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Article

Microbial Interactions as a Sustainable Tool for Enhancing PGPR Antagonism against Phytopathogenic Fungi

by
Ana M. Santos
1,
Ana Soares
1,2,
João Luz
3,
Carlos Cordeiro
3,
Marta Sousa Silva
3,
Teresa Dias
1,
Juliana Melo
1,
Cristina Cruz
1,* and
Luís Carvalho
1,2
1
cE3c—Centre for Ecology, Evolution and Environmental Changes & Change—Global Change and Sustainability Institute, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, Bloco C2, 1749-016 Lisboa, Portugal
2
BioScale, Rua Nova da CEE, 2005-008 Santarém, Portugal
3
Laboratório de FTICR e Espectrometria de Massa Estrutural, Departamento de Química e Bioquímica, Biosystems and Integrative Sciences Institute (BioISI), Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal
*
Author to whom correspondence should be addressed.
Sustainability 2024, 16(5), 2006; https://doi.org/10.3390/su16052006
Submission received: 29 January 2024 / Revised: 19 February 2024 / Accepted: 20 February 2024 / Published: 29 February 2024
(This article belongs to the Special Issue Environmental Microbiology and Biotechnology)
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Abstract

:
Microbial interactions, which regulate the dynamics of eco- and agrosystems, can be harnessed to enhance antagonism against phytopathogenic fungi in agriculture. This study tests the hypothesis that plant growth-promoting rhizobacteria (PGPR) can also be potential biological control agents (BCAs). Antifungal activity assays against potentially phytopathogenic fungi were caried out using cultures and cell-free filtrates of nine PGPR strains previously isolated from agricultural soils. Cultures of Bacillus sp. BS36 inhibited the growth of Alternaria sp. AF12 and Fusarium sp. AF68 by 74 and 65%, respectively. Cell-free filtrates of the same strain also inhibited the growth of both fungi by 54 and 14%, respectively. Furthermore, the co-cultivation of Bacillus sp. BS36 with Pseudomonas sp. BS95 and the target fungi improved their antifungal activity. A subsequent metabolomic analysis using Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) identified fengycin- and surfactin-like lipopeptides (LPs) in the Bacillus sp. BS36 cell-free filtrates, which could explain their antifungal activity. The co-production of multiple families of LPs by Bacillus sp. BS36 is an interesting feature with potential practical applications. These results highlight the potential of the PGPR strain Bacillus sp. BS36 to work as a BCA and the need for more integrative approaches to develop biocontrol tools more accessible and adoptable by farmers.

1. Introduction

Plant diseases cause major economic losses and pose threats to food and environmental safety. Phytopathogenic fungi are responsible for 70 to 80% of plant diseases, negatively affecting crop growth and yield [1]. The current management of plant diseases comprises an overuse of synthetic fungicides, which has several undesirable effects on the environment, including on beneficial microorganisms and human health [2,3]. Over recent years, limitations on the use of pesticides in European agriculture have been implemented. For instance, the EU Farm to Fork strategy aims to reduce the use of synthetic pesticides on crops by 50% by 2030 [4]. In this context, the use of plant growth-promoting rhizobacteria (PGPR) for biological control of phytopathogenic fungi is a valuable and sustainable alternative to the use of synthetic fungicides [5].
The antagonistic action of PGPR often relies on their production of bioactive metabolites. Pseudomonas and Bacillus are commonly used as biological control agents (BCAs) due to their ability to produce metabolites with different structures and broad-spectrum antifungal activity [6]. Pseudomonas species are able to produce a wide array of antifungal metabolites, including 2,4-diacetylphloroglucinol [7,8], cell-wall-degrading enzymes (CWDEs) [9,10], phenazines [11,12], pyoluteorin [8], pyrrolnitrin [13,14], and volatile organic compounds (VOCs) [15,16]. Bacillus species also produce important antifungal compounds, including CWDEs [17,18] and VOCs [19,20]. In addition, both genera are known for their ability to produce lipopeptides (LPs) [21,22,23,24,25]. LPs are amphiphilic secondary metabolites with a low molecular weight synthesised by multi-enzyme complexes known as non-ribosomal peptide synthetases (NRPSs). Pseudomonas and Bacillus species produce a variety of LPs with different structural characteristics. These differences comprise variations in the amino acids of the peptide domain and variations in the length and composition of the lipid tail [26]. Bacillus is the most studied genus capable of secreting LPs [27]. Bacillus produces LPs that are commonly classified into three main families: fengycin [28], iturin [29], and surfactin [30]. Compounds from different families vary in their structure and therefore in their antimicrobial properties [31]. Fengycin and iturin are the main LPs with strong antifungal activity against phytopathogens [28,32,33,34].
In nature, the biosynthesis and accumulation of natural products, such as LPs and other metabolites, are triggered by interactions between microorganisms, such as competition for nutrients and space. However, certain biosynthetic pathways responsible for producing secondary metabolites are silenced in laboratory conditions where axenic cultures are maintained [35]. Thus, mimicking the natural microbial environment using the mixed fermentation of different microorganisms (co-cultivation) promotes microbial interactions. This can increase the accumulation of certain metabolites or activate the expression of silent biosynthetic gene clusters, leading to the production of new metabolites [36]. Therefore, microbial co-cultivation has been used to elicit the synthesis of antimicrobial secondary metabolites in an attempt to increase antifungal activity [36,37,38].
The aims of this study were to (1) compare a set of PGPR strains for their ability to supress the growth of potentially phytopathogenic fungi in an attempt to select suitable candidates for BCAs; (2) establish microbial interactions through co-cultivation as an approach to increasing the production of existing and new metabolites and enhancing the antifungal activity of the PGPR; and (3) determine the nature and stability of extracellular metabolites with antimicrobial activity.

2. Materials and Methods

2.1. PGPR Strains

Nine PGPR strains, previously isolated from agricultural soils and characterised for their plant growth-promoting potential, were obtained from a culture collection maintained in the company Bioscale (Santarém, Portugal). The strains had been stored in Nutrient Broth (NB, Biokar Diagnostics, Allonne, France) supplemented with 20% (w/v) glycerol at −80 °C and were revitalized on Nutrient Agar (NA, Biokar Diagnostics, Allonne, France) at 28 °C for 24 h or until visible growth. The incubation time in NB was optimised to reach a minimum concentration of 107 CFU mL−1. For the antifungal activity assays, the strains were sub-cultured on NB at 28 °C, 160 rpm, for 24–48 h, depending on the PGPR strain, with the initial concentration set to 106 CFU mL−1.

2.2. Fungal Strains

Alternaria sp. AF12 and Fusarium sp. AF68 were obtained from a fungal collection maintained in Bioscale (Santarém, Portugal). Four additional fungal strains, Alternaria sp. FP3, Botrytis sp. SM-D1, Fusarium sp. SM-D3, and Stemphylium sp. FP5, were recently isolated from leaves of Pyrus communis L. cv. “Rocha” with brown spots. All fungal strains had been stored in glycerol 10% (w/v) at −80 °C and were grown on Potato Dextrose Agar (PDA, Biokar Diagnostics, Allonne, France) at 28 °C for 5–6 days prior to their use in the antifungal activity assays. Fungal strains were identified based on conidia morphology and ITS sequence analysis.

2.3. Genomic DNA Extraction, PCR, and Phylogenetic Analysis of PGPR Strains

To identify the PGPR strains, the genomic DNA (gDNA) of the bacterial strains was extracted following a modified and optimised version of the guanidium thiocyanate method described by Pitcher et al. [39]. The partial 16S rRNA gene was amplified using the forward primer PA/8F (5′-AGAGTTTGATCCTGGCTCAG-3′) [40,41] and the reverse primers 907R (5′-CCGTCAATTCMTTTRAGTTT-3′) [42] or 1392R (5′-ACGGGCGGTGTGTRC-3′) [43]. PCR reactions were performed in a Biometra Uno II thermal cycler (Biometra GmbH, Göttingen, Germany) under the following conditions: initial denaturation at 94 °C for 3 min, followed by 35 cycles at 94 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min, and final extension at 72 °C for 3 min. The amplified products were sequenced by Eurofins Genomics (Ebersberg, Germany) and the newly generated 16S rRNA gene sequences were blasted against GenBank. Maximum likelihood (ML) trees were constructed using MEGA X (v. 10.2.6) and the General Time Reversible (GTR) nucleotide substitution model. The best-scoring ML tree was estimated by conducting a bootstrap analysis of 1000 replicates.

2.4. Antifungal Activity in Dual Culture Assay

The antifungal activity of the PGPR strains was tested using in vitro dual culture assays in Petri plates (60 mm diameter) containing 10 mL of PDA. A mycelial disc with a 6 mm diameter from the target fungal strain was inoculated on the centre of the Petri plate, whereas four aliquots (5 µL) of the fresh bacterial monoculture were inoculated on the periphery. Control plates were prepared using sterile ultrapure water aliquots. After incubation at 28 °C for 6 days, the radial fungal growth was measured in the control and treated plates. Mycelial growth inhibition (MGI, in%) was expressed as the radial reduction in the fungal mycelium observed in the treated plates compared to the corresponding control plates, and was calculated using the following equation:
M G I   ( % ) = ( R 1 − R 2 ) R 1 ∗ 100
where R1 is the radial growth of the fungal mycelium in the control plates and R2 is the radial growth of the fungal mycelium in the treated plates. The experiment was carried out with four replicates for the control and for each bacterium–fungus combination.

2.5. Antifungal Activity of Extracellular Metabolites in Cell-Free Filtrates

Bacterial cells were removed by centrifugation of the fresh bacterial monoculture (after 24–48 h, as described in Section 2.1) three times at 3220× g for 15 min at 4 °C. The supernatant was filtered through a 0.22 µm pore size membrane (Merck Millipore, Darmstadt, Germany). The resulting cell-free filtrate was added to PDA at a final concentration of 10% (v/v) (1 mL of bacterial filtrate and 9 mL of PDA). A mycelial disc with a 6 mm diameter from the target fungal strain was inoculated on the centre of the Petri plate. Control plates were prepared using PDA with sterile ultrapure water. The plates were incubated at 28 °C for 6 days. The radial fungal growth was measured in the control and treated plates, and the MGI was determined as described above (see Section 2.4). The experiment was carried out with four replicates for the control and for each bacterium–fungus combination.

2.6. Bacterial Co-Cultivation including Bacillus sp. BS36

To increase the antifungal activity of the cell-free filtrates, Bacillus sp. BS36 was co-cultured with additional PGPR strains. A total of eight pairwise interactions were tested. From fresh bacterial subcultures, each pair was inoculated on NB, with the initial inoculum of each strain set at 5 × 105 CFU mL−1 (1:1). The cultures were incubated at 28 °C and 160 rpm for 48 h. The antifungal activity of the cultures’ filtrates against Alternaria sp. AF12 and Fusarium sp. AF68 was determined as described above (see Section 2.4).

2.7. Co-Cultivation of Bacillus sp. BS36 and Inactivated Fungal Cells

To increase the antifungal activity of the cell-free filtrates, Bacillus sp. BS36 was cultivated in the presence of thermally inactivated cells of the target microorganisms, Alternaria sp. AF12 and Fusarium sp. AF68. Fungal strains were grown on Brain Heart Infusion (BHI, Biokar Diagnostics, Allonne, France) at 28 °C and 160 rpm for 3 days. The cultures were centrifuged three times at 12,000× g for 20 min at 4 °C. Each pellet was resuspended in an equal volume of NB and autoclaved at 121 °C for 30 min. The resulting suspension was added to NB at a final concentration of 10% (v/v), and Bacillus sp. BS36 was inoculated with an initial concentration set to 106 CFU mL−1. The cultures were incubated at 28 °C and 160 rpm for 48 h. The antifungal activity of the cultures’ filtrates against Alternaria sp. AF12 and Fusarium sp. AF68 was determined as described above (see Section 2.4).

2.8. Effects of Proteinase K Treatment and Heating on Antifungal Activity of Bacillus sp. BS36 Cell-Free Filtrates

To evaluate the stability of the antifungal metabolites produced by Bacillus sp. BS36, the cell-free filtrates were exposed to physicochemical treatments, with 0.1 mg mL−1 proteinase K (Invitrogen, Carlsbad, CA, USA) at 37 °C for 60 min or incubated at 80 °C for 30 min, before being used for antifungal activity assays. The antifungal activity against Alternaria sp. AF12 and Fusarium sp. AF68 was determined as described above (see Section 2.4).

2.9. Antifungal Activity of Crude Extracts of LPs from Bacillus sp. BS36

The antifungal activity of the LPs present in the Bacillus sp. BS36 cell-free filtrates was investigated. The LPs were extracted by acid precipitation and solvent extraction. The filtrates were precipitated by adjusting the pH to 2.0 with concentrated HCl and were stored overnight at 4 °C. The precipitates were collected by centrifugation at 12,000× g for 30 min at 4 °C and extracted with methanol at room temperature and 160 rpm. The solution was filtered through Whatman filter paper No. 1 (Whatman, London, UK) and dried with a rotary vacuum evaporator SyncorePlus (Buchim, Flawil, Switzerland) at 56 °C, 278 bar, 160 rpm, for 3–4 h. The remaining extract of the LPs was dissolved in 1× PBS buffer (pH 7.3–7.4) (Invitrogen, Carlsbad, CA, USA) and filtered through a 0.22 µm pore size membrane (Merck Millipore, Darmstadt, Germany). The antifungal activity against Alternaria sp. AF12 and Fusarium sp. AF68 was determined as described above (see Section 2.4).

2.10. FTICR-MS and Data Analysis

The presence of LPs in the cell-free filtrates of Bacillus sp. BS36 was screened following an untargeted metabolomics approach using Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS). Two samples were prepared by diluting the filtrate 2-fold in methanol (500 µL of filtrate in 500 µL of methanol). For internal mass spectrum calibration, human leucine enkephalin was added ([M + H]+ = 556.27657 Da) at a concentration of 0.1 µg mL−1. Formic acid (MS grade, Sigma-Aldrich, St. Louis, MO, USA) was also added to the samples at a final concentration of 0.1% (v/v). The samples were ionized by electrospray ionization in positive mode (ESI+) and spectra were acquired between 200 and 1500 m/z. The mass spectra were analysed using the software package Data Analysis 5.0 and MetaboScape 5.0 (Brüker Daltonics, Bremen, Germany), considering peaks with a minimum signal-to-noise ratio of 4. The spectra were aligned and compound identification was performed using the Human Metabolome Database (HMDB, from 27 May 2022) [44] and LOTUS (from 16 September 2022) [45], uploaded to MetaboScape 5.0, considering the adducts H+, Na+, and K+, and a mass deviation of less than 1 ppm.

2.11. Statistical Analysis

The MGI values are presented as the means of four independent replicates with standard deviation (SD). The data were analysed statistically by one-way ANOVA, the Tukey post hoc test, and the independent samples t-test using the software IBM SPSS Statistics (v. 29).

3. Results

3.1. PGPR Strains Identification

In this study, we amplified the 16S rRNA gene of the nine PGPR strains and an ML analysis was generated from 38 aligned sequences (Figure 1). Based on these molecular data, the PGPR strains were clustered with three different genera, including Bacillus (BS36 and BS84), Priestia (BS1 and BS90), and Pseudomonas (BS2, BS3, BS27, BS94, and BS95), with moderate or high bootstrap values (Figure 1).

3.2. Screening of Antifungal Activity of PGPR Strains

The nine PGPR strains showed a wide range of MGI against both potentially phytopathogenic fungi, Alternaria sp. AF12 and Fusarium sp. AF68 (Table 1). The antifungal activity against Alternaria sp. AF12 varied from 0 to 74.0% (higher for Bacillus sp. BS36), and against Fusarium sp. AF68, varied from 0 to 65.4% (higher for Bacillus sp. BS36). Bacillus sp. BS36 (74.0 and 65.4%), Pseudomonas sp. BS95 (38.5 and 61.8%), and Pseudomonas sp. BS2 (20.2 and 7.8%) were the most efficient strains in the MGI of Alternaria sp. AF12 and Fusarium sp. AF68, respectively. Pseudomonas sp. BS94 also slightly inhibited the growth of both fungi. All Pseudomonas (BS2, BS3, BS27, BS94, and BS95) strains were able to inhibit the growth of Fusarium sp. AF68. The Priestia (BS1 and BS90) strains were the only ones that did not inhibit the growth of any of the fungal strains (Table 1).
Bacillus sp. BS36 and Pseudomonas sp. BS95 were selected for additional studies of antifungal activity against four potentially phytopathogenic fungi recently isolated from Pyrus communis L. cv. “Rocha” brown spots (Figure 2). Bacillus sp. BS36 revealed the highest MGI of Alternaria sp. FP3 (82.7 ± 4.3%), Botrytis sp. SM-D1 (60.6 ± 1.7%), Fusarium sp. SM-D3 (69.2 ± 0.0%), and Stemphylium sp. FP5 (76.9 ± 0.0%). Pseudomonas sp. BS95 only inhibited the growth of Alternaria sp. FP3 (38.5 ± 4.7%) and Stemphylium sp. FP5 (29.8 ± 1.7%) (Figure 2).

3.3. Antifungal Activity of Extracellular Metabolites in Cell-Free Filtrates

The cell-free filtrates derived from cultures of Bacillus sp. BS36 and Pseudomonas sp. BS95 were also tested for their antifungal activity against Alternaria sp. AF12 and Fusarium sp. AF68 (Figure 3). The Bacillus sp. BS36 filtrates inhibited the growth of both fungal strains (53.9% MGI for Alternaria sp. AF12 and 14.4% MGI for Fusarium sp. AF68). However, the Pseudomonas sp. BS95 filtrates only inhibited the growth of Alternaria sp. AF12 (5.8%). The MGI induced by the cell cultures of both PGPR strains was significantly higher (p ≤ 0.05) than that induced by the corresponding cell-free filtrates. For instance, the MGI of Fusarium sp. AF68 significantly increased (p ≤ 0.05) from 0 to 61.8% in the presence of Pseudomonas sp. BS95 cell cultures when compared to the respective cell-free filtrates (Figure 3). The results showed that Bacillus sp. BS36 was the PGPR strain with the best performance in suppressing the growth of the tested fungi. Therefore, experiments were carried out to induce and characterise the antifungal activity of the strain.

3.4. Bacterial Co-Culturing

The liquid co-cultivation of Bacillus sp. BS36, the strain with higher antifungal activity, with the remaining eight PGPR strains was evaluated through pairwise interactions. The cell-free filtrates of the co-cultures were tested for their antifungal activity against Alternaria sp. AF12 and Fusarium sp. AF68. The interspecific co-culture of Bacillus sp. BS36 and Pseudomonas sp. BS95 was the only one that exhibited improved antifungal activity. The MGI of Fusarium sp. AF68 induced by the cell-free filtrates of the co-cultures (20.5%) was significantly higher (p ≤ 0.05) than those induced by the Bacillus sp. BS36 and Pseudomonas sp. BS95 monocultures’ filtrates (14.4 and 0%, respectively). Yet, the high antifungal activity against Alternaria sp. AF12 detected for the Bacillus sp. BS36 monocultures’ filtrates (53.9%) was not verified in the co-culture assay (30.7%) (Table 2).

3.5. Effect of Target Microorganism on Increasing Antifungal Activity

The MGI of Alternaria sp. AF12 and Fusarium sp. AF68 induced by Bacillus sp. BS36 changed with the addition of heat-inactivated cells of the target microorganisms (Table 3). The addition of inactivated cells of Alternaria sp. AF12 and Fusarium sp. AF68 significantly increased (p ≤ 0.05) the growth inhibition of Fusarium sp. AF68. However, only the addition of inactivated cells of Fusarium sp. AF68 significantly increased (p ≤ 0.05) the growth inhibition of Alternaria sp. AF12 (Table 3).

3.6. Characterisation of Antifungal Metabolites Produced by Bacillus sp. BS36

In order to obtain an insight into the chemical nature of the antifungal metabolites, the Bacillus sp. BS36 cell-free filtrates were tested for their antifungal activity against Alternaria sp. AF12 and Fusarium sp. AF68 before and after physicochemical treatments. As shown in Figure 4, proteinase K digestion and heat treatment did not significantly (p > 0.05) affect the antifungal activity of the Bacillus sp. BS36 filtrates against Fusarium sp. AF68. However, heating the cell-free filtrates at 80 °C for 30 min significantly increased (p ≤ 0.05) the growth inhibition of Alternaria sp. AF12 compared to the untreated filtrates (from 59.6 to 71.2%). The antifungal activity against Alternaria sp. AF12 produced by the Bacillus sp. BS36 filtrates was resistant to enzymatic digestion by proteinase K (Figure 4).
LPs were extracted from the cell-free filtrates of Bacillus sp. BS36 by acid precipitation and methanol extraction. The antifungal activity of the crude extracts of the LPs was tested against Alternaria sp. AF12 and Fusarium sp. AF68 and compared with that of the untreated filtrates. The extracts of the LPs inhibited the growth of both fungal strains (65.4% MGI for Alternaria sp. AF12, and 7.7% MGI for Fusarium sp. AF68), and there was no significant difference (p > 0.05) between the untreated and extracted filtrates (Figure 4).

3.7. LPs Detection by FTICR-MS

The cell-free filtrates from Bacillus sp. BS36 were analysed with FTICR-MS. The FTICR-MS analysis revealed that fengycin- and surfactin-like LPs were produced by Bacillus sp. BS36. A peak with an m/z ratio of 1485.78515 corresponds to the mass of a [M + Na]+ ion of fengycin-C16 (C72H110N12O20) with a molecular weight of 1462.79538 Da. A peak with an m/z ratio of 1074.64629 corresponds to the mass of a [M + K]+ ion of surfactin-C15 (C53H93N7O13) with a molecular weight of 1035.68259 Da. No significant peaks for other LPs were detected in the FTICR-MS analysis.

4. Discussion

Our data suggest that PGPR, including some of selected strains of Bacillus and Pseudomonas, may also work as BCAs. Antagonism is a significant mechanism for suppressing pathogen growth through the production of antifungal compounds [46,47]. The use of antagonistic strains is one of the most important biological control technologies for disease management due to its environmental and biological safety.
A phylogenetic analysis of 16S rRNA showed the cluster of the PGPR strains under study with three different genera, namely Bacillus, Priestia, and Pseudomonas. The identification of the strains at the species level was not possible and would only be accomplished with additional analyses using other suitable DNA barcodes [48,49,50].
It was found that most Bacillus and Pseudomonas strains under study had direct antifungal activity against Alternaria sp. AF12 and/or Fusarium sp. AF68 using dual culture assays. These results are in line with previous studies reporting the high antifungal efficiency of several strains of Bacillus and Pseudomonas spp. against relevant phytopathogenic species of Alternaria and Fusarium [20,25,51,52,53,54,55,56]. In contrast, none of the Priestia strains showed antifungal activity against both fungi. Bacillus sp. BS36 and Pseudomonas sp. BS95 were the most efficient strains in inhibiting the growth of Alternaria sp. AF12 and Fusarium sp. AF68. Moreover, Bacillus sp. BS36 showed high antifungal activity against Alternaria sp. FP3, Botrytis sp. SM-D1, Fusarium sp. SM-D3, and Stemphylium sp. FP5, acting as a broad-spectrum antagonistic strain.
Cell-free filtrates of Bacillus sp. BS36 showed antifungal activity against both Alternaria sp. AF12 and Fusarium sp. AF68. These results suggest that this strain secreted antifungal metabolites that directly inhibited the phytopathogenic fungi. Bacillus spp. are known to produce a wide range of peptide and non-peptide antimicrobial compounds [26,57,58]. However, higher antifungal activity was recorded when cell cultures were used in dual culture assays, as also shown by Elshafie et al. [59]. As reviewed by Zhang et al. [60], microorganisms can influence the environment and induce the production of specific metabolites by other microorganisms. Thus, the interactions between antagonistic bacteria and targeted fungi may stimulate the antagonistic bacteria to produce or over-express certain antifungal compounds.
Considering the importance of microbial interactions in the production of new antimicrobial compounds [61,62], Bacillus sp. BS36 was co-cultured with the other PGPR strains in an attempt to obtain cell-free filtrates enriched with new antifungal metabolites and, therefore, with increased antifungal activity. The co-culture of Bacillus sp. BS36 and Pseudomonas sp. BS95 showed improved antifungal activity against Alternaria sp. AF12. Wu et al. [63] showed that microbial interactions through the co-culturing of biocontrol microorganisms enhanced antifungal activity against B. cinerea due to the production of specific compounds. Li et al. [64] demonstrated similar effects against F. graminearum. Thus, the present results suggest that the interaction between Bacillus sp. BS36 and Pseudomonas sp. BS95 under co-culture conditions induces the production of specific antifungal compounds able to inhibit the growth of Alternaria sp. AF12. These cell-free filtrates obtained by bacterial co-culture would have an advantage in the production of BCAs.
Although there is evidence that co-culturing with heat-inactivated cells, cell-free filtrates, or extracts may not always be sufficient to induce the production of secondary metabolites [61], Bacillus sp. BS36 was also co-cultured with heat-inactivated cells of the target microorganisms, Alternaria sp. AF12 and Fusarium sp. AF68, in an attempt to obtain cell-free filtrates with increased antifungal activity. The co-culture of Bacillus sp. BS36 with heat-inactivated cells of Fusarium sp. AF68 showed improved antifungal activity against both fungi, while the co-culture of Bacillus sp. BS36 with heat-inactivated cells of Alternaria sp. AF12 showed improved antifungal activity only against Fusarium sp. AF68. It can be hypothesised that the secretion of antifungal metabolites by Bacillus sp. BS36 was induced by a stimulus from the target microorganism, which resulted in cell-free filtrates with increased antifungal activity.
Microbial interactions play a crucial role in shaping the dynamics of biological communities [65]. This study shows that these interactions can be exploited in vitro to enhance antagonism against phytopathogenic fungi, which is instrumental in the transition to more sustainable agriculture. Moreover, the study provides evidence that PGPR may also be efficient BCAs and that their biological control activity may be mediated through the production of antimicrobial metabolites. These metabolites’ production can be enhanced through specific co-culturing conditions. Understanding the intricate microbial interactions and their potential to enhance antagonism against phytopathogenic fungi requires a holistic approach that considers the ecological and molecular aspects of these relationships.
The extracellular metabolites of Bacillus sp. BS36 had the same antifungal activity against Alternaria sp. AF12 and Fusarium sp. AF68, even after proteinase K treatment. However, antifungal activity against Alternaria sp. AF12 significantly increased after heating to 80 °C as compared to the untreated control filtrate. These findings suggest that the antifungal activity of cell-free filtrates against Alternaria sp. AF12 may be due, in part, to heat labile compounds. It can be hypothesised that the high temperature may have caused the degradation of certain compounds, resulting in the maturation of BCA or formation of new bioactive compounds with stronger antifungal properties. Alternatively, it may have affected metabolites that negatively interacted with the antimicrobial metabolites.
Moreover, the untreated cell-free filtrates had the same antifungal activity against Alternaria sp. AF12 and Fusarium sp. AF68 as the crude extracts of LPs. These results suggest that the LPs’ secretion was involved in the in vitro inhibition of the growth of both fungi by Bacillus sp. BS36, as antifungal activity did not decrease in the presence of crude extracts of the LPs. LPs are secondary metabolites of particular interest due to their unique structure and bioactivity. LPs can be resistant to hydrolysis by peptidases and proteases and can withstand relatively high temperatures [66].
According to the FTICR-MS analysis, Bacillus sp. BS36 co-produced two types of LPs, fengycin and surfactin. The presence of these metabolites in the cell-free filtrates could explain their high efficiency in suppressing the growth of the potentially phytopathogenic fungi. Fengycin and surfactin are produced by several Bacillus strains and have antagonistic activity against different microorganisms [22,67,68,69,70]. Fengycin is known for its high fungitoxicity, specifically against filamentous fungi [28,32,71]. Surfactin is not fungitoxic by itself, but it can contribute to indirect protection processes that involve the induced systemic resistance of the host plant [72,73,74]. Previous studies have described the co-production of fengycin and surfactin by Bacillus strains and their synergistic effects in the control of plant diseases [72]. The co-production of multiple families of LPs by Bacillus sp. BS36 is an interesting and potentially useful feature. Further characterisation of these molecules could be relevant in reducing the use of synthetic pesticides.

5. Conclusions

Our data show that the PGPR Bacillus sp. BS36 was the most effective strain in suppressing the growth of the two potentially phytopathogenic fungi, Alternaria sp. AF12 and Fusarium sp. AF68. Additionally, this strain exhibited strong antifungal activity against four other potentially phytopathogenic fungi, namely Alternaria sp. FP3, Botrytis sp. SM-D1, Fusarium sp. SM-D3, and Stemphylium sp. FP5. Although the cell cultures of Bacillus sp. BS36 inhibited fungal growth more than its cell-free filtrates, the latter also revealed high antifungal activity. It was shown that these cell-free filtrates contained fengycin- and surfactin-like lipopeptides, which may be responsible for its antifungal activity. This study also highlights the potential of microbial co-culturing to enhance the antifungal activity of Bacillus sp. BS36, and therefore supports that it should be further explored as a candidate to develop new BCA products.
To characterise Bacillus sp. BS36 as a potential BCA, it is essential to identify it at the species level. Therefore, additional molecular analyses using alternative primers/probes or long-read sequencing should be carried out. Evaluating the potential of PGPR to work as BCAs under field conditions is crucial to understanding how biotic and abiotic factors affect their activity and interactions. Moreover, the successful implementation of BCAs requires sustainable agricultural practices that promote the establishment and persistence of beneficial microorganisms in the plant ecosystem.

Author Contributions

Conceptualization, A.M.S., C.C. (Cristina Cruz) and L.C.; methodology, A.M.S., C.C. (Cristina Cruz) and L.C.; investigation, A.M.S., A.S. and J.M.; resources, C.C. (Cristina Cruz) and L.C.; data acquisition: A.M.S. and A.S.; data curation, A.M.S. and T.D.; statistical analysis, A.M.S. and L.C.; FTICR-MS analysis, C.C. (Carlos Cordeiro), M.S.S. and J.L.; writing—original draft preparation, A.M.S.; writing—review and editing, A.M.S., C.C. (Cristina Cruz), L.C. and T.D.; supervision, C.C. (Cristina Cruz) and L.C.; project administration, C.C. (Cristina Cruz) and L.C.; funding acquisition, C.C. (Cristina Cruz) and L.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by (i) Portuguese funds from Fundação para a Ciência e a Tecnologia through the project UIDB/00329/2020 (DOI 10.54499/UIDB/00329/2020), the Researcher contract to T.D. (DOI 10.54499/DL57/2016/CP1479/CT0009), the PhD grant to J.L. (2023.05150.BDANA), the Portuguese Mass Spectrometry Network (LISBOA-01-0145-FEDER-022125) and the BioISI research centre (UIDB/04046/2020-DOI: 10.54499/UIDB/04046/2020 and UIDP/04046/2020-DOI: 10.54499/UIDP/04046/2020). We also acknowledge the support from the European project EU_FT-ICR_MS, funded by the European research and innovation programme Horizon 2020 (project no. 731077). The APC was funded by BioScale.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Conflicts of Interest

Authors Ana Soares and Luís Carvalho were employed by the company BioScale, Rua Nova da CEE. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. Maximum likelihood (ML) phylogenetic tree based on partial sequences of 16S rRNA gene. Bootstrap values >70% are shown at the nodes. Type strains are noted with T superscript (T). The strains from this study are indicated in bold font. The scale bars represent the expected number of nucleotide changes per site. Escherichia coli ATCC 11,775 strain was used as outgroup.
Figure 1. Maximum likelihood (ML) phylogenetic tree based on partial sequences of 16S rRNA gene. Bootstrap values >70% are shown at the nodes. Type strains are noted with T superscript (T). The strains from this study are indicated in bold font. The scale bars represent the expected number of nucleotide changes per site. Escherichia coli ATCC 11,775 strain was used as outgroup.
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Figure 2. Antifungal activity of Bacillus sp. BS36 and Pseudomonas sp. BS95 against Alternaria sp. FP3, Botrytis sp. SM-D1, Fusarium sp. SM-D3, and Stemphylium sp. FP5, in dual culture assay. Bacillus sp. BS36 and Pseudomonas sp. BS95, respectively: MGI = 82.7 ± 4.3 and 38.5 ± 4.7% for Alternaria sp. FP3, MGI = 60.6 ± 1.7 and 0.0 ± 0.0% for Botrytis sp. SM-D1, MGI = 69.2 ± 0.0 and 0.0 ± 0.0% for Fusarium sp. SM-D3, MGI = 79.6 ± 0.0 and 29.8 ± 1.7% for Stemphylium sp. FP5.
Figure 2. Antifungal activity of Bacillus sp. BS36 and Pseudomonas sp. BS95 against Alternaria sp. FP3, Botrytis sp. SM-D1, Fusarium sp. SM-D3, and Stemphylium sp. FP5, in dual culture assay. Bacillus sp. BS36 and Pseudomonas sp. BS95, respectively: MGI = 82.7 ± 4.3 and 38.5 ± 4.7% for Alternaria sp. FP3, MGI = 60.6 ± 1.7 and 0.0 ± 0.0% for Botrytis sp. SM-D1, MGI = 69.2 ± 0.0 and 0.0 ± 0.0% for Fusarium sp. SM-D3, MGI = 79.6 ± 0.0 and 29.8 ± 1.7% for Stemphylium sp. FP5.
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Figure 3. Antifungal activity of cultures and cell-free filtrates of Bacillus sp. BS36 and Pseudomonas sp. BS95 against Alternaria sp. AF12 (A) and Fusarium sp. AF68 (B), expressed by MGI. Values represent the mean ± SD (n = 4). Asterisks (*) indicate mean values significantly different (p ≤ 0.05) according to independent samples t-test ((A): t (3) = 10.99 and p < 0.001 for Bacillus sp. BS36, and t (3.6) = 8.87 and p < 0.001 for Pseudomonas sp. BS95; (B): t (6) = 21.03 and p < 0.001 for Bacillus sp. BS36, and t (3) = 63.36 and p < 0.001 for Pseudomonas sp. BS95).
Figure 3. Antifungal activity of cultures and cell-free filtrates of Bacillus sp. BS36 and Pseudomonas sp. BS95 against Alternaria sp. AF12 (A) and Fusarium sp. AF68 (B), expressed by MGI. Values represent the mean ± SD (n = 4). Asterisks (*) indicate mean values significantly different (p ≤ 0.05) according to independent samples t-test ((A): t (3) = 10.99 and p < 0.001 for Bacillus sp. BS36, and t (3.6) = 8.87 and p < 0.001 for Pseudomonas sp. BS95; (B): t (6) = 21.03 and p < 0.001 for Bacillus sp. BS36, and t (3) = 63.36 and p < 0.001 for Pseudomonas sp. BS95).
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Figure 4. Effects of proteinase K, heat, and acidification (HCl) and methanol extraction on the antifungal activity of cell-free filtrates of Bacillus sp. BS36 against Alternaria sp. AF12 (A) and Fusarium sp. AF68 (B). Values represent the mean ± SD (n = 4). Bars with different letters indicate mean values significantly different (p ≤ 0.05) according to Tukey test, and “ns” indicate not significant (p > 0.05) (ANOVA, (A): F = 8.42 and p = 0.003, (B): F = 0.43 and p = 0.735).
Figure 4. Effects of proteinase K, heat, and acidification (HCl) and methanol extraction on the antifungal activity of cell-free filtrates of Bacillus sp. BS36 against Alternaria sp. AF12 (A) and Fusarium sp. AF68 (B). Values represent the mean ± SD (n = 4). Bars with different letters indicate mean values significantly different (p ≤ 0.05) according to Tukey test, and “ns” indicate not significant (p > 0.05) (ANOVA, (A): F = 8.42 and p = 0.003, (B): F = 0.43 and p = 0.735).
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Table 1. Antifungal activity of PGPR strains against Alternaria sp. AF12 and Fusarium sp. AF68, expressed by MGI.
Table 1. Antifungal activity of PGPR strains against Alternaria sp. AF12 and Fusarium sp. AF68, expressed by MGI.
PGPR StrainsMycelial Growth Inhibition (MGI,%) 1
Alternaria sp. AF12Fusarium sp. AF68
Bacillus sp.BS3674.0 ± 3.265.4 ± 3.0
BS843.9 ± 2.70.0 ± 0.0
Priestia sp.BS10.0 ± 0.00.0 ± 0.0
BS900.0 ± 0.00.0 ± 0.0
Pseudomonas sp.BS220.2 ± 1.77.8 ± 4.4
BS30.0 ± 0.07.8 ± 4.4
BS270.0 ± 0.06.9 ± 1.7
BS942.9 ± 1.77.8 ± 2.0
BS9538.5 ± 6.161.8 ± 1.7
1 Values represent the mean ± SD (n = 4).
Table 2. Antifungal activity of co-culture and monocultures filtrates of Bacillus sp. BS36 and Pseudomonas sp. BS95 against Alternaria sp. AF12 and Fusarium sp. AF68, expressed by MGI.
Table 2. Antifungal activity of co-culture and monocultures filtrates of Bacillus sp. BS36 and Pseudomonas sp. BS95 against Alternaria sp. AF12 and Fusarium sp. AF68, expressed by MGI.
Treatment 1Mycelial Growth Inhibition (MGI,%) 2
Alternaria sp. AF12Fusarium sp. AF68
BP30.8 ± 0.0 b20.2 ± 1.7 a
B53.8 ± 0.0 a14.4 ± 3.2 b
P5.8 ± 1.9 c0.0 ± 0.0 c
1 BP indicates co-culture of Bacillus sp. BS36 and Pseudomonas sp. BS95; B indicates monoculture of Bacillus sp. BS36; P indicates monoculture of Pseudomonas sp. BS95. 2 Values represent the mean ± SD (n = 4). Values represent the mean ± SD (n = 4). Different letters in the same column indicate mean values significantly different (p ≤ 0.05) according to Tukey test (ANOVA, F = 1366.98 and p < 0.001 for Alternaria sp. AF12, and F = 74.36 and p < 0.001 for Fusarium sp. AF68).
Table 3. Effects of the target microorganism on the antifungal activity of Bacillus sp. BS36 filtrates against Alternaria sp. AF12 and Fusarium sp. AF68, expressed by MGI.
Table 3. Effects of the target microorganism on the antifungal activity of Bacillus sp. BS36 filtrates against Alternaria sp. AF12 and Fusarium sp. AF68, expressed by MGI.
Treatment 1Mycelial Growth Inhibition (MGI,%) 2
Alternaria sp. AF12Fusarium sp. AF68
BA57.7 ± 0.0 b18.3 ± 1.7 b
BF63.5 ± 1.9 a36.5 ± 5.8 a
B59.6 ± 1.9 b7.7 ± 3.8 c
1 BA indicates co-culture of Bacillus sp. BS36 and inactivated Alternaria sp. AF12; BF indicates co-culture of Bacillus sp. BS36 and inactivated Fusarium sp. AF68; B indicates monoculture of Bacillus sp. BS36. 2 Values represent the mean ± SD (n = 4). Different letters in the same column indicate mean values significantly different (p ≤ 0.05) according to Tukey test (ANOVA, F = 10.42 and p = 0.005 for Alternaria sp. AF12, and F = 37.74 and p < 0.001 for Fusarium sp. AF68).
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Santos, A.M.; Soares, A.; Luz, J.; Cordeiro, C.; Sousa Silva, M.; Dias, T.; Melo, J.; Cruz, C.; Carvalho, L. Microbial Interactions as a Sustainable Tool for Enhancing PGPR Antagonism against Phytopathogenic Fungi. Sustainability 2024, 16, 2006. https://doi.org/10.3390/su16052006

AMA Style

Santos AM, Soares A, Luz J, Cordeiro C, Sousa Silva M, Dias T, Melo J, Cruz C, Carvalho L. Microbial Interactions as a Sustainable Tool for Enhancing PGPR Antagonism against Phytopathogenic Fungi. Sustainability. 2024; 16(5):2006. https://doi.org/10.3390/su16052006

Chicago/Turabian Style

Santos, Ana M., Ana Soares, João Luz, Carlos Cordeiro, Marta Sousa Silva, Teresa Dias, Juliana Melo, Cristina Cruz, and Luís Carvalho. 2024. "Microbial Interactions as a Sustainable Tool for Enhancing PGPR Antagonism against Phytopathogenic Fungi" Sustainability 16, no. 5: 2006. https://doi.org/10.3390/su16052006

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