A careful selection of beneficial microorganisms to create synthetic microbial consortia was performed focusing on the main groups of rhizospheric beneficial microorganisms such as PGPR, mycoparasitic fungi, and AMF. An extensive literature review on biocontrol studies of known BCAs was performed, taking also into account as potential candidates the microbial strains available at Koppert Biological Systems. The most relevant studies considered are summarized in Supplementary Table 1.

As a result, we chose two Bacillus amyloliquefaciens strains CECT 8238 and CECT 8237, formerly known as Bacillus subtilis UMAF6614 and UMAF6639, respectively (Magno-Perez-Bryan et al., 2015), and Pseudomonas chlororaphis MA 342 and Pseudomonas azotoformans F30A (Abuamsha et al., 2011a; Levenfors et al., 2014). From fungi, we selected Trichoderma harzianum strains T22 and ESALQ1306 (Geraldine et al., 2013; Coppola et al., 2019), and for the ISR bioassay, we included additionally the AMF R. irregularis MUCL 57021.

Bacillus amyloliquefaciens strains were grown on tryptone soya agar (TSA, Oxoid, Basingstoke, United Kingdom) for 24 h at 28°C. After that, a single colony from TSA culture was inoculated in 25 ml of Difco sporulation medium (DSM; Nicholson and Setlow, 1990) and incubated for 48 h at 28°C in a rotatory shaker (200 rpm). Spores were quantified using a Bürker-Türk counting chamber, then centrifuged at 5,000 rpm for 15 min, and after discarding the supernatant, the pellet containing the spores was resuspended in sterile tap water to a final concentration of 1 × 10⁷ spores/ml.

Pseudomonas azotoformans and P. chlororaphis were grown on TSA for 24 h at 28°C. Liquid pre-culture was prepared using tryptone soya broth (TSB, Oxoid, Basingstoke, United Kingdom) inoculated with a single bacterial colony from TSA culture and incubated overnight at 28°C with rotary shaking at 200 rpm. After that, 1 ml of pre-culture was inoculated in 25 ml of TSB medium and placed in a rotatory shaker (200 rpm) at 28°C. After 150 min of incubation, with bacterial growth in exponential phase, the cell concentration was calculated measuring the O.D. (620 nm) of the bacterial culture on Shimadzu UVmini-1240 Spectrophotometer. The bacterial culture was centrifuged at 5,000 rpm for 15 min, and after discarding the supernatant, the pellet containing the bacterial cells was resuspended in sterile tap water to a final concentration of 1 × 10⁷ colony forming unit (cfu)/ml.

Trichoderma harzianum strains were cultured on potato dextrose agar (PDA, Difco, Le Pont de Claix, France) for 7 days at room temperature. Spores were collected from sporulating plates in sterile tap water, and the concentration of the spore suspension was quantified using a Bürker-Türk counting chamber and adjusted to 1 × 10⁷ spores/ml.

Rhizophagus irregularis was grown in a monoxenic culture on a minimal (M) medium and using Agrobacterium rhizogenes-transformed carrot (Daucus carota) roots as a host root (St-Arnaud et al., 1996). To extract the AMF spores, citrate buffer 0.01 M (pH = 6) was added to a sporulating AMF culture in a proportion of 3:1 (v/v) and placed in a rotary shaker for 1 h to dissolve the agar. AMF spores were recovered from the solution using sieves of different sizes (250 and 53 μm) and resuspended in sterile tap water at final concentrations of 1,000 spores/ml.

Two major fungal pathogens causing important crop losses worldwide were tested: F. oxysporum f.sp. radicis-lycopersici as soil pathogen and the necrotrophic shoot pathogen B. cinerea strain B05.10.

Fusarium oxysporum was grown on PDA at 25°C for 4 days. For spore production, 25 plugs of 4 mm diameter with new growing mycelia were removed from the PDA plates and transferred to 500 ml Erlenmeyer containing 200 ml of Czapek Dox Broth (Oxoid, Basingstoke, United Kingdom) and placed in a rotary shaker (110 rpm) at room temperature. After 4 days of incubation, the liquid culture was filtered using a sterile miracloth filter, and the spore concentration was quantified using a Bürker-Türk counting chamber. The resulting spore suspension was centrifuged at 9,500 rpm for 15 min and after discarding the supernatant, the pellet containing the spores was resuspended in sterile tap water to a final concentration of 1 × 10⁸ spores/ml.

Botrytis cinerea was cultured on PDA at 20°C. Spores were collected from sporulating 14 days old plates in potato dextrose broth (PDB, Difco, Le Pont de Claix, France), and the concentration of the spore suspension was quantified using a Bürker-Türk counting chamber and adjusted to 1 × 10⁶ spores/ml.

The antagonistic activity of the individual strains Bacillus amyloliquefaciens CECT 8238 and CECT 8237, P. azotoformans, P. chlororaphis, and T. harzianum T22 and ESALQ1306 were initially evaluated in vitro, in confrontation assays against F. oxysporum and B. cinerea. For Trichoderma, one PDA plug (4 mm) of Trichoderma culture and one of the pathogen cultures were placed on PDA plates with 4 cm of distance from each other. For Bacillus and Pseudomonas, 10 μl drop of TSB liquid culture grown overnight was used instead of PDA plugs. As a control, a plug of the pathogen culture was placed in the Petri dish without any antagonist. All treatments were replicated three times. All plates were incubated at 25°C for 7 days. The radius of the pathogen colony in the confrontation plates was measured and compared to the radius of the pathogen colony in the control plates.

Biocontrol potential was tested in planta through several bioassays including diverse inoculation methods and targeting different pathogens. This strategy allows testing in vivo different modes of action ranging from direct antagonism to indirect plant-mediated effects. Thus, we tested through seed inoculation suppression of the root pathogen F. oxysporum and ISR against the foliar pathogen B. cinerea, and suppression of B. cinerea by foliar spray application.

Data were analyzed using R statistical language, version 4.0.5 (R Core Team, 2021), and figures were produced using the package ggplot2 (Wickham, 2009). The effect of microbial treatments (single strains and synthetic communities) on the necrotic lesions caused by B. cinerea, microbial colonization after single and combined inoculations, and the effect of single strains on B. cinerea and F. oxysporum radial growth was assessed using a general linear model with blocks as an error term and microbial treatments as a fixed effect. To examine whether microbial treatments influenced the probability of the tomato seedlings to survive to the soil pathogenic fungus F. oxysporum, a generalized linear model with binomial distribution and logit link function and blocks as an error term was performed. Post hoc comparisons among microbial treatments were based on the Tukey honestly significant difference (HSD). Model validation was performed graphically by inspecting the residuals and fitted values (Zuur and Ieno, 2016).

Upon a thorough literature review, we selected bacterial and fungal groups/genera with well-documented potential to control plant pathogens, trying to compile diverse mechanisms including antibiosis, competition for iron and other nutrients, and colonization sites, mycoparasitism, and induction of plant resistance. Strains from the selected groups available at the Koppert microbial collection were: Bacillus amyloliquefaciens strains CECT 8238 and CECT 8237, P. chlororaphis MA 342, and P. azotoformans F30A, T. harzianum strains T22 and ESALQ1306, and the AMF R. irregularis MUCL 57021 (Supplementary Table 1). Two synthetic communities were designed, one combining one strain for each genera (SynCom1), and another in which all selected microbes were included (SynCom2).

As a first screening to move into the biocontrol potential of the selected individual strains, their antagonistic activity was tested in an in vitro dual confrontation assay. All selected BCA strains decreased F. oxysporum radial growth compared to the control plates (p < 0.05; Supplementary Figure 2A). Both T. harzianum strains showed the strongest antifungal activity, with about 80% reduction of the pathogen radial growth (p < 0.05; Supplementary Figure 2A). Similarly, all individual strains reduced B. cinerea radial growth compared to the control, and T. harzianum T22 was the most effective strain with a 90% reduction of pathogen growth (p < 0.05; Supplementary Figure 2B).

The research was scaled up using a tomato-Fusarium-soil system, comparing the biocontrol activity of the individual microbial strains and the differently designed consortia (SynCom1, SynCom2). The pathogen fully compromised plant survival, as no plants survived in the disease control, while almost 100% survival was found in the absence of the pathogen (non-diseased control) (Figure 1A). None of the individual bacterial strains significantly increased plant survival compared to the disease control. In contrast, both T. harzianum strains and all of the SynComs were able to efficiently suppress F. oxysporum, increasing plant survival above 80% (p < 0.05, Figure 1A). In fact, plant survival in the T. harzianum and consortia treatments reached the levels of the non-diseased control (p < 0.05, Figure 1A). These results not only show the potential of T. harzianum but also indirectly the compatibility/tolerance of the other isolates as this high protection level was maintained in the consortia treatments (Figures 1A,B).

The antagonistic potential of single strains and consortia against the foliar pathogen B. cinerea was also tested in planta, applying the BCA treatments by spraying the leaves before B. cinerea infection. Among single microbial treatments, P. chlororaphis, P. azotoformans, and T. harzianum T22 were able to reduce the area of the necrotic lesion caused by B. cinerea by 56, 45, and 38%, respectively, compared to the control treatment (p < 0.05, Figure 2A). Remarkably, all the microbial consortia treatments reduced B. cinerea lesion area by about 50% as compared to the disease control, reaching up to a 70% reduction in SynCom2B (p < 0.05, Figure 2A). The higher antagonistic effect against B. cinerea was therefore achieved by P. chlororaphis (56%) and the SynCom2B (Supplementary Figure 3).

In addition to the direct antagonistic effect of the foliar application against B. cinerea, we evaluated the capacity of the microbial treatments to activate plant systemic resistance. We tested the potential plant-mediated effects by avoiding direct contact between the BCAs and the pathogen. In this experiment, the AMF R. irregularis was included both individually and in the consortia due to the reported capacity of AMF to induce ISR and their current interest as inoculants in agriculture. Among the individual treatments, only Bacillus amyloliquefaciens CECT 8238 and R. irregularis were able to induce ISR against B. cinerea, reducing the area of the necrotic lesions by 38 and 44%, respectively, as compared to the control treatment (p < 0.05, Figure 2B). The consortia also achieved significant plant-mediated protection against B. cinerea, with SynCom1A, SynCom1B, and SynCom2A reducing lesions by 33–37% as compared to the control (p < 0.05, Figure 2B). Again, a similar reduction in disease symptoms was achieved by the consortia and the best performing individual treatments in this pathosystem.

Taking into account all the bioassays performed, SynComs was more versatile than the individual strains, showing effective biocontrol across the different pathosystems and inoculation methods, as summarized in Table 1.

The repeated success of the consortia across all the in planta bioassays supports the strains compatibility. In fact, the SynComs performance in biocontrol was not significantly different from that achieved by the best performing BCA strain in any of the experiments. We further investigated the compatibility of the components in a new experiment aiming to compare the colonization of each microorganism in the single or SynCom treatments after interacting in the tomato rhizosphere for 15 days. The absence in the soil of indigenous species from any of the inoculated genera (Bacillus, Pseudomonas, and Trichoderma) was confirmed in the control treatment plates (Supplementary Figure 1A). Each microbial strain (except R. irregularis) was initially inoculated at a total concentration of 4 × 10⁵ cfu/g of soil for each strain (both in the individual microbial treatments and in consortia).

Bacillus spp. abundance at the end of the experiment was similar to that initially inoculated both in single strain and SynCom1 treatments (Figure 3A). In SynCom2 treatments, both Bacillus strains were co-inoculated, and the abundance of Bacillus spp. in the soil was even higher, around 6 × 10⁵ cfu/g of soil (Figure 3A). These results confirm the successful establishment of both Bacillus amyloliquefaciens strains, both when inoculated individually and in consortia.

In the single strain treatments, Pseudomonas spp. abundance increased compared to the initial inoculation (up to 1.5 × 10⁶ and 1.2 × 10⁶ cfu/g of soil in P. azotoformans and P. chlororaphis, respectively) (Figure 3B), evidencing the good colonization ability of Pseudomonas spp. Remarkably, Pseudomonas spp. abundance in soil increased more than four times in SynCom1 (containing P. azotoformans) compared to the individual P. azotoformans treatment (around 6.5 × 10⁶ cfu/g of soil) (Figure 3B). Regarding SynCom2 treatments in the absence of AMF Pseudomonas spp., abundance was 2.7 × 10⁶ cfu/g of soil, corresponding to the sum of both inoculated Pseudomonas species in SynCom2, whereas in SynCom2 + AMF, their abundance was more than double (6.9 × 10⁶ cfu/g of soil), pointing to a potential positive effect of AMF presence in this consortium (Figure 3B).

Trichoderma spp. abundance in the individual treatments was 9.3 × 10⁵ and 6.3 × 10⁵ cfu/g of soil in T. harzianum T22 and ESALQ1306, respectively, which in the case of T22 is more than double of the concentration inoculated (Figure 3C). Regarding the consortia, Trichoderma spp. abundance was in a similar range than the individual inoculations: 1 × 10⁶ cfu/g of soil in SynCom1 (where only T22 was present) and around 1.5 × 10⁶ cfu/g of soil in SynCom2 treatments equivalent to the sum of both Trichoderma strains co-inoculated in this consortium (Figure 3C).

Finally, the percentage of root length colonized by R. irregularis was 1.2% when applied individually (Figure 3D). Root colonization was similar in both consortia treatments, (SynCom1 + AMF and SynCom2 + AMF) (Figure 3D), confirming that mycorrhizal colonization was not significantly affected when inoculated in consortia. The low percentages are common in the early stages of the colonization (only 2 weeks upon AMF inoculation). To compare the treatments in more advanced stages of the mycorrhizal symbiosis, mycorrhizal colonization was quantified in the roots of the ISR bioassay, corresponding to plants growing with the AMF for 5 weeks. Mycorrhizal colonization reached 40% in the individual treatment, and these levels remained unaltered in both SynCom1 and SynCom2 treatments at any of the tested doses (Supplementary Figure 4).

Across the different experiments, different individual microorganisms were the most effective in the different scenarios, depending on the type of pathogen or the strategy used for its control. Remarkably, the consortia effectively controlled all pathogens in all different bioassays, both through direct antagonism by seed or foliar application, or inducing plant systemic resistance against foliar pathogens by seed inoculation (results summarized in Figure 4). The bioprotection achieved by the consortia was always similar to that of the best performing single strains. Although no significant synergism was detected, no negative interactions were observed, in contrast to some studies reporting positive and negative effects by the combination of BCAs (Freeman et al., 2004; Abo-Elyousr et al., 2009; Elliott et al., 2009; Ruano Rosa et al., 2014). Our results illustrate the topical dilemma of selecting single beneficial microbes versus SynComs for biological control. Strictly from the potential efficacy point of view, SynComs offered the widest protection after comparing the single components and several consortia across the soil and foliar threats and through direct and indirect actions. Yet, the efficacy was not higher than that of the best performing single strain and, in most cases, more than one individual microbe provided effective control. Considering the current high costs and outstanding long process for registering microbial products, targeting single strain or SynCom products is a tough dilemma to face from the commercial point of view. Nevertheless, the advantage of SynComs as a more versatile tool may become more apparent under field conditions, considering the variability of growing conditions and the uncertainty of the potential challenges to be faced—what pathogens or pests would be threatening the crop. We postulate that in the field, under commercial conditions, the benefits for the SynComs would further differentiate to the individual components. Thorough validation of results in field conditions will give the answer.

For the design of the synthetic microbial consortia, we selected different strains aiming to combine different mechanisms for biocontrol from the production of diverse antimicrobial metabolites through mycoparasitism to ISR. Bacillus amyloliquefaciens strains CECT 8238 and CECT 8237 have been shown to promote plant growth and effectively control diverse microbial pathogens through direct antagonism or indirectly through ISR (Romero et al., 2007; García-Gutiérrez et al., 2012, 2013; Magno-Perez-Bryan et al., 2015). P. chlororaphis MA342 has been described to effectively control seed and soil pathogens via direct antagonism (Tombolini et al., 1999; Abuamsha et al., 2011a) and protecting against leaf pathogens through seed priming (Abuamsha et al., 2011b). P. azotoformans F30A effectively enhance plant emergency and growth (Levenfors et al., 2014) and can also induce ISR to leaf pathogens (Sang et al., 2014; Bouaoud et al., 2018). T. harzianum strain T22 is one of the best characterized and commercialized Trichoderma strains. It effectively antagonizes soil pathogens (Wilson et al., 2008; Percival et al., 2011; Roberti et al., 2015; Fatouros et al., 2018) and can trigger ISR against diverse above- and belowground attackers (Tucci et al., 2011; Vitti et al., 2016; Coppola et al., 2017, 2019; Debode et al., 2018; Pocurull et al., 2020; Alınç et al., 2021). Besides promoting plant growth, T. harzianum ESALQ1306 has been shown to highly reduce Sclerotinia sclerotiorum disease severity through parasitism and to induce ISR against spider mites (Geraldine et al., 2013; De Oliveira et al., 2018; Barroso et al., 2019; Canassa et al., 2020). In contrast, R. irregularis is not a direct antagonist of plant pathogens, but is able to induce ISR against root and foliar pathogens (Martínez-Medina et al., 2011; Sanchez-Bel et al., 2016; Bidellaoui et al., 2019; Campo et al., 2020; Sanmartín et al., 2020). All in all, we selected a potentially powerful pool of microbes, already well characterized in multiple aspects. A number of them are being already exploited commercially either under development into microbial products, or, like T. harzianum, already commercialized as BCA by Koppert Biological Systems all over the world from vegetable and ornamental to field and row crops.

Most studies focusing on the use of microbial consortia for disease control are looking for synergistic or additive effects, aiming to achieve a higher pest or disease control than their components. While some of these studies have indeed reported positive effects (Guetsky et al., 2001, 2002; Srivastava et al., 2010; Singh et al., 2013; Ruano Rosa et al., 2014; Sylla et al., 2015), many others showed similarly or even less effective in disease control when applying consortia as compared to the application of the individual microbes (Freeman et al., 2004; Abo-Elyousr et al., 2009; Elliott et al., 2009; Ruano Rosa and López Herrera, 2009). However, most of these studies focused on one model system. In contrast, we intended to extend the scope by including an array of target diseases—soil and foliar—and possible mechanisms—direct and indirect control via ISR. The SynComs performed consistently well across the different pathosystems. Yet, differences between the SynComs and the individual components were relatively mild in terms of efficacy/degree of control.

Microbial compatibility is a key factor when designing a microbial consortium, essential for the successful establishment and functionality of the included microorganisms and the success of SynCom products. In our study, the conservation of the biocontrol effectiveness in the SynComs to the same levels as the best performing individual isolates supported the compatibility between the coexisting microorganisms. We further tested their compatibility in our consortia by assessing the microbial survival in a plant–soil-based experiment, and we did not find any negative interaction between them. Instead, Bacillus and Trichoderma performed in the consortia as good as when individually inoculated, and Pseudomonas even benefited from the combination with the other organisms, as they performed better in the SynComs than when inoculated alone. It is important to note that R. irregularis was neither negatively affected in early nor late symbiosis stages by the presence of Trichoderma spp., as demonstrated by the similar mycorrhizal colonization in roots inoculated with the AMF alone or as part of the consortia. This is remarkable, as the compatibility of Trichoderma spp. with mycorrhizal fungi is frequently questioned because of the high mycoparasitic potential of these biocontrol fungi. In fact, Trichoderma is able to parasite AMF in vitro (Rousseau et al., 1996), but other studies proved their compatibility under more realistic scenarios (i.e., rhizospheric soil) as observed here (Martínez-Medina et al., 2011). Even more, Trichoderma-AMF synergistic effects have been reported (Poveda et al., 2019). Although microbe compatibility remains poorly studied, understanding the compatibility between groups or key BCA genera is required for informed decisions in the selection of suitable candidates for SynComs development in biocontrol programs in agriculture.

Overall, our findings highlight the potential multifunctionality of SynComs for biological control. Combining compatible beneficial microorganisms with complementary effects on different targets, direct and indirect mechanisms of control and/or effective under different conditions will lead to the development of biocontrol products with increased versatility. To became commercial products, consistency of the outcomes needs to be tested and finally validated across multiple field trials in the geographical regions where is aimed to be used. This is a key step for the successful application of this sustainable technology in agriculture.