were also examined for their effect on bacterial biomass production, and the results are shown in Figure 4C, Figure 4D, and Figure 4E, respectively. Overall, P. polymyxa EB.KN35 grew best using a 1.75% SPBPs/LB substrate (7:3 ratio), 0.05% FeSO4, and 0.1% KH2PO4 with pH = 6.75 at 30 °C in 20 h of culture. This work reports the reuse of SPBPs as a new low-cost and effective substrate for the growth of P. polymyxa EB.KN35. The results of this study concerning the effect of temperature and the initial pH of medium cultivation are similar to those of the previous reports. In the report by Gong et al. [25], P. polymyxa BY-28 fermented using bean cake powder at 30 °C reached 3.1–3.3 × 108 CFU/mL. P. polymyxa M-1 fermented using GSC at 30 °C reached 4 × 107 CFU/mL [27]. Similarly, P. polymyxa reached 105–107 CFU/mL when cultured at 25–30 °C [28]. Gao et al. [32] reported that P. polymyxa Pp-7250 produced a biomass of 107 CFU/mL at 30 °C, pH 6–8, with wheat starch and soybean flour as the main substrate sources, while P. polymyxa NSY50 reached a density of 2.5 × 108 CFU /mL at 28 °C using LB medium [20]. However, the growth of P. polymyxa on the substrate of soybean byproducts combined with FeSO4 and KH2PO4 achieving high biomass in a short time (20 h) is a new finding of this work. In summary, the growth of P. polymyxa strains depends on the substrate and the bacterial strain.

3.3. Scaling up Biomass Production of P. polymyxa EB.KN35 via Fermentation in a 14 L Bioreactor

For enhancing P. polymyxa EB.KN35 biomass productivity and shortening the fermentation time, a 14 L bioreactor system was applied for fermentation from 0 h to 10 h in this experiment. Monitoring the fermentation process every 2 h, the data were recorded and illustrated in Figure 5. Accordingly, the bacterial density increased gradually over time and reached the optimum after 8 h of fermentation, with a maximum density of 3.46 × 1011 CFU/mL. Thus, compared to fermentation at the small flask scale (Figure 4E), scaling up to a 14 L bioreactor reduced the fermentation time from 20 h to 8 h while increasing biomass yield.
In an earlier report by Kaziūnienė et al. [34], P. polymyxa MVY-024 was cultivated in an EDF-5.4 bioreactor (with the working volume of up to 4.5 L out of the total volume of 6.2 L) using yeast extract and molasses to reach a density of 2.2 × 109 CFU/mL−1. The research on P. polymyxa fermentation on a large scale has been rarely reported. To our current knowledge, this is the first work to report the fermentation of P. polymyxa using a 14 L bioreactor system with a working volume of 8 L. In addition, the study demonstrates the use of SPBPs in the fermentation of increased biomass of P. polymyxa strain EB.KN35 in a low-cost, safe, effective, and environmentally friendly manner.

3.4. The Bioactive Compounds Produced by P. polymyxa EB.KN35

P. polymyxa has been reported to promote plant growth by producing a variety of phytohormones such as auxins, cytokinins, indole-3-acetic acid (IAA), etc., and its fungicidal effect is closely related to the production of volatile compounds (VOCs) [46,47,48]. For a better understanding of the plant-promoting effect and fungicidal activity of P. polymyxa EB.KN35, we used HPLC and GC–MS to identify the bioactive compounds produced by P. polymyxa EB.KN35.
HPLC fingerprinting shown in Figure 6 indicated that P. polymyxa EB.KN35 produced 4 plant growth-promoting compounds, including GA3, zeatin, IAA, and kinetin. Of these, GA3 and zeatin were produced at a high-level yield in the culture medium with concentrations of 1168 µg/mL and 38.78 µg/mL, while IAA and kinetin were produced in minor amounts. The report by Tokmakova et al. [49] recorded that another P. polymyxa KB strain also produced these four growth stimulants with a content of 2.48–81.15 μg/g dry biomass. Additionally, some reports have recorded the IAA production activity of other P. polymyxa strains such as P. polymyxa SK1 and P. polymyxa SC2 producing IAA at concentrations from 13 to 31 μg/mL and P. polymyxa ZYPP18 producing IAA at a concentration of 3050 μg/mL [50,51,52].
VOCs produced by microorganisms have been described as having the ability to inhibit the growth of bacteria and fungi [46,47,48,53,54,55,56]. VOCs produced by P. polymyxa were detected and identified using GC–MS, and the results are shown in Table 2. P. polymyxa was found to produce 11 VOCs. Of these, glycine, N-2-naphthalenyl-, 2-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]hydrazide (9) was found in the most abundant amount with an area of 77.58%, metanephrine (1) was found to be present in a small amount (0.21%), equal to bexarotene (3). Meanwhile, glafenin (2) was found to produce an amount twice that of metanephrine (1). By contrast, cholestan-3-ol, 4-methyl-, (3β,4α,5α)-(10) and estra-1,3,5(10)-trien-17-one, 3-hydroxy-, O-methyloxime (CAS) (11) were produced in moderate amounts (5.01–9.87% of the area), while the remaining VOCs (4, 5, 6, 7, and 8) were found as minor compounds in the culture medium fermented by P. polymyxa EB.KN35. Of these, some VOCs, such as 3 and 9, have been shown to be potential fungicidal compounds [57]. The VOCs produced by P. polymyxa were also found to be effective against some nematodes [58,59,60]. P. polymyxa J2-4 also produced 14 VOCs, which exhibited nematicidal activity Meloidogyne incognita [59]. P. polymyxa Sb3-1 created 2-nonanone and 3-hydroxy-2-butanone against Verticillium longisporum [60]. The chemical structures of these VOCs are presented in Figure 7.

3.5. Molecular Docking and DFT Analysis

Up to date, several studies have reported the potential effect of P. polymyxa against F. oxysporum [17,18,19,20,21,22,23,24]; however, exploring the mechanism action of P. polymyxa’s bioactive compounds against F. oxysporum via a docking study has not been reported. In this study, we conducted molecular docking to investigate the interaction and binding energy of the identified compounds produced by P. polymyxa toward the protein 1TRY targeting F. oxysporum. 1TRY is a crucial metabolic enzyme involved in glycolysis and gluconeogenesis. In Fusarium oxysporum, 1TRY is an important enzyme with multiple functions. It is a key metabolic enzyme that also contributes to fungal virulence.
The structure of F. oxysporum 1TRY was investigated using a locally developed image plate scanner and reported by Rypniewski et al. (1995) [36]. A single crystal of 1TRY was analyzed on the X31 beamline at EMBL, Hamburg. The final protein model was found to consist of 1557 protein atoms, 400 water molecules, 1 molecule of isopropanol, and 1 molecule of an isopropyl phosphoryl inhibitor group, covalently bound to the catalytic Ser195. There are some fundamental differences in the structures of F. oxysporum 1TRY compared to mammalian and Streptomyces trypsins. The specificity pocket of F. oxysporum 1TRY is larger than in bovine trypsin, which results in the preference of F. oxysporum trypsin for the bulkier arginine over lysine. The capacity for binding on the C-terminal side of the substrate is more restricted in F. oxysporum trypsin than in mammalian and Streptomyces griseus trypsins, leading to the relative inactivation of F. oxysporum trypsin toward peptide-pNA substrate analogs.
It has been reported that the possible mechanism of action of a bacterial strain inhibits F. oxysporum by producing plant growth-promoting compounds [61], producing fungal cell wall-degrading enzymes [20,21,22,23,24], enhancing the effect of some enzymes, such as catalase, phenylalanine ammonia-lyase, superoxide dismutase, and peroxidase, in plant roots [62], and producing siderophores [63] and antifungal lipopeptides [61]. It has also been evidenced that the anti-F. oxysporum effect of bacterial strains is associated with the synthesis of VOCs [19,64]. However, the interaction mechanism of major VOCs has not been reported. Thus, we identified the VOCs produced by P. polymyxa EB.KN35 and elucidated the interaction mechanism of these identified VOCs toward the protein 1TRY targeting F. oxysporum in this work.
The structural data of 1TRY were obtained from the RCSB Protein Data Bank, its 3D structure was optimized using the MOE software (Figure 8A), and the possible binding sites (BS) of 1TRY were searched using the site finder function of MOE. Four BS were found on this protein 1TRY (Figure 8B), and detailed information on the size and residues of these BS is presented in Table 3. The ligands (11 VOCs identified in this work and asparagine—a commercial 1TRY inhibitor) were docked into all binding sites of the protein 1TRY; for each compound, only the highest interaction was collected as data and described. In molecular docking studies, RMSD and docking score (DS) have been widely used for checking the potential successful interaction of a ligand with its target protein. When a ligand binds to its target protein with an RMSD value under 2.0Å and a DS value under −3.20 kcal/mol, this interaction is accepted, and this compound may be suggested as a possible inhibitor [65].
As shown in Table 4, all the checked ligands were found to be interacting with 1TRY with RMSD values in the accepted range (0.87–1.95Å) and, as such, were eligible for further investigation. In addition, these ligands bound to 1TRY with DS values in the range from −7.6 to −10.6 kcal/mol, suggesting that these compounds may be potential inhibitors of the protein 1TRY targeting F. oxysporum. In a comparison of DS values, including the positive inhibitor asparagine (AS), the effective binding ability of these ligands toward 1TRY was ranked as follows: 9 > 2 > 1 > 4 ≥ AS (commercial TRY inhibitor) > 5 > 3 > 7 > 8 > 6 > 11. Based on the comparison of DS values, VOC 4 showed a comparable DS value to AS, while VOCs 1, 2, and 9 demonstrated a more efficient interaction with 1TRY than that of the positive 1TRY inhibitor. In addition, VOC 9 was produced by P. polymyxa EB.KN35 (Table 2) with the highest productivity, occupying an area of 77.58%, indicating that the strong anti-F. oxysporum activity of P. polymyxa EB.KN35 may be due to secreting a high content of VOC 9. For a deeper understanding of the interaction of the most active ligands (VOCs 1, 2, 4, and 9) with protein 1TRY, the detailed interaction of these VOCs with 1TRY at its binding sites is shown in Figure 9.
VOC 1 and 2 are effectively bound to 1TRY at binding site 2 with low DS values of −9.5 and −9.6 kcal/mol, respectively. Among these, VOC 1 interacted with 1TRY via four linkages (1 H-acceptor, three pi-H) by binding to amino acids Arg165 and Ala132 to generate 1 H-acceptor and one pi-H linkage and binding to Val131 to create two pi-H linkages. VOC 2 was found to bind to 1TRY by interacting with amino acids Pro130, Cys182, Asp129, and Val131 to create up to five linkages (2 H-donor, 1 H-acceptor, and two pi-H). Meanwhile, VOC 4, VOC 9, and AS were found to be strongly bound to 1TRY at binding site 1 with low DS values of −9.4, −10.6, and −9.4 kcal/mol, respectively. Among these, AS was only bound to 1TRY through an interaction with one amino acid, Gly25, and one H-acceptor linkage was formed. VOC 4 interacted with 1TRY via linking to Gly116 and Leu71, forming 1 H-donor and 1 H-acceptor, respectively. Similar to AS, VOC 9 was also found to effectively interact with the target protein through one H-donor linkage formed between the ligand and amino acid Asn154.
Trypsin-like proteases such as F. oxysporum 1TRY are key virulence factors that degrade plant defense proteins, facilitate invasion, and support fungal growth. Thus, inhibiting these enzymes or genetic modifications may provide potential strategies for controlling F. oxysporum [37,38]. In this study, VOCs 1, 2, 4, and 9 showed effective binding to F. oxysporum 1TRY. Thus, they may be suggested as potential 1TRY inhibitors, as such, having promising use as control agents of F. oxysporum. However, these results were obtained based on screening in silico studies. Thus, further screening tests in vitro for these VOCs need to be conducted for confirmation. In a previous report, compounds such as VOC bexarotene and VOC glycine, N-2-naphthalenyl-, 2-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]hydrazide, identified as VOCs 3 and 9, respectively, in this study, were recognized as potential fungicidal compounds via in silico docking studies against protein 3QPC targeting F. solani [57]. However, no data have reported the fungicidal effect of the identified compounds against F. oxysporum. Thus, this result may indicate a novel potential fungicidal effect of VOCs 1, 2, 4, and 9 against F. oxysporum via molecular docking elucidation.
Some of the most potent compounds (VOCs 1, 2, 4, and 9) and AS were further checked for their highest-energy molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) using density functional theory (DFT) at the B3LYP/6-31G theoretical level. The data are presented in Figure 10. The structures of these ligands possess low EHomo values (from 4.91 to −6.85 eV), indicating these molecules have significant electronic stability (commonly accepted when the EHomo value is under −5 eV) [65]. In this investigation, all the checked ligands showed an energy gap in the range of 3.00 eV to 6.01 eV. As such, almost all of these compounds have potential intermolecular binding capability to the target protein.

3.6. Implications and Recommendations

This study contains some significant new findings and recommendations.
In this study, the durian endophytic P. polymyxa EB.KN35 is recorded as being effective against F. oxysporum (79.58%) for the first time. This finding broadens the application of P. polymyxa as a potential source of biofungicides against the durian pathogenic fungal strain F. oxysporum. In addition, EB.KN35 was also found to be secreting some plant growth-promoting compounds such as GA3, IAA, kinetin, and zeatin. As such, this endophytic bacterium could be used as a biofertilizer. However, further investigation is needed, such as evaluating the potential antifungal activity, plant growth-promoting effect, as well as the potential impact on microbial soil systems of P. polymyxa EB.KN35 in greenhouse tests and field cultivations for developing this endophytic bacterial strain as an applicable biocontrol agent.
Regarding eco-friendly and effective EB.KN35 biomass production, some agricultural processing byproducts were used as C/N sources for fermentation. This study is the first to report the reuse of a soybean processing byproduct as a novel, low-cost, and friendly substrate for producing P. polymyxa via fermentation. The utilization of a 14 L bioreactor system for P. polymyxa EB.KN35 fermentation achieved higher biomass productivity in a shorter fermentation time. To achieve higher biomass productivity, optimal fermentation in a bioreactor should be further investigated, and large-scale fermentation may be performed.
The possible mechanism of action of the VOCs produced by P. polymyxa against F. oxysporum was also explored for the first time via docking with the protein 1TRY targeting anti-pathogenic fungi. The molecular docking results indicated that some VOCs (1, 2, 4, and 9) may play a significant role in inhibiting F. oxysporum. This result suggested that these VOCs may be potential fungicidal agents for further investigation.

4. Conclusions

P. polymyxa EB.KN35 was screened as having the most effective fungicidal effect against F. oxysporum, and optimal fermentation conditions for its biomass production were investigated. Soybean processing byproducts were newly found to be a suitable substrate for P. polymyxa EB.KN35 fermentation. In addition, this bacterial strain was also successfully fermented in a 14 L bioreactor system and achieved a high biomass yield (3.46 × 1011 CFU/mL) in a short fermentation time (8 h). This study also demonstrated that P. polymyxa EB.KN35 secretes several plant growth-promoting compounds (GA3, IAA, kinetin, and zeatin) and 11 VOCs. Furthermore, based on the molecular docking analysis, some VOCs (1, 2, 4, and 9) were suggested as bioactive compounds playing an important role in inhibiting F. oxysporum. This study demonstrates the feasibility of using soybean processing byproducts as a cost-effective C/N source for P. polymyxa EB.KN35 fermentation, facilitating sustainable biocontrol of F. oxysporum. Future research should explore field-scale applications and optimize fermentation parameters for commercial production.

Author Contributions

Conceptualization, V.B.N., S.-L.W. and A.D.N.; methodology, V.A.N., A.D.N., S.-L.W. and V.B.N.; software and validation, V.A.N. and T.Q.P.; formal analysis, S.-L.W., A.D.N., D.S.N. and V.B.N.; investigation, V.A.N., T.H.T.T. and T.Q.P.; resources, V.A.N., V.B.N. and A.D.N.; data curation, S.-L.W. and A.D.N.; writing—original draft preparation, T.H.T.T. and V.B.N.; writing—review and editing, V.B.N.; supervision, V.B.N. and A.D.N.; visualization and project administration, V.A.N., V.B.N. and A.D.N. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by a grant from the Ministry of Education and Training, Vietnam (B2023-TTN-02).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article; further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflict of interest.

Appendix A

Table A1. Evaluation of the antifungal potential of endophytic bacteria against pathogenic fungus F. oxysporum.
Table A1. Evaluation of the antifungal potential of endophytic bacteria against pathogenic fungus F. oxysporum.
No.StrainAntifungal Activity (%)No.StrainAntifungal Activity (%)
1 EB.CK1 - 56 EB.KN27 17.44
2 EB.CK2 48.72 57 EB.KN28 48.72
3 EB.CK3 - 58 EB.KN29 -
4 EB.CK4 - 59 EB.KN30 -
5 EB.CK5 28.21 60 EB.KN31 28.97
6 EB.CK6 - 61 EB.KN32 -
7 EB.CK7 10.26 62 EB.KN33 34.87
8 EB.CK8 - 63 EB.KN34 -
9 EB.CK9 50.83 64 EB.KN35 79.58
10 EB.CK10 - 65 EB.KN36 -
11 EB.CK11 43.59 66 EB.KN37 -
12 EB.CK12 - 67 EB.KN38 32.31
13 EB.CK13 27.69 68 EB.KN39 -
14 EB.CK14 48.97 69 EB.KN40 -
15 EB.CK15 - 70 EB.KN41 48.97
16 EB.CK16 - 71 EB.KN42 -
17 EB.CK17 - 72 EB.KN43 48.46
18 EB.CK18 42.31 73 EB.KN44 -
19 EB.CK19 47.69 74 EB.KN45 11.79
20 EB.CK20 - 75 EB.KN46 -
21 EB.CK21 48.72 76 EB.KN47 -
22 EB.CK22 23.59 77 EB.EH1 42.31
23 EB.CK23 - 78 EB.EH2 41.79
24 EB.CK24 - 79 EB.EH3 -
25 EB.CK25 39.74 80 EB.EH4 23.59
26 EB.CK26 34.62 81 EB.EH5 -
27 EB.CK27 - 82 EB.EH6 40.77
28 EB.CK28 - 83 EB.EH7 46.92
29 EB.CK29 32.05 84 EB.EH8 -
30 EB.KN1 21.28 85 EB.EH9 -
31 EB.KN2 - 86 EB.EH10 -
32 EB.KN3 - 87 EB.EH11 -
33 EB.KN4 48.72 88 EB.EH12 44.36
34 EB.KN5 41.28 89 EB.EH13 -
35 EB.KN6 - 90 EB.EH14 -
36 EB.KN7 - 91 EB.EH15 19.49
37 EB.KN8 8.97 92 EB.EH16 -
38 EB.KN9 - 93 EB.EH17 45.90
39 EB.KN10 51.25 94 EB.EH18 56.67
40 EB.KN11 37.44 95 EB.EH19 48.97
41 EB.KN12 31.28 96 EB.EH20 -
42 EB.KN13 - 97 EB.EH21 49.23
43 EB.KN14 48.72 98 EB.EH22 -
44 EB.KN15 48.97 99 EB.EH23 -
45 EB.KN16 - 100 EB.EH24 -
46 EB.KN17 16.92 101 EB.EH25 37.44
47 EB.KN18 - 102 EB.EH26 -
48 EB.KN19 36.67 103 EB.EH27 -
49 EB.KN20 - 104 EB.EH28 22.82
50 EB.KN21 - 105 EB.EH29 -
51 EB.KN22 - 106 EB.EH30 -
52 EB.KN23 - 107 EB.EH31 -
53 EB.KN24 49.23 108 EB.EH32 -
54 EB.KN25 31.28 109 EB.EH33 48.46
55 EB.KN26 - 110 EB.EH34 55.83

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Figure 1. Images of the antifungal assays of the durian endophytic bacterial strain. In the control dish (C), the pathogenic fungus was grown on potato dextrose agar (PDA), and in the experiment dish (T), also on PDA, the pathogenic fungus was treated with an endophytic bacterial strain. After five days of cultivation, the radii (cm) of the fungal mycelial spread on the control dish (D1) and the experimental dish (D2) were measured for the calculation of antifungal activity.
Figure 1. Images of the antifungal assays of the durian endophytic bacterial strain. In the control dish (C), the pathogenic fungus was grown on potato dextrose agar (PDA), and in the experiment dish (T), also on PDA, the pathogenic fungus was treated with an endophytic bacterial strain. After five days of cultivation, the radii (cm) of the fungal mycelial spread on the control dish (D1) and the experimental dish (D2) were measured for the calculation of antifungal activity.
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Microorganisms 13 00800 g002
Figure 2. Evaluation of the antifungal potential of 5 endophytic strains against the pathogenic fungus F. oxysporum isolated in the Central Highlands, Vietnam. Control is the control plate and (A) B. amyloliquefaciens EB.CK9, (B) B. amyloliquefaciens EB.EH18, (C) B. methylotrophicus EB.EH34, (D) B. siamensis EB.KN10, and (E) P. polymyxa EB.KN35.
Figure 2. Evaluation of the antifungal potential of 5 endophytic strains against the pathogenic fungus F. oxysporum isolated in the Central Highlands, Vietnam. Control is the control plate and (A) B. amyloliquefaciens EB.CK9, (B) B. amyloliquefaciens EB.EH18, (C) B. methylotrophicus EB.EH34, (D) B. siamensis EB.KN10, and (E) P. polymyxa EB.KN35.
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Microorganisms 13 00800 g003
Figure 3. The effect of the substrate source on the fermentation of the endophytic bacterial strain P. polymyxa EB.KN35. (A) The effect of four substrate sources, including soybean processing byproducts (SPBPs), rice bran processing byproducts (RBPBPs), cassava processing byproducts (CPBPs), and Luria broth (LB) used at a concentration of 1.5%. (B) The effect of different ratios of agricultural processing byproducts (SPBPs) and LB. (C) The effect of the different concentrations of the substrate (SPBPs/LB = 7/3). Means followed by different letters (a, b, c) are significantly different according to Duncan’s Multiple Range Test at p < 0.05.
Figure 3. The effect of the substrate source on the fermentation of the endophytic bacterial strain P. polymyxa EB.KN35. (A) The effect of four substrate sources, including soybean processing byproducts (SPBPs), rice bran processing byproducts (RBPBPs), cassava processing byproducts (CPBPs), and Luria broth (LB) used at a concentration of 1.5%. (B) The effect of different ratios of agricultural processing byproducts (SPBPs) and LB. (C) The effect of the different concentrations of the substrate (SPBPs/LB = 7/3). Means followed by different letters (a, b, c) are significantly different according to Duncan’s Multiple Range Test at p < 0.05.
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Microorganisms 13 00800 g004
Figure 4. The effect of mineral salts and fermentation parameters on the cell growth of P. polymyxa EB.KN35 using a 1.75% SPBPs/LB substrate (7:3 ratio). (A) The effect of sulfate salts tested at the concentration of 0.05% (B). The effect of phosphate salts tested at a concentration of 0.01%. (C) The effect of the initial pH of the culture medium. (D) The effect of cultivation temperature. (E) The effect of cultivation time. Means followed by different letters (a, b, c) are significantly different according to Duncan’s Multiple Range Test at p < 0.05.
Figure 4. The effect of mineral salts and fermentation parameters on the cell growth of P. polymyxa EB.KN35 using a 1.75% SPBPs/LB substrate (7:3 ratio). (A) The effect of sulfate salts tested at the concentration of 0.05% (B). The effect of phosphate salts tested at a concentration of 0.01%. (C) The effect of the initial pH of the culture medium. (D) The effect of cultivation temperature. (E) The effect of cultivation time. Means followed by different letters (a, b, c) are significantly different according to Duncan’s Multiple Range Test at p < 0.05.
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Microorganisms 13 00800 g005
Figure 5. The results of P. polymyxa EB.KN35 biomass production in a larger fermentation system.
Figure 5. The results of P. polymyxa EB.KN35 biomass production in a larger fermentation system.
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Microorganisms 13 00800 g006
Figure 6. The HPLC profiles of plant growth-promoting compounds biosynthesized by P. polymyxa EB.KN35. The bacterial strains were cultivated in King’s B medium at 28 °C. After 2 days, the fermentation broth was collected and centrifuged at 6000 rpm/10 min, and the residue was removed. The supernatant was filtered through a 0.45 µm membrane and used for the analysis of plant growth stimulants by the HPLC system. Five µL of the culture filtrate were injected into the HPLC system and separated using a C18 column before detecting PGPCs at 254 nm.
Figure 6. The HPLC profiles of plant growth-promoting compounds biosynthesized by P. polymyxa EB.KN35. The bacterial strains were cultivated in King’s B medium at 28 °C. After 2 days, the fermentation broth was collected and centrifuged at 6000 rpm/10 min, and the residue was removed. The supernatant was filtered through a 0.45 µm membrane and used for the analysis of plant growth stimulants by the HPLC system. Five µL of the culture filtrate were injected into the HPLC system and separated using a C18 column before detecting PGPCs at 254 nm.
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Microorganisms 13 00800 g007
Figure 7. Chemical structures of the volatile compounds (1–11) biosynthesized by P. polymyxa EB.KN35.
Figure 7. Chemical structures of the volatile compounds (1–11) biosynthesized by P. polymyxa EB.KN35.
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Microorganisms 13 00800 g008
Figure 8. The 3D structure (A) and four binding sites (B) of 1TRY targeting F. oxysporum.
Figure 8. The 3D structure (A) and four binding sites (B) of 1TRY targeting F. oxysporum.
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Microorganisms 13 00800 g009
Figure 9. The detailed interaction of the most active ligands (VOCs 1, 2, 4, and 9) produced by P. polymyxa EB.KN35 and asparagine (AS) at the binding sites of 1TRY targeting F. oxysporum.
Figure 9. The detailed interaction of the most active ligands (VOCs 1, 2, 4, and 9) produced by P. polymyxa EB.KN35 and asparagine (AS) at the binding sites of 1TRY targeting F. oxysporum.
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Microorganisms 13 00800 g010
Figure 10. HOMO and LUMO of the most active ligands (VOCs 1, 2, 4, and 9) produced by P. polymyxa EB.KN35 and asparagine (AS) analyzed using DFT at the theoretical level of B3LYP/6–31G.
Figure 10. HOMO and LUMO of the most active ligands (VOCs 1, 2, 4, and 9) produced by P. polymyxa EB.KN35 and asparagine (AS) analyzed using DFT at the theoretical level of B3LYP/6–31G.
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Table 1. Antifungal potential of P. polymyxa against F. oxysporum in crops.
Table 1. Antifungal potential of P. polymyxa against F. oxysporum in crops.
StrainOriginBiocontrolAntifungal Effect (%)Ref.
P. polymyxa EB.KN35Root of the durian plantF. oxysporum79.58This study
P. polymyxa SQR-21Rhizosphere of healthy watermelon plantsF. oxysporum70[17]
P. polymyxa CF05Cryptomeria fortuneiF. oxysporum f. sp. lycoersici78.24[18]
P. polymyxa WR-2-F. oxysporum f. sp. niveum 36–40[19]
P. polymyxa NSY50Vinegar waste compostF. oxysporum f. sp. cucumerinum [20]
P. polymyxa SR19Root of the Urtica dioica plantF. oxysporum≥60[21]
P. polymyxa HX-140Rhizosphere soil of rapeF. oxysporum f. sp. cucumerinum 55.6[22]
P. polymyxa hg18Healthy cucumber plantF. oxysporum f. sp. cucumerinum 69.57[23]
P. polymyxa PJH16Cucumber soil F. oxysporum f. sp. cucumerinum Foc FJH3688.36[24]
Table 2. Identification of the volatile compounds produced by P. polymyxa EB.KN35 using GC–MS.
Table 2. Identification of the volatile compounds produced by P. polymyxa EB.KN35 using GC–MS.
No.RT (min)Area (%)Compounds
15.230.21Metanephrine
27.940.48Glafenin
311.220.2Bexarotene
426.010.05Cyclopenta[b]pyran-5-octanoic acid, octahydro-ε,2,6-trioxo-, (4aR,5R,7aS)-
526.710.05Tetranorprostaglandin J2
628.710.05N-butyryl-DL-homoserine lactone
736.660.04Epitestosterone
838.170.03Crinan-11-ol, 1,2-didehydro-3-methoxy-, (3β,5α,11S,13β,19α)- (CAS)
938.6777.58Glycine, N-2-naphthalenyl-, 2-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]hydrazide
1039.599.87Cholestan-3-ol, 4-methyl-, (3β,4α,5α)-
1144.625.01Estra-1,3,5(10)-trien-17-one, 3-hydroxy-, O-methyloxime (CAS)
Table 3. Sizes and residues of the binding sites on the protein 1TRY.
Table 3. Sizes and residues of the binding sites on the protein 1TRY.
Site No.SizeResidues
127Ala24, Gly25, Asp26, Phe27, Pro28, Ser70, Leu71, Ser72, Gly116, Asn117, Asn154
224Asp129, Pro130, Val131, Ala132, Ile162, Val163, Ser164, Arg165, Cys168, Met180, Phe181, Cys182,
316Pro130, Val131, Ser134, Ser135, Ala136, Ile162, Asp201, Ser201a, Ile210
415Asp26, Phe27, Pro28, Phe29, Thr137, Lys157, Val200, Asp201, Asn203
Table 4. Docking study data of the docking of ligands (L, volatile compounds) and asparagine (AS) binding with the protein (P) 1TRY targeting F. oxysporum.
Table 4. Docking study data of the docking of ligands (L, volatile compounds) and asparagine (AS) binding with the protein (P) 1TRY targeting F. oxysporum.
Ligands Symbol of the L–P
Complex		Binding SiteRMSD
(Å)		DS
(kcal/mol)		Linkages Interactions
(Distance (Å)/E (kcal/mol) /
Linkage Type)
11-1TRY21.36−9.54 (1 H-acceptor, 3 pi-H)Arg165 (3.06/−2.4/H-acceptor)
Val131 (4.44/−0.6/pi-H)
Val131 (4.69/−1.0/pi-H)
Ala132 (4.69/−1.0/pi-H)
22-1TRY21.82−9.65 (2 H-donor, 1 H-acceptor, 2 pi-H)Pro130 (3.32/−1.0/H-donor)
Cys182 (3.14/−1.1/H-donor)
Cys182 (3.21/−0.4/H-acceptor)
Asp129 (4.46/−0.8/pi-H)
Val131 (4.78/−0.6/pi-H)
33-1TRY11.21−9.02 (1 H-donor, 1 pi-H)Ser70 (2.96/−0.9/H-donor)
Ser75 (3.96/−1.2/pi-H)
44-1TRY10.87−9.42 (1 H-donor, 1 H-acceptor)Gly116 (3.04/−1.5/H-donor)
Leu71 (3.21/−2.0/H-acceptor)
55-1TRY21.80−9.15 (3 H-donor, 2 H-acceptor)Asp129 (3.04/−1.4/H-donor)
Pro130 (2.83/−1.3/H-donor)
Asp129 (3.12/−5.0/H-donor)
Ala132 (3.29/−0.6/H-acceptor)
Arg165 (2.95/−3.1/H-acceptor)
66-1TRY21.86−7.862 (1 H-donor, 1 pi-H)Asp129 (2.94/−4.3/H-donor)
Ala132 (3.02/−2.9/H-acceptor)
77-1TRY21.66−8.71 H-donorTry120 (3.37/−0.5/H-donor)
88-1TRY11.90−8.51 H-donorAsn117 (2.95/−0.6/H-donor)
99-1TRY11.95−10.61 H-donorAsn154 (3.42/−0.8/H-donor)
1010-1TRY41.86−8.21 H-donorAsp26 (3.13/−0.6/H-donor)
1111-1TRY41.47−7.6H-piPhe27 (4.21/−0.7/H-pi)
ASAS-1TRY11.81−9.41 H-acceptorGly25 (3.15/−1.9/H-acceptor)
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Ngo, V.A.; Nguyen, A.D.; Wang, S.-L.; Phan, T.Q.; Tran, T.H.T.; Nguyen, D.S.; Nguyen, V.B. Eco-Friendly Biomass Production and Identification of Active Compounds of Paenibacillus polymyxa EB.KN35 with Potent Anti-Fusarium oxysporum Effect. Microorganisms 2025, 13, 800. https://doi.org/10.3390/microorganisms13040800

AMA Style

Ngo VA, Nguyen AD, Wang S-L, Phan TQ, Tran THT, Nguyen DS, Nguyen VB. Eco-Friendly Biomass Production and Identification of Active Compounds of Paenibacillus polymyxa EB.KN35 with Potent Anti-Fusarium oxysporum Effect. Microorganisms. 2025; 13(4):800. https://doi.org/10.3390/microorganisms13040800

Chicago/Turabian Style

Ngo, Van Anh, Anh Dzung Nguyen, San-Lang Wang, Tu Quy Phan, Thi Ha Trang Tran, Dinh Sy Nguyen, and Van Bon Nguyen. 2025. "Eco-Friendly Biomass Production and Identification of Active Compounds of Paenibacillus polymyxa EB.KN35 with Potent Anti-Fusarium oxysporum Effect" Microorganisms 13, no. 4: 800. https://doi.org/10.3390/microorganisms13040800

APA Style

Ngo, V. A., Nguyen, A. D., Wang, S.-L., Phan, T. Q., Tran, T. H. T., Nguyen, D. S., & Nguyen, V. B. (2025). Eco-Friendly Biomass Production and Identification of Active Compounds of Paenibacillus polymyxa EB.KN35 with Potent Anti-Fusarium oxysporum Effect. Microorganisms, 13(4), 800. https://doi.org/10.3390/microorganisms13040800

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