Cell-free supernatants from plant growth - promoting rhizobacteria Bacillus albus strains control Aspergillus flavus disease in peanut and maize seedlings

doi:10.21203/rs.3.rs-5091759/v1

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Cell-free supernatants from plant growth - promoting rhizobacteria Bacillus albus strains control Aspergillus flavus disease in peanut and maize seedlings

https://doi.org/10.21203/rs.3.rs-5091759/v1

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published 17 Jan, 2025

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Background: Aspergillus flavus, a seed-borne fungal pathogen, can colonize host plants and exploit nutrients, hindering the growth of seedlings such as peanut and maize. This study investigated the effectiveness of cell-free supernatants (CFSs) from plant growth - promoting rhizobacteria (PGPR) Bacillus albus NNK24 and NDP61 in suppressing the growth of A. flavus AF1.

Results: The antifungal activity of these CFSs was attributed to their surfactant properties and chemical profile, characterized through rapid chemical assays and UHPLC-ESI-QTOF/MS combined with bioinformatic analysis using GNPS and npatlas. Identified putative antifungal compounds included two diketopiperazines (cyclo(pro-leu) and cyclo(2-hydroxy-Pro-R-Leu)), four macrolactins (7-O-succinyl macrolactin A, 7-O-methyl-5'-hydroxy-3'-heptenoate-macrolactin, macrolactin C, and macrolactin B), two siderophores (bacillibactin and petrobactin), and three lipopeptides (kurstakin 1, 2 or 3, and 4).

Both CFSs exhibited strong suppression of the harmful effects of A. flavus AF1 and seed-borne A. flavus on peanut and maize seedlings, as evidenced by significantly reduced Disease Incidence (DI) and Disease Severity Index (DSI) compared to the control. The Disease Control Efficacy (DCE) of CFS treatments was equivalent to that of commercial fungicide treatments. Additionally, all CFS treatments stimulated seed germination, vigor, seedling length, and weight in both peanut and maize, with Vigor Index (VI) values increasing by 222.4%–286% and 181.7%–216.4% relative to the negative control for peanut and maize seedlings at 7 days after treatment (DAT), respectively.

Conclusion: These results suggest that the CFSs of PGPR B. albusNNK24 and NDP61 hold promise as effective bioprotection agents in sustainable agriculture.

Aspergillus flavus

Bioprotection agents

Biosurfactant

GNPS

PGPR

UHPLC-ESI-QTOF/MS

Aspergillus flavus is notorious for its ability to produce aflatoxins on agricultural products. During its saprophytic life cycle, it exploits host plant nutrients for proliferation and metabolite production [1–3]. This fungus induces yellow mold disease (afla root) in peanuts, leading to seed necrosis, abortion, and rot, reduces or eliminates germination capacity, and damages seedling [4]. Additionally, it is well-established that A. flavus can colonize maize silks and infect kernels without any previous damage, adversely affecting maize seed germination and seedling health [5].

To combat aflatoxin-producing molds, farmers typically resort to chemical agents [6, 7]. Subsequently, biological control strategies, such as competitive displacement by atoxigenic A. flavus and antagonism by Trichoderma spp. or Bacillus spp., have gained attention [8–11]. While the use of atoxigenic A. flavus faces challenges in selecting non-pathogenic strains, antagonistic microbes offer a promising alternative. These microbes can produce various antifungal compounds, controlling phytopathogens more effectively than chemical fungicides, without harming the plant environment [12].

When encountering ecological competitors like phytopathogens, antagonistic microbes deploy an effective strategy, producing synergistic compounds that target various sites within harmful competitor organisms [13]. For example, Streptomyces species produce a large number of secondary metabolites that act synergistically or contingently against biological competitors, protecting themselves when confronted with saprophytic enemies [14]. The synergistic action of antimicrobial compounds produced by bacteria can aid humans in controlling resistant phytopathogens [15].

In recent decades, interest in plant growth-promoting rhizobacteria (PGPR) has grown significantly. These root-associated bacteria, natural inhabitants of plant roots, perform activities vital for plant health, including secreting compounds that directly or indirectly enhance plant defenses [16]. Similar to other antagonistic microbes, PGPR produce a diverse and dynamic array of antimicrobial compounds to combat ecological competitors [17]. Evidence from numerous studies has revealed that various plant-associated strains of Bacillus cereus group are rich sources of antimicrobial compounds [18], including renowned hybrid polyketide–non-ribosomal peptide zwittermicin A and lipopeptide kurstakin produced by B. cereus [19, 20], as well as lipopeptides like fengycin, surfactin, and iturin, along with polyketides like bacillaene produced by B. thuringiensis CHGP12 [21]. In addition to secondary metabolites, cell-wall-degrading enzymes such as chitinase, β-glucanase, and protease secreted by strain SBA12 of B. cereus group contributed to the inhibition of fungal cells [22].

However, safety concerns arise when novel isolated PGPR strains belong to genera Stenotrophomonas, Serratia, Enterobacter, Acinetobacter, Burkholderia and the Bacillus cereus group [23]. Additionally, challenges arise with the formulation and effectiveness of PGPR cells in the field, leading to low bacterial survival during product shelf life and after application within agroecosystems [24]. Consequently, an alternate approach to harness PGPR is to utilize their cell-free supernatants, containing a mixture of their metabolites, including antimicrobial compounds, to maintain the benefice of synergistic effects against phytophathogens.

In a previous study, two bacterial strains of the Bacillus cereus group, namely B. albus NNK24 and NDP61, were isolated from peanut roots, effectively reduced yellow mold disease caused by A. flavus CDP2 and promoted the growth of peanut seedlings through seed treatment. These PGPR can serve as alternatives to chemical fungicides in Vietnam, where drug resistance is often associated with their overuse [25]. To explore a safe method for developing bioprotection agents against aflatoxigenic A. flavus, this study investigated the potential use of cell-free supernatants from PGPR B. albus NNK24 and NDP61, which contain a combination of antifungal compounds, to combat Aspergillus flavus infection in peanuts and maize seedlings.

2.1. Materials

PGPR Bacillus albus strains NNK24 (GenBank accession no. ON329036) and NDP61 (GenBank accession no. ON329038) were previously isolated from peanut roots [10]. Additionally, the mold indicator, aflatoxigenic Aspergillus flavus AF1 (Genbank accession no. ON974733), was isolated from greenish-yellow moldy peanuts and was demonstrated to cause yellow mold disease in peanut seedlings [25].

2.2. In-vitro antifungal assay of PGPR against A. flavus AF1

The antifungal activities of bacterial strains against A. flavus AF1 were assayed using four different culture media (refer to Supplementary material 1) through a dual-culture technique [25].

2.3. Bacterial culture and their cell-free supernatant recovery

A. flavus AF1 was cultured in shaked conical flasks and Petri dishes to obtain micelia and spores respectively. As described by [25], 1 ml of A. flavus AF1 culture broth containing 1 g/ml mycelia and 1 ml of A. flavus AF1 spore suspension (10⁷ spores/ml) were added to 100 ml of fermentation medium (Nutrient broth). Following sterilization, each culture medium was inoculated with 5% of the respective inoculum and agitated at 27 ± 2°C, 200 rpm for 7 days. After fermentation, cell-free supernatants were obtained by centrifuging the culture broths at 4000 rpm for 15 minutes, and then underwent sterilization via filtration (using a 0.22 µm pore size membrane) for future investigations.

2.4. Turbidimetric antifungal assay of bacterial cell-free supernatants

The modified EUCAST-Aspergillus procedure (turbidimetric antifungal assay) was utilized to evaluate the effectiveness of the CFSs in inhibiting A. flavus AF1 [25].

2.5. Determination of physicochemical characteristics of bacterial cell-free supernatants

The pH of the CFSs was measured using a pH meter. The detection of fungal cell wall-degrading enzymes, including protease, chitinase, and β-1,3-glucanase in the CFSs, was conducted via the agar well diffusion method. Nutrient-free agar plates consisting of 15.0 g/L agar, supplemented with specific substrates (1% skim-milk, 2% colloidal chitin, 1% β-1,3-glucan) [26, 27], were prepared. Then, 0.1 ml of CFS was applied to the wells. Subsequently, all plates were incubated at 27 ± 2°C for 24 hours to estimate enzymatic activity by determining halo zones around the wells on agar plates.

Carbohydrates were identified through Molisch's and Barfoed’s tests. Peptides were detected using the Biuret test, while free amino groups were identified via the Ninhydrin test. Lipids in bacterial CFSs were detected through saponification reaction and Coomassie brilliant blue staining. Terpenoids and steroids were identified using the Salkowski test. Mayer's and Dragendorff's tests were employed to detect alkaloids, while phenols were identified using ferric chloride’s reagent [28–30]. Biosurfactant screening was performed using drop collapse assay, oil spread assay, and emulsification index (E24) determination [31, 32]. The E24 index (%) was calculated using the following formula:

$$\:E24\:\left(\%\right)=\frac{Heigℎt\:of\:tℎe\:emulsion\:layer}{Total\:ℎeigℎt\:of\:tℎe\:liquid}\times\:100$$

2.6. Untargeted metabolite mining of cell-free supernatants using the UHPLC-ESI-QTOF/MS instrument and molecular networking analysis

2.6.1. UHPLC-ESI-QTOF/MS analysis

An HPLC Agilent 1290 (USA) was connected to a 6545 quadrupole-time of flight mass spectrometer (LC-QTOF-MS, Agilent Technologies, USA) using an electrospray ionization (ESI) source in positive mode. Detailed information can be found in [25].

2.6.2. Data processing, molecular networking, and compounds profiling

Mass spectral data in ESI positive mode were converted using Reify's ABF converter for compatibility with MS-DIAL 4.9. The processed ABF files, with contaminants excluded [33], were exported for GNPS analysis in MGF and GNPS Sample Table formats via WinSCP [34]. Analysis was performed using the FBMN approach at GNPS [35]. Antifungal compounds were identified using DEREPLICATOR[36] and SNAP-MS, leveraging the NPAtlas microbial extract library [37]. Network Annotation Propagation (NAP) was applied with searches across multiple databases [38]. Compound classification was conducted in R (v4.2.2) using the RMolNetEnhancer package, integrating FBMN, DEREPLICATOR, NAP, and SNAP-MS outputs [39]. An external chemical classification platform was also used [40]. The network was visualized and analyzed in Cytoscape 3.9.1 [41], with details in Supplementary material 2.

2.7. Use of bacterial CFSs for peanut and maize seedling protection against A. flavus

Peanut and maize seeds were rinsed with distilled water, soaked in warm distilled water for 4 hours, and then immersed in bacterial CFSs for 30 minutes. For controls, seeds were treated with distilled water (negative control) or a commercial fungicide, Cruiser plus 312.5FS (positive control), and a blank control involved untreated seeds not exposed to A. flavus AF1. Treated seeds (30 per batch) were placed in sterilized boxes with one SDA plug/box containing 10⁵ A. flavus AF1 spores/plug and incubated at 27 ± 2°C for 7 days, following protocols [10, 25, 42]. After 7 days, parameters such as Germination Percentage (GP), Vigor Index (VI), seedling Disease Incidence (DI), Disease Severity Index (DSI), and Disease Control Efficacy (DCE) were evaluated [10, 25].

2.8. Data analysis

All experiments were conducted in 5 replicates, with the exception of the in-vivo antifungal assay on peanut seedlings, which was performed with 10 replicates. Statistical analysis of the assay data was carried out using one-way ANOVA and the TukeyHSD method to compare means. Data processing was performed using R software (version 4.2.2), and graphical representation was generated using OriginPro 2021 software.

3.1. In-vitro antifungal activity of PGPR against aflatoxigenic Aspergillus flavus AF1

Both strain Bacillus albus NNK24 and NDP61 demonstrated the capability to inhibit the growth of A. flavus AF1 across various culture media, exhibiting significant mold inhibition rates compared to the negative control (p < 0.0001) (Fig. 1). Notably, YPDA did not support bacterial growth; therefore, they could not effectively combat filamentous fungi when faced with the nutrient competitor, A. flavus AF1, resulting in a mold inhibition rate of approximately 30%. Conversely, on TSA, TGYE-A, and NA, inhibition rates exceeding 50% against A. flavus AF1 were observed. Interestingly, both strains exhibited similar efficacy without any significant difference between them (p > 0.05).

3.2. Antifungal activity of bacterial cell-free supernatants

The bacterial cell-free supernatants (CFSs) obtained from both B. albus strains demonstrated notable inhibition against A. flavus AF1 growth in Sabouraud Liquid Medium (SLM) following a 48-hour incubation period, exhibiting a significant difference compared to the control (p < 0.0001). Among the bacterial strains, NNK24 and NDP61 displayed similar antifungal inhibition rates of approximately 40%, with no statistically significant difference (p > 0.05) observed between them (Fig. 2).

3.3. Physicochemical characteristics of bacterial CFSs

Both CFSs exhibited pH levels exceeding 9.0 and lacked fungal cell wall-degrading enzymes such as protease, chitinase, and β-1,3-glucanase. Conversely, they were found to contain a diverse array of compounds spanning multiple classes, including carbohydrates, amino acids, peptides, lipids, terpenoids and/or steroids, alkaloids, and phenols. Interestingly, both CFSs demonstrated their surfactant properties, such as the collapse of their drops on oil-coated surfaces, the oil displacement clear zones compared to the negative control and E24 values of more than 50% (Table 1).

Table 1

Physicochemical characteristics of bacterial CFSs
Constituents and characteristics	Analytical methods	NNK24’s CFS	NDP61’s CFS
pH	pH meter	9.87 ± 0.07	9.25 ± 0.08
Protease	Well diffusion assay	-	-
Chitinase		-	-
β-1,3-glucanase		-	-
Carbohydrates	Molisch’s test	+	+
Carbohydrates	Barfoed’s test	+	+
Amino acids and peptides	Ninhydrin test	+	+
Amino acids and peptides	Biuret test	+	+
Lipids	CBB* staining	+	+
Lipids	Saponification test	+	+
Alkaloids	Dragendorff’s test	+	+
Alkaloids	Mayer’s test	+	+
Phenols	Ferric chloride test	+	+
Terpenoids Steroids	Salkowski test	+	+
Biosurfactants	Drop collapse’s test	+	+
	Oil spreading test	+	+
	Emulsification 24h	51.90 ± 1.78%	52.38 ± 1.35%
* Coomassie brilliant blue

3.4. Chemical classification and profiling antifungal compounds through UHPLC-ESI-QTOF/MS and bioinformatic analysis

The spectral data obtained from all bacterial CFSs were utilized to construct a molecular network (MN) based on their spectral similarities. This resulted in the formation of an MN comprising 960 nodes interconnected by 2634 edges, delineating 768 connected components. To elucidate the chemical composition of the CFSs, significant nodes within the MN were subjected to classification using Network Annotation Propagation (NAP), thereby providing structural insights for 440 nodes. Additionally, DEREPLICATOR identified one node corresponding to one compound, SNAP-MS identified six nodes for six compounds, and manual prediction identified four nodes representing five compounds. In total, structural information was obtained for 11 nodes.

As a result, the chemical structural information was classified into 22 distinct classes. Analysis revealed that lipids constituted the highest proportion of the total compounds, comprising 37.4% and 48.4% in the CFSs of NDP61 and NNK24, respectively. Notably, lipid compounds such as prenol lipids (including terpenoids and macrolactams), fatty acyls, and glycerol lipids were predominant. Conversely, compounds such as steroids and derivatives, glycerophospholipids, saccharolipids, and sphingolipids were present in minimal quantities. Carboxylic acids and derivatives, primarily composed of peptides, amino acids, and derivatives, accounted for 14% and 11.8% of the total composition, respectively. Organooxygens, consisting mainly of monosaccharides and ketones, represented only 3.6% and 4% of the total composition. Benzene and substituted derivatives constituted 3.8% and 3.6%, respectively, while peptidomimetics, indicating cyclic depsipeptides, comprised 3.1% and 1.8%, respectively, as depicted in Fig. 3. Other secondary metabolites, including total alkaloids (such as alkaloids, diazines, isoquinolines and derivatives, pyridines and derivatives, quinolines and derivatives), macrolides, macrolactams, azoles, and phenols, were present in varying proportions, most of which were less than 1%. Unfortunately, approximately 20–30% of the relative abundance (area, %) remained unclassified, appearing as "no matches". The observed variation between NNK24’s and NDP61’s CFS is noteworthy. Specifically, NNK24’s CFS exhibited higher levels of fatty acyls and glycerolphospholipids compared to NDP61’s, whereas NDP61’s CFS showed elevated levels of carboxylic acids and derivatives, glycerolipids, and peptidomimetics compared to NNK24’s CFS, as illustrated in Fig. 3.

Putative compounds in the CFSs were identified through DEREPLICATOR and SNAP-MS, utilizing spectral data and MN output, respectively. DEREPLICATOR suggested the presence of lipopeptide kurstakin 4 (C₄₁H₆₇N₁₁O₁₂, [M + H]⁺ = 906.8469). SNAP-MS data revealed the presence of two diketopiperazines, cyclo(pro-leu) (C₁₁H₁₈N₂O₂, [M + H]⁺ = 211.1442) and cyclo(2-hydroxy-Pro-R-Leu) (C₁₁H₁₈N₂O₃, [M + H]⁺ = 227.1394), alongside various members of the macrolactin family. This included 7-O-succinyl macrolactin A (C₂₈H₃₈O₈, [M + H]⁺ = 503.2658), 7-O-methyl-5'-hydroxy-3'-heptenoate-macrolactin (C₃₁H₄₄O₇, [M + H]⁺ = 529.3129), macrolactin C (C₃₁H₄₆O₉, [M + Na]⁺ = 585.2982), and macrolactin B (C₃₀H₄₄O₁₀, [M-H₂O + H]⁺ = 547.2924). Additionally, two siderophores, petrobactin (C₃₄H₅₀N₆O₁₁, [M + H]⁺ = 719.3516) and bacillibactin (C₃₉H₄₂N₆O₁₈, [M + H]⁺ = 883.6971), along with three lipopeptides, kurstakin 1 (C₃₉H₆₃N₁₁O₁₂, [M + H]⁺ = 878.8167) and kurstakin 2 (C₄₀H₆₅N₁₁O₁₂, [M + H]⁺ = 892.8292) or kurstakin 3 (C₄₀H₆₅N₁₁O₁₂, [M + H]⁺ = 892.8292), were manually annotated based on the relevance of molecular networking nodes. The entire repertoire of putatively annotated compounds is showcased in the MN analysis (Fig. 4).

3.5. Use of bacterial CFSs for peanut and maize seedling protection against A. flavus

Aspergillus flavus disease initiated with the germination of A. flavus conidia and the proliferation of its mycelia on the surface of germinating seeds. Subsequently, new conidia were generated, and as it progressed onto the seedlings, necrotic lesions appeared on cotyledons, hypocotyls, and roots (Fig. 5A1 - A2).

The blank control showed a Disease Incidence (DI) of 37.7% and a Disease Severity Index (DSI) of 0.22 in peanut seedlings, and a DI of 53.7% with a DSI of 0.40 in maize seedlings at 7 days post-germination, indicating the presence of seed - borne A. flavus-induced disease in both plant species (Fig. 6A1 - A2). A. flavus AF1 infection resulted in similar disease symptoms in both peanut and maize seedlings, although the incidence and severity varied. Notably, in the negative control group (seeds treated with H₂O), inoculating A. flavus AF1 during peanut seed germination significantly elevated the Disease Incidence (DI) and Disease Severity Index (DSI) values by 73.5% and 106.1% (p < 0.05), respectively, compared to the blank control (lacking both CFS treatment and mold infection) (Fig. 6A1). Meanwhile, A. flavus AF1 increased the DSI and DI values in maize seedlings at 7 days post-inoculation, although the difference was not statistically significant (p > 0.05) (Fig. 6A2).

Seed treatment with bacterial CFSs effectively protected the growth of both peanut and maize seedlings, significantly reducing DI and DSI compared to the negative control (p < 0.05). Specifically, bacterial CFSs decreased DI and DSI values in peanut seedlings by 46.1% − 63.8% and 45.9% − 65.8%, respectively. In maize seedlings, the CFSs exhibited slightly weaker protective effects, reducing DI and DSI values by 37.1% − 39.3% and 41.1% − 42.1%, respectively. Notably, in peanut seedlings, the DSI values for both CFS and chemical fungicide treatments did not significantly differ from the blank control (p > 0.05), as did the DI values for NNK24’s CFS and the chemical fungicide treatments. However, the DI value was lower than the blank control (p < 0.05) for NDP61’s CFS treatment. In maize seedlings, all CFS and chemical fungicide treatments resulted in significantly lower DI and DSI values compared to the blank control (p < 0.05). Consequently, the Disease Control Efficacy (DCE, %) of bacterial CFS treatments was comparable to that of chemical fungicide treatment (p > 0.05) in maize seedlings, ranging from 41.1–50.3% (Fig. 6B2). For peanut seedlings treated with NNK24’s CFS, the DCE value was 45.9%, falling within the same range as maize seedlings. Notably, NDP61’s CFS, as well as the chemical fungicide, protected peanut seedlings with DCE values of 65.8% and 57.8%, respectively, demonstrating better disease control efficacy than NNK24’s CFS (Fig. 6B1). These findings indicate that despite some differences in disease control efficacy, both CFSs and the commercial fungicide effectively mitigate A. flavus infestation in peanut and maize seedlings (Fig. 5B1 - B2).

Contrary to disease parameters such as DI and DSI, growth parameters including Germination Percentage (GP), Vigor Index (VI), and biomass (length and weight of seedlings) in the negative control were comparable to those of the blank control (p > 0.05) (Fig. 6C1 - C2, 6D1 - D2). Antifungal treatments not only shielded plants from fungal diseases but also enhanced the growth of peanut and maize seedlings. This was evident from the higher GP, VI, and biomass observed in treated seedlings compared to those in the negative and blank controls. Specifically, in peanut seedlings treated with all antifungal agents, including the commercial fungicide and bacterial CFSs, GP values increased by 43.1% − 47.9% compared to the negative control and by 27.1% – 31.4% relative to the blank control. Notably, the Vigor Index (VI) of peanut seedlings treated with bacterial CFSs significantly exceeded those treated with the commercial fungicide, increasing by 222.4% – 286% relative to the negative control and by 143.5% − 191.6% relative to the blank control, whereas the commercial fungicide increased VI by only 118.2% relative to the negative control and 64.8% relative to the blank control.

Similarly, in maize seedlings, antifungal treatments with the commercial fungicide as well as bacterial CFSs significantly increased VI values compared to the negative control by 181.7% − 216.4% and to the blank control by 142.6% − 172.4%, whereas the commercial fungicide increased VI by 153.8% relative to the negative control and 118.2% relative to the blank control (p < 0.05), while maintaining GP at similar levels to the negative control, except for NNK24’s CFS treatment, which increased GP values (p < 0.05).

Moreover, the biomass of peanut and maize seedlings treated with bacterial CFSs exhibited an improvement trend compared to those treated with H₂O and the blank control (Fig. 6D1 - D2). This enhancement was particularly noticeable in the weight of peanut seedlings and the length of both plant species (p < 0.05). Notably, both bacterial CFSs demonstrated similar, or, in some cases, superior plant growth-promoting effects compared to the commercial fungicide.

4.1. Antifungal activity of rhizobacterial CFSs against A. flavus

Extensive research has underscored the potential of Bacillus species as biological control agents against aflatoxigenic Aspergillus strains. For instance, studies have demonstrated that Bacillus species can significantly curb the growth of A. flavus and A. parasiticus on maize [43]. Similarly, various plant growth-promoting rhizobacteria (PGPR) from the Bacillus genus have proven effective in combating Aspergillus-induced diseases in peanut seedlings [44]. A previous study identified the PGPR strains B. albus NNK24 and NDP61 as potent inhibitors of A. flavus CDP2 on both agar plates and peanut plants [10]. To further substantiate their antifungal properties, these strains were tested against another aflatoxigenic strain, A. flavus AF1. Consistent with expectations, both NNK24 and NDP61 strains were able to suppress the growth of A. flavus AF1 in four distinct culture media.

To avoid the direct application of bacterial cells, we assessed the antifungal capabilities of their cell-free supernatants (CFSs). To enhance the efficacy of strains NNK24 and NDP61 against the competitor A. flavus AF1, we introduced A. flavus AF1 mycelia and spores into the fermentation medium. The findings revealed that the crude CFSs derived from B. albus NNK24 and NDP61 inhibited the growth of A. flavus AF1 by approximately 40%, a significantly higher inhibition rate compared to the control, which only included A. flavus AF1 without CFSs.

4.2. Antifungal metabolites in rhizobacterial CFSs

To outcompete their ecological competitors, bacteria typically employ diverse strategies, including the secretion of cell-wall degrading enzymes and/or secondary metabolites. Many antagonistic bacteria produce hydrolytic enzymes such as chitinase, β-glucanase, and protease, which play a crucial role in suppressing fungal growth [45]. However, in both examined CFSs, these essential fungal cell wall-degrading enzymes - namely chitinase, glucanase, and protease - were notably absent despite the addition of fungal biomass as enzyme inducers. Therefore, the antifungal activity of both CFSs was likely reliant on their secondary metabolites. Similarly, in the fermentation broth of Paenibacillus polymyxa HT16, the addition of chitin and β-glucan as inducers did not result in the presence of cell wall-degrading enzymes against fungal pathogens. However, antifungal low molecular weight compounds were detected in the culture broth supplemented with fungal biomass [46]. Another study indicated that bacteria release secondary metabolites upon interaction with fungal pathogens. For instance, rhizosphere B. subtilis was found to produce significant levels of small molecular weight compounds, effectively inhibiting the growth of competitors such as Setophoma terrestri [47]. Moreover, when exposed to phytopathogen biomass, Trichoderma asperellum produced various secondary antifungal compounds [48]. Co-cultivation of microbes is a well-known method to activate silent biosynthetic gene clusters for novel metabolite production, even when a member microbe is autoclaved [49].

The surfactant properties of bacterial cell-free supernatants (CFSs) suggest the presence of compounds with amphiphilic structures [50]. Bacillus spp. are known to synthesize many low molecular weight biosurfactants such as lipopeptides and glycolipids, where peptides or disaccharides are conjugated with long-chain fatty acids or hydroxy fatty acids [51]. These biosurfactants interact with cell membranes and can disrupt the membranes of various fungal species [52].

The composition of the CFSs, characterized by a high proportion of fatty acyls, glycerolipids, prenol lipids, cyclic depsipeptides, macrolactams, and macrolides, elucidated their surfactant nature. Several putative antifungal compounds were identified from CFS spectral data using both in-silico tools and manual predictions.

Firstly, the diketopiperazine cyclo(Pro-Leu), a compound produced by B. cereus, has demonstrated strong inhibition of mycelia growth in phytopathogenic A. flavus and A. niger, thereby impacting aflatoxin production [53].

Secondly, four macrolides from the macrolactin family were detected in both CFSs, including 7-O-succinyl macrolactin A, 7-O-methyl-5'-hydroxy-3'-heptenoate-macrolactin, macrolactin C, and macrolactin B. Remarkably, 7-O-succinyl macrolactin A, derived from B. amyloliquefaciens ELI149, exhibits robust antifungal activity against various significant filamentous fungal phytopathogens [54]. Additionally, macrolactin B and C, alongside 7-O-methyl-5'-hydroxy-3'-heptenoate-macrolactin, have been identified in B. subtilis MTCC10403 and Bacillus sp. AH159-1, respectively, showcasing broad-spectrum antibacterial activity [54, 55]. While their antifungal activity against plant phytopathogens remains unrecorded, a structurally similar compound, macrolactin R (also glycosylated similar to B and C), impacted the membrane structure of Botrytis cinerea, resulting in changes in membrane permeability, protein and nucleic acid leakage, and ultimately, cell death [56].

Thirdly, catecholate siderophores of Bacillus cereus group, petrobactin and bacillibactin, found in both CFSs, exhibit remarkable iron affinity, competitively inhibiting the growth of plant pathogens with less efficient iron-uptake systems [57, 58]. Bacillibactin produced by B. amyloliquefaciens MBI600 has been demonstrated to inhibit the growth of various fungi, including A. flavus, in iron-containing media. This suggests that its antibiotic function relies not only on iron scavenging to starve competing species but also on direct antibiosis against phytopathogenic fungi [59]. Additionally, microbial siderophores are recognized as a potential source of iron for plant survival and growth [60].

Finally, lipopeptides kurstakins (NRPs), identified in the CFSs of both NNK24 and NDP61 strains, have been previously reported to be linked to fatty acids ranging from C10 to C14, which may be β-hydroxylated or not, with two isoforms (n-, iso-). They are partially cyclic, forming a lactone bond between Ser/4 and the C-terminal Gln/7, and may also be linear [61, 62]. These amphiphilic lipopeptides act as biosurfactants, essential for swarming and biofilm formation, preventing the colonization and growth of competing microorganisms, and having a high affinity for cell membranes, through which they can form pores [20, 61, 63]. Consequently, they exhibit notable antimicrobial activities, including against various fungi. For examples, purified kurstakins 1–4 (isoC11, nC12, isoC12, isoC13), produced by B. thuringiensis kurstaki HD-1, demonstrating antifungal activity against Stachybotrys chartarum [64]. Subsequently, kurstakins (C10, C11, 12) were found to be produced by several biocontrol agents Bacillus spp. and Enterobacter cloacae C3 [62, 65]. Multiple kurstakin producers, such as B. cereus, B. thuringiensis, and B. pumilus, inhibited a certain number of fungi [66].

Taken together, it becomes evident that the majority of the identified compounds interacted with fungal cells. This observation leads us to hypothesize that the presence of fungal cells may prompt bacteria to develop strategies for producing various antifungal compounds that act through different mechanisms.

4.3. Potential to use rhizobacterial CFSs as bioprotection agents in agriculture

The infection of peanut and maize seeds with aflatoxigenic Aspergillus flavus AF1 during the germination experiment highlighted the severe impact of mycotoxigenic molds on seedling health. In both crops, the proliferation of mold mycelia and conidia, coupled with the formation of necrotic lesions on cotyledons, hypocotyls, and roots, inevitably leads to reduced productivity. The persistence of conidia throughout the plant life cycle can result in aflatoxin contamination in harvested products and during storage.

This study found that even in the blank control (seeds without A. flavus AF1 infection and without antifungal treatment), symptoms of A. flavus disease appeared on seedlings, indicating the presence of seed-borne A. flavus strains. This finding aligns with numerous reports on A. flavus in peanuts and maize [67, 68].

Treatment of seeds with CFSs of Bacillus albus NNK24 and B. albus NDP61 significantly mitigated the harmful effects caused not only by A. flavus AF1 but also by other seed-borne A. flavus strains that were indistinguishable from AF1 in peanut and maize seedlings, similar to the commercial fungicide Cruiser plus 312.5FS. This was evidenced by a notable reduction in Disease Incidence (DI) and Disease Severity Index (DSI). Moreover, these treatments promoted seedling growth, especially in peanuts, as indicated by various growth parameters, particularly the Vigor Index (VI).

However, the disease control efficacy of Cruiser Plus 312.5FS at the recommended concentration was limited (50.3–57.8%) in both seedlings. This limitation could be due to the potential resistance to the commercial fungicide despite containing two active ingredients, Difenoconazole and Fludioxonil, which have different antifungal mechanisms. This finding underscores the need for ongoing research into novel antifungal agents.

Using complex microbial metabolites, such as CFSs or their crude solvent extracts, to control phytopathogens offers several advantages. This approach avoids the safety concerns associated with using whole cells and harnesses the synergy of multiple antimicrobial compounds, thereby reducing the likelihood of early resistance development. For example, B. subtilis culture filtrate has shown inhibitory effects on A. flavus growth on peanuts [69]. Similarly, crude extracts from the CFSs of Bacillus sp. 16060, Serratia marcescens SE1, and S. marcescens SE2 have demonstrated antifungal activity against A. flavus in peanut seedlings [25]. In addition to controlling A. flavus, Bacillus extracts containing a complex mixture of antifungal compounds can reduce other plant fungal diseases [70]. As a recent trend, microbial CFSs are being explored as biostimulants and biocontrol agents in sustainable agricultures [71–74]. Furthermore, our previous study showcased the potent antifungal activity of bacterial cells of PGPR B. albus NNK24 and NDP61 in peanut seeds and plants [10]. Typically, bacterial endophytes confer various benefits upon their hosts [75]. Our study emphasizes the potential of utilizing endophyte CFSs to combat A. flavus not only in their host peanut but also in maize. This could significantly expand the application of bacterial products in sustainable agriculture, providing a safer and more effective alternative to chemical fungicides.

PGPR Bacillus albus NNK24 and NDP61 have demonstrated significant antifungal activity against A. flavus AF1on agar plates. Similarly, cell-free supernatants (CFSs) from these strains effectively inhibited its growth. The CFSs were found to predominantly contain lipid and lipid-like molecules and exhibited biosurfactant properties. Bioinformatic analysis revealed several putative antifungal compounds within the CFSs, including three cyclic lipopeptides (kurstakin 1, 2 or 3, and 4), two siderophores (petrobactin and bacillibactin), four macrolactins (7-O-succinyl macrolactin A, 7-O-methyl-5'-hydroxy-3'-heptenoate-macrolactin, macrolactin C, and macrolactin B), and two diketopiperazines (cyclo(Pro-Leu) and cyclo(2-hydroxy-Pro-Leu)). Furthermore, the CFSs significantly suppressed disease caused by A. flavus in both peanut and maize seedlings, demonstrating effectiveness comparable to the commercial fungicide while also promoting healthy seedling growth. This suggests the promising potential of these CFSs as an alternative approache to chemical fungicides in agriculture.

In this study, the bacterial CFSs were used in their crude state, maintaining the original concentrations of bioactive compounds from the culture broths. Future research will aim to enhance these bioprotection agents through downstream processes such as concentration, solvent extraction, and drying. A key objective will be determining the optimal concentration for these biological preparations. The findings of this study underscore the potential of Bacillus albus CFSs as sustainable and effective agents for crop protection, paving the way for their integration into agricultural practices.

CFS

Cell-free supernatant

DCE

Disease Control Efficacy

Disease Incidence

DSI

Disease Severity Index

FBMN

Feature-Based Molecular Networking

GNPS

Global Natural Product Social Molecular Networking

Germination Percentage

Molecular Networking

NAP

Network Annotation Propagation

NRPs

Nonribosomal peptides

PGPR

Plant growth - promoting rhizobacteria

SNAP-MS

Structural similarity Network Annotation Platform for Mass Spectrometry

UHPLC-ESI-QTOF/MS

Ultra High Performance Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry

Vigor index

Funding sources

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Ethics statements

Not applicable.

Author Contribution

T.L.L.: Conceptualization, Methodology, Investigation, Formal analysis, Data curation, Writing - Original draft preparation, Visualization.L.K.N. and L.L.H.A: Investigation and Data curation. N.H.H: Conceptualization, Methodology, Validation, Writing - Review & Editing, Supervision.

Acknowledgement

The authors express their gratitude to the Hutech Institute of Applied Science, HUTECH University, for providing the facilities essential for the successful completion of this research.

Data Availability

Data is provided within the manuscript or supplementary information files.Spectral data processed for GNPS analysis: ftp://[email protected] Molecular Networking (FBMN): ID=b579ffb703c34ffd85a41423fd974be1Dereplicator analysis: ID=d29ddb3f116c4ba7b12321253de78d76 Network Annotation Propagation (NAP) analysis: ID=c741601e3a094458a6028c6d88e5db92Chemical classification and NAP-MS analysis: https://github.com/loi032/SNAP-MS-and-Classification_NNK24-and-NDP61.git

Mellon JE, Cotty PJ, Dowd MK (2007) Aspergillus flavus hydrolases: Their roles in pathogenesis and substrate utilization. Appl Microbiol Biotechnol 77:497–504. https://doi.org/10.1007/S00253-007-1201-8
Moore GG (2021) Practical considerations will ensure the continued success of pre-harvest biocontrol using non-aflatoxigenic Aspergillus flavus strains. Crit Rev Food Sci Nutr 62:4208–4225. https://doi.org/10.1080/10408398.2021.1873731
Passone MA, Rosso LC, Etcheverry M (2012) Influence of sub-lethal antioxidant doses, water potential and temperature on growth, sclerotia, aflatoxins and aflD (= nor-1) expression by Aspergillus flavus RCP08108. Microbiol Res 167:470–477. https://doi.org/10.1016/J.MICRES.2011.11.004
Subrahmanyam P, Smith DH, Raber RA, Shepherd E (1987) An outbreak of yellow mold of peanut seedlings in Texas. Mycopathologia 1987 100:2 100:97–102. https://doi.org/10.1007/bf00467101
Gorman DP, Kang MS (1991) Preharvest Aflatoxin Contamination in Maize: Resistance and Genetics 1. Plant Breeding 107:1–10. https://doi.org/10.1111/J.1439-0523.1991.TB00522.X
Jayaprakashvel M, Mathivanan N (2011) Management of Plant Diseases by Microbial Metabolites. Bacteria in Agrobiology: Plant Nutrient Management 237–265. https://doi.org/10.1007/978-3-642-21061-7_10
Kumar A, Rabha J, Jha DK (2021) Antagonistic activity of lipopeptide-biosurfactant producing Bacillus subtilis AKP, against Colletotrichum capsici, the causal organism of anthracnose disease of chilli. Biocatal Agric Biotechnol 36:102133. https://doi.org/10.1016/J.BCAB.2021.102133
Lavkor I, Ay T, Sobucovali S, et al (2023) Non-Aflatoxigenic Aspergillus flavus: A Promising Biological Control Agent against Aflatoxin Contamination of Corn. ACS Omega 8:16779–16788. https://doi.org/10.1021/ACSOMEGA.3C00303
Siahmoshteh F, Hamidi-Esfahani Z, Spadaro D, et al (2018) Unraveling the mode of antifungal action of Bacillus subtilis and Bacillus amyloliquefaciens as potential biocontrol agents against aflatoxigenic Aspergillus parasiticus. Food Control 89:300–307. https://doi.org/10.1016/J.FOODCONT.2017.11.010
Trinh LL, Le Nguyen AM, Nguyen HH (2023) Root-associated bacteria Bacillus albus and Bacillus proteolyticus promote the growth of peanut seedlings and protect them from the aflatoxigenic Aspergillus flavus CDP2. Biocatal Agric Biotechnol 47:102582. https://doi.org/10.1016/j.bcab.2022.102582
Ren X, Zhang Q, Zhang W, et al (2020) Control of Aflatoxigenic Molds by Antagonistic Microorganisms: Inhibitory Behaviors, Bioactive Compounds, Related Mechanisms, and Influencing Factors. Toxins 12:24. https://doi.org/10.3390/TOXINS12010024
Maksimov I V., Abizgil’dina RR, Pusenkova LI (2011) Plant growth promoting rhizobacteria as alternative to chemical crop protectors from pathogens (review). Appl Biochem Microbiol 47:333–345. https://doi.org/10.1134/S0003683811040090
Jayaprakashvel M, Chitra C, Mathivanan N (2019) Metabolites of plant growth-promoting rhizobacteria for the management of soilborne pathogenic fungi in crops. Secondary Metabolites of Plant Growth Promoting Rhizomicroorganisms: Discovery and Applications 293–315. https://doi.org/10.1007/978-981-13-5862-3_15
Challis GL, Hopwood DA (2003) Synergy and contingency as driving forces for the evolution of multiple secondary metabolite production by Streptomyces species. Proceedings of the National Academy of Sciences 100:14555–14561. https://doi.org/10.1073/PNAS.1934677100
Yousfi S, Krier F, Deracinois B, et al (2024) Characterization of Bacillus velezensis 32a metabolites and their synergistic bioactivity against crown gall disease. Microbiol Res 280:127569. https://doi.org/10.1016/J.MICRES.2023.127569
Adeleke BS, Babalola OO, Glick BR (2021) Plant growth-promoting root-colonizing bacterial endophytes. Rhizosphere 20:100433. https://doi.org/10.1016/J.RHISPH.2021.100433
Subramanian S, Smith DL (2015) Bacteriocins from the rhizosphere microbiome – From an agriculture perspective. Front Plant Sci 6:150323. https://doi.org/10.3389/FPLS.2015.00909
Vater J, Tam LTT, Jähne J, et al (2023) Plant-Associated Representatives of the Bacillus cereus Group Are a Rich Source of Antimicrobial Compounds. Microorganisms 11:2677. https://doi.org/10.3390/MICROORGANISMS11112677/S1
He H, Silo-Suh LA, Handelsman J, Clardy J (1994) Zwittermicin A, an antifungal and plant protection agent from Bacillus cereus. Tetrahedron Lett 35:2499–2502. https://doi.org/10.1016/S0040-4039(00)77154-1
Yu YY, Zhang YY, Wang T, et al (2023) Kurstakin Triggers Multicellular Behaviors in Bacillus cereus AR156 and Enhances Disease Control Efficacy Against Rice Sheath Blight. Plant Dis 107:1463–1470. https://doi.org/10.1094/PDIS-01-22-0078-RE
Fatima R, Mahmood T, Moosa A, et al (2023) Bacillus thuringiensis CHGP12 uses a multifaceted approach for the suppression of Fusarium oxysporum f. sp. ciceris and to enhance the biomass of chickpea plants. Pest Manag Sci 79:336–348. https://doi.org/10.1002/PS.7203
Salwan R, Sharma V (2020) Genome wide underpinning of antagonistic and plant beneficial attributes of Bacillus sp. SBA12. Genomics 112:2894–2902. https://doi.org/10.1016/J.YGENO.2020.03.029
Keswani C, Prakash O, Bharti N, et al (2019) Re-addressing the biosafety issues of plant growth promoting rhizobacteria. Science of The Total Environment 690:841–852. https://doi.org/10.1016/J.SCITOTENV.2019.07.046
Pellegrini M, Pagnani G, Bernardi M, et al (2020) Cell-Free Supernatants of Plant Growth-Promoting Bacteria: A Review of Their Use as Biostimulant and Microbial Biocontrol Agents in Sustainable Agriculture. Sustainability 2020, Vol 12, Page 9917 12:9917. https://doi.org/10.3390/SU12239917
Trinh LL, Nguyen Ngoc MD, Nguyen HH (2024) Cell-free supernatant crude extracts of mold-competing bacteria protect peanut crops against yellow mold disease caused by Aspergillus flavus AF1. Biocatal Agric Biotechnol 103028. https://doi.org/10.1016/J.BCAB.2024.103028
Rasool A, Imran Mir M, Zulfajri M, et al (2021) Plant growth promoting and antifungal asset of indigenous rhizobacteria secluded from saffron (Crocus sativus L.) rhizosphere. Microb Pathog 150:104734. https://doi.org/10.1016/J.MICPATH.2021.104734
Zhou H, Ren ZH, Zu X, et al (2021) Efficacy of Plant Growth-Promoting Bacteria Bacillus cereus YN917 for Biocontrol of Rice Blast. Front Microbiol 12:684888. https://doi.org/10.3389/FMICB.2021.684888
Nakamura K, Handa S (1984) Coomassie brilliant blue staining of lipids on thin-layer plates. Anal Biochem 142:406–410. https://doi.org/10.1016/0003-2697(84)90484-6
Aslam I, Afridi MSK (2018) Pharmacognostic characterization of Beaumontia grandiflora (Roxb.) Wall. leaf for taxonomic identification for quality control of a drug. J Appl Res Med Aromat Plants 8:53–59. https://doi.org/10.1016/J.JARMAP.2017.11.002
Gibbs HD (1926) Phenol tests. I. A classification of the tests and a review of the literature. Chem Rev 3:291–319. https://doi.org/10.1021/CR60011A003
Rani M, Weadge JT, Jabaji S (2020) Isolation and Characterization of Biosurfactant-Producing Bacteria From Oil Well Batteries With Antimicrobial Activities Against Food-Borne and Plant Pathogens. Front Microbiol 11:64. https://doi.org/10.3389/FMICB.2020.00064
Płaza GA, Zjawiony I, Banat IM (2006) Use of different methods for detection of thermophilic biosurfactant-producing bacteria from hydrocarbon-contaminated and bioremediated soils. J Pet Sci Eng 50:71–77. https://doi.org/10.1016/J.PETROL.2005.10.005
Tsugawa H, Cajka T, Kind T, et al (2015) MS-DIAL: data-independent MS/MS deconvolution for comprehensive metabolome analysis. Nature Methods 2015 12:6 12:523–526. https://doi.org/10.1038/nmeth.3393
Aron AT, Gentry EC, McPhail KL, et al (2020) Reproducible molecular networking of untargeted mass spectrometry data using GNPS. Nature Protocols 2020 15:6 15:1954–1991. https://doi.org/10.1038/s41596-020-0317-5
Nothias LF, Petras D, Schmid R, et al (2020) Feature-based molecular networking in the GNPS analysis environment. Nature Methods 2020 17:9 17:905–908. https://doi.org/10.1038/s41592-020-0933-6
Mohimani H, Gurevich A, Mikheenko A, et al (2016) Dereplication of peptidic natural products through database search of mass spectra. Nature Chemical Biology 2016 13:1 13:30–37. https://doi.org/10.1038/nchembio.2219
Morehouse NJ, Clark TN, McMann EJ, et al (2023) Annotation of natural product compound families using molecular networking topology and structural similarity fingerprinting. Nature Communications 2023 14:1 14:1–10. https://doi.org/10.1038/s41467-022-35734-z
da Silva RR, Wang M, Nothias LF, et al (2018) Propagating annotations of molecular networks using in silico fragmentation. PLoS Comput Biol 14:e1006089. https://doi.org/10.1371/JOURNAL.PCBI.1006089
Ernst M, Kang K Bin, Caraballo-Rodríguez AM, et al (2019) MolNetEnhancer: Enhanced Molecular Networks by Integrating Metabolome Mining and Annotation Tools. Metabolites 2019, Vol 9, Page 144 9:144. https://doi.org/10.3390/METABO9070144
Djoumbou Feunang Y, Eisner R, Knox C, et al (2016) ClassyFire: automated chemical classification with a comprehensive, computable taxonomy. J Cheminform 8:1–20. https://doi.org/10.1186/S13321-016-0174-Y
Shannon P, Markiel A, Ozier O, et al (2003) Cytoscape: A Software Environment for Integrated Models of Biomolecular Interaction Networks. Genome Res 13:2498–2504. https://doi.org/10.1101/GR.1239303
Patel S, Jinal HN, Amaresan N (2017) Isolation and characterization of drought resistance bacteria for plant growth promoting properties and their effect on chilli (Capsicum annuum) seedling under salt stress. Biocatal Agric Biotechnol 12:85–89. https://doi.org/10.1016/J.BCAB.2017.09.002
Bluma R V., Etcheverry MG (2006) Influence of Bacillus spp. isolated from maize agroecosystem on growth and aflatoxin B1 production by Aspergillus section Flavi. Pest Manag Sci 62:242–251. https://doi.org/10.1002/PS.1154
Syed S, Tollamadugu NVKVP, Lian B (2020) Aspergillus and Fusarium control in the early stages of Arachis hypogaea (groundnut crop) by plant growth-promoting rhizobacteria (PGPR) consortium. Microbiol Res 240:126562. https://doi.org/10.1016/j.micres.2020.126562
Shin JH, Lee HK, Lee SC, Han YK (2023) Biological Control of Fusarium oxysporum, the Causal Agent of Fusarium Basal Rot in Onion by Bacillus spp. Plant Pathol J 39:600–613. https://doi.org/10.5423/PPJ.OA.08.2023.0118
Zhou WW, Huang JX, Niu TG (2008) Isolation of an antifungal Paenibacillus strain HT16 from locusts and purification of its medium-dependent antagonistic component. J Appl Microbiol 105:912–919. https://doi.org/10.1111/J.1365-2672.2008.03822.X
Albarracín Orio AG, Petras D, Tobares RA, et al (2020) Fungal–bacterial interaction selects for quorum sensing mutants with increased production of natural antifungal compounds. Communications Biology 2020 3:1 3:1–9. https://doi.org/10.1038/s42003-020-01342-0
De La Cruz-Quiroz R, Ascacio-Valdés JA, Rodríguez-Herrera R, et al (2019) Phytopathogen Biomass as inducer of antifungal compounds by Trichoderma asperellum under solid-state fermentation. Secondary Metabolites of Plant Growth Promoting Rhizomicroorganisms: Discovery and Applications 113–124. https://doi.org/10.1007/978-981-13-5862-3_6
Tomm HA, Ucciferri L, Ross AC (2019) Advances in microbial culturing conditions to activate silent biosynthetic gene clusters for novel metabolite production. J Ind Microbiol Biotechnol 46:1381–1400. https://doi.org/10.1007/S10295-019-02198-Y
Vieira IMM, Santos BLP, Ruzene DS, Silva DP (2021) An overview of current research and developments in biosurfactants. Journal of Industrial and Engineering Chemistry 100:1–18. https://doi.org/10.1016/J.JIEC.2021.05.017
De Giani A, Zampolli J, Di Gennaro P (2021) Recent Trends on Biosurfactants With Antimicrobial Activity Produced by Bacteria Associated With Human Health: Different Perspectives on Their Properties, Challenges, and Potential Applications. Front Microbiol 12:655150. https://doi.org/10.3389/FMICB.2021.655150
Naughton PJ, Marchant R, Naughton V, Banat IM (2019) Microbial biosurfactants: current trends and applications in agricultural and biomedical industries. J Appl Microbiol 127:12–28. https://doi.org/10.1111/JAM.14243
Nishanth Kumar S, Mohandas C, Nambisan B (2013) Purification of an antifungal compound, cyclo(l-Pro-d-Leu) for cereals produced by Bacillus cereus subsp. thuringiensis associated with entomopathogenic nematode. Microbiol Res 168:278–288. https://doi.org/10.1016/J.MICRES.2012.12.003
Salazar F, Ortiz A, Sansinenea E (2020) A Strong Antifungal Activity of 7-O-Succinyl Macrolactin A vs Macrolactin A from Bacillus amyloliquefaciens ELI149. Curr Microbiol 77:3409–3413. https://doi.org/10.1007/S00284-020-02200-2/METRICS
Zheng CJ, Lee S, Lee CH, Kim WG (2007) Macrolactins O-R, glycosylated 24-membered lactones from Bacillus sp. AH159-1. J Nat Prod 70:1632–1635. https://doi.org/10.1021/NP0701327
Ni J, Yu L, Li F, et al (2023) Macrolactin R from Bacillus siamensis and its antifungal activity against Botrytis cinerea. World J Microbiol Biotechnol 39:1–9. https://doi.org/10.1007/S11274-023-03563-X
Wilson MK, Abergel RJ, Raymond KN, et al (2006) Siderophores of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis. Biochem Biophys Res Commun 348:320–325. https://doi.org/10.1016/J.BBRC.2006.07.055
Yu X, Ai C, Xin L, Zhou G (2011) The siderophore-producing bacterium, Bacillus subtilis CAS15, has a biocontrol effect on Fusarium wilt and promotes the growth of pepper. Eur J Soil Biol 47:138–145. https://doi.org/10.1016/J.EJSOBI.2010.11.001
Dimopoulou A, Theologidis I, Benaki D, et al (2021) Direct Antibiotic Activity of Bacillibactin Broadens the Biocontrol Range of Bacillus amyloliquefaciens MBI600. mSphere 6:. https://doi.org/10.1128/MSPHERE.00376-21
Saha M, Sarkar S, Sarkar B, et al (2015) Microbial siderophores and their potential applications: a review. Environmental Science and Pollution Research 2015 23:5 23:3984–3999. https://doi.org/10.1007/S11356-015-4294-0
Béchet M, Caradec T, Hussein W, et al (2012) Structure, biosynthesis, and properties of kurstakins, nonribosomal lipopeptides from Bacillus spp. Appl Microbiol Biotechnol 95:593–600. https://doi.org/10.1007/S00253-012-4181-2
Dimkic I, Stankovic S, Nišavic M, et al (2017) The profile and antimicrobial activity of Bacillus lipopeptide extracts of five potential biocontrol strains. Front Microbiol 8:259438. https://doi.org/10.3389/FMICB.2017.00925
Dubois T, Faegri K, Perchat S, et al (2012) Necrotrophism Is a Quorum-Sensing-Regulated Lifestyle in Bacillus thuringiensis. PLoS Pathog 8:e1002629. https://doi.org/10.1371/JOURNAL.PPAT.1002629
Hathout Y, Ho YP, Ryzhov V, et al (2000) Kurstakins: A new class of lipopeptides isolated from Bacillus thuringiensis. J Nat Prod 63:1492–1496. https://doi.org/10.1021/NP000169Q
Jemil N, Hmidet N, Manresa A, et al (2019) Isolation and characterization of kurstakin and surfactin isoforms produced by Enterobacter cloacae C3 strain. Journal of Mass Spectrometry 54:7–18. https://doi.org/10.1002/JMS.4302
Labiadh M, Dhaouadi S, Chollet M, et al (2021) Antifungal lipopeptides from Bacillus strains isolated from rhizosphere of Citrus trees. Rhizosphere 19:100399. https://doi.org/10.1016/J.RHISPH.2021.100399
Achar PN, Hermetz K, Rao S, et al (2009) Microscopic studies on the Aspergillus flavus infected kernels of commercial peanuts in Georgia. Ecotoxicol Environ Saf 72:2115–2120. https://doi.org/10.1016/J.ECOENV.2009.04.002
Goko ML, Murimwa JC, Gasura E, et al (2021) Identification and Characterisation of Seed-Borne Fungal Pathogens Associated with Maize (Zea mays L.). Int J Microbiol 2021:. https://doi.org/10.1155/2021/6702856
Zhang T, Shi ZQ, Hu L Bin, et al (2008) Antifungal compounds from Bacillus subtilis B-FS06 inhibiting the growth of Aspergillus flavus. World J Microbiol Biotechnol 24:783–788. https://doi.org/10.1007/S11274-007-9533-1
da Silva Junior AL, Borges Á V., da Silva HAO, et al (2023) Lipopeptide-enriched extracts of Bacillus velezensis B157 for controlling tomato early blight. Crop Protection 172:106317. https://doi.org/10.1016/J.CROPRO.2023.106317
Morcillo RJL, Baroja-Fernández E, López-Serrano L, et al (2022) Cell-free microbial culture filtrates as candidate biostimulants to enhance plant growth and yield and activate soil- and plant-associated beneficial microbiota. Front Plant Sci 13:1040515. https://doi.org/10.3389/FPLS.2022.1040515
Pellegrini M, Pagnani G, Bernardi M, et al (2020) Cell-Free Supernatants of Plant Growth-Promoting Bacteria: A Review of Their Use as Biostimulant and Microbial Biocontrol Agents in Sustainable Agriculture. Sustainability 12:9917. https://doi.org/10.3390/SU12239917
Puttawong K, Beesa N, Kasem S, et al (2024) Potential of Bacillus spp. against root-knot nematode, Meloidogyne enterolobii parasitizing chili (Capsicum annuum L.). Crop Protection 106780. https://doi.org/10.1016/J.CROPRO.2024.106780
Izurdiaga D, Sánchez-López ÁM, Fernández-San Millán A, Poveda J (2024) Cell-free filtrates from plant pathogens: Potential new sources of bioactive molecules to improve plant health. Crop Protection 176:106477. https://doi.org/10.1016/J.CROPRO.2023.106477
Afzal I, Shinwari ZK, Sikandar S, Shahzad S (2019) Plant beneficial endophytic bacteria: Mechanisms, diversity, host range and genetic determinants. Microbiol Res 221:36–49. https://doi.org/10.1016/j.micres.2019.02.001

No competing interests reported.

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Cell-free supernatants from plant growth - promoting rhizobacteria Bacillus albus strains control Aspergillus flavus disease in peanut and maize seedlings

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Journal Publication

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Abstract

Figures

1. Background

2. Materials and methods

2.1. Materials

2.2. In-vitro antifungal assay of PGPR against A. flavus AF1

2.3. Bacterial culture and their cell-free supernatant recovery

2.4. Turbidimetric antifungal assay of bacterial cell-free supernatants

2.5. Determination of physicochemical characteristics of bacterial cell-free supernatants

2.6. Untargeted metabolite mining of cell-free supernatants using the UHPLC-ESI-QTOF/MS instrument and molecular networking analysis

2.6.1. UHPLC-ESI-QTOF/MS analysis

2.6.2. Data processing, molecular networking, and compounds profiling

2.7. Use of bacterial CFSs for peanut and maize seedling protection against A. flavus

2.8. Data analysis

3. Results

3.1. In-vitro antifungal activity of PGPR against aflatoxigenic Aspergillus flavus AF1

3.2. Antifungal activity of bacterial cell-free supernatants

3.3. Physicochemical characteristics of bacterial CFSs

3.4. Chemical classification and profiling antifungal compounds through UHPLC-ESI-QTOF/MS and bioinformatic analysis

3.5. Use of bacterial CFSs for peanut and maize seedling protection against A. flavus

4. Discussion

4.1. Antifungal activity of rhizobacterial CFSs against A. flavus

4.2. Antifungal metabolites in rhizobacterial CFSs

4.3. Potential to use rhizobacterial CFSs as bioprotection agents in agriculture

5. Conclusion, limitations, and further prospects

Abbreviations

Declarations

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Supplementary Files

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