1. Introduction
Plant fungal diseases are responsible for the emergence of different symptoms, such as wilting, scabs, moldy coatings, rusts, blotches and rotted tissue, on a wide variety of crops causing heavy economic losses [
1]. In recent decades, many control strategies, such as chemical-based tools, plant breeding and agronomic techniques (crop rotations, soil tillage, solarization, weed control, organic amendment, etc.), have been developed in order to reduce deleterious disease effects. Among them, synthetic fungicides still remain the most adopted means for reliability reasons [
1]. However, ecofriendly alternatives are needed because of increasing public request for organic and chemical-free vegetables and the enactment of restrictive regulations about sustainable fungicide use, aimed at reducing risks for the environment and human health. Furthermore, the application of chemicals over the years may contribute to the development of resistance in pathogens, thereby impacting the real effectiveness of the control solution [
2]. In the last few decades, many efforts have been made by scientists to find alternative tools, paying attention particularly to antagonistic microorganisms, such as bacteria belonging to
Pseudomonas and
Bacillus genera [
3,
4,
5], as potential biological control agents (BCAs). Several authors have described the ability of many
Bacillus rhizosphere-competent strains to inhibit the growth of a plethora of fungal plant pathogens through different direct mechanisms [
6,
7,
8,
9,
10,
11,
12]. It was demonstrated that some antagonistic
Bacillus strains can, moreover, elicit systemic resistance by increasing the level of PR-proteins [
13] or salicilic acid [
14].
Bacillus spp. may exert antagonistic activity through the release of antifungal lipopeptides, such as surfactin, iturin, fengycin and other bioactive molecules with surfactant-like properties. These molecules are implicated both in the biocontrol of diseases and in the promotion of plant growth [
15,
16].
Bacillus spp. can produce endospores: such structures confer resistance to desiccation, have a threshold temperature of 90 °C and have extreme pH and osmotic tolerances. So, these bacterial cells are particularly suitable for commercial purposes for their potential to adapt to different environmental situations [
17].
Composting is a biochemical process that involves a large variety of mesophilic and thermophilic aerobic microorganisms, including bacteria, actinomycetes, yeasts and fungi, in transforming low-value undecomposed materials into a high-value humified products [
18]. A wide range of biowaste can be composted, including materials generated by the agriculture, food and wood processing, sewage treatment, industry and municipal waste [
19,
20]. Microorganisms such as bacteria, actinomycetes and fungi, play a fundamental role in the organic matter decomposing processes that takes place during composting, and moreover, confer specific biological characteristics to the compost, such as suppressiveness [
21]. Several bacterial strains, belonging to species of
Bacillus,
Micrococcus,
Clostridium,
Staphylococcus,
Citrobacter,
Serratia,
Klebsiella,
Pseudomonas,
Enterobacter and
Escherichia, were isolated from composts [
22]. Among the aerobic prokaryotes isolated from compost,
Bacillus spp. are among the most important potential biocontrol agents. Spore-forming bacteria are abundant in the compost and are promising for the suppression of soil-borne phytopathogens, especially through the production of antifungal proteins, antibiotics and lipopeptides [
23].
The aim of this work was to isolate and characterize spore-forming bacteria from a set of composted aromatic plant residues for the in vitro and in vivo ability to control Sclerotinia minor and Rhizoctonia solani on rocket. Our investigation was directed to a stepwise antagonistic screening program as a general criterion to recruit new Bacillus biocontrol agents for agricultural applications by using composts as suitable sources of beneficial microbes.
4. Discussion
Soil-borne fungal diseases are difficult to manage, and the causal pathogens are among the main threats which farmers must be face, due to their ability to survive in soil for a long time. Integrated approaches with the use of microbial biocontrol agents are welcome. In the last few decades, the use of eco-friendly control means of soil-borne pathogens has become a popular alternative to conventional chemical treatments in a lot of cropping systems [
37]. In fact, several studies and reviews highlighted the possibility to isolate new potential BCAs and use them to reduce crop losses [
12,
38,
39,
40,
41]. In this study, we carried out a step-by-step selection for the
S. minor and
R. solani controllability of spore-forming bacterial isolates, obtained from aromatic plant residue-based composts. A general assessment of the selecting program showed that composts obtained with defatted matrices, such as P9 and P11, respectively, oil-free rosemary and sage, gave the largest number of putative BCAs for the succeeding steps.
Phylogenetic analysis revealed that the best biocontrol agents of
Sclerotinia damping-off selected here, are related to
Bacillus amyloliquefaciens and
B. subtilis species. Many reports indicate that microbiota present in compost can exert a suppressive effect on some phytopathogens [
42,
43,
44].
Bacillus genus is a group of microorganisms widely present in soils and compost-amended soils [
42,
45], and well known for their beneficial effects exerted on plant growth, health and fitness [
7]. Moreover, a consistent number of
Bacillus species have been reported to behave both as direct antagonists of pathogens [
46] and may function as elicitors of induced resistance mechanisms [
47,
48].
The in vitro evaluation through dual antagonist-pathogen assay, may indicate the potential of microorganisms to act as BCAs [
49]. In the current study, to individuate the isolates with the best in vitro activities, a total of 133 candidates were found at beginning of this stepwise selection. This result suggests the possible production of diffusible metabolites in the media that are inhibitory for the in vitro development of the target pathogens, thereby indicating a possible antibiosis-like mechanism based on delivering antimicrobials outside the cells. Despite many
Bacillus species having been shown to antagonize microbial pathogens that way [
16,
50,
51], the contemporary presence of other types of interactions, such as competition for the space and/or resources, and predation, cannot be excluded. The percentage of the in vitro fungal growth reduction, as compared to untreated control, ranged from 10% to 56.1% for
R. solani and was between 13.9% and 59.4% for
S. minor. Thirty-five out of 104 BCAs candidates were able to inhibit mycelial growth of both pathogens. It is well established that
Bacillus spp. can inhibit fungal pathogens under in vitro conditions by producing a plethora of active molecules, such as diffusible metabolites, volatile compounds and cyclic lipopeptides (LPs) belonging to different families [
52]. LPs produced by many BCAs are responsible for the suppression of several phytopathogens belonging to different genera, such as
Sclerotium,
Fusarium,
Rhizoctonia and
Aspergillus [
7]. In the present study, the most promising bacterial isolates harbor at least two of lipopeptide genes in the genome, as revealed by PCR investigation. This agrees with several authors [
9,
11,
53,
54,
55] who described the presence of multiple LP-genes in
Bacillus strains exhibiting antagonistic activity. The antifungal properties of lipopeptides have been deeply investigated. For example, the activities of Iturin A and Surfactin rely on the surfactant properties of these molecules which induce the disruption of the pathogens’ membranes by pore formation [
56,
57] in a dose-dependent manner, so as to lose cellular contents [
9].
Among the seven most promising bacteria, two, namely, B. subtilis isolates (P9-2 and P10-6) and a B. amyloliquefaciens strain (P9-4), confirmed their biocontrol activity in the rocket/S. minor pathosystem. However, they failed in the R. solani/rocket experiments. The difficulty of controlling Rhizoctonia damping-off, probably, is linked to cruelty, high fitness and the development capability of the pathogen in the telluric environments.
For the selected strains, the LP screening revealed in all the isolates the contemporary presence of surfactin, bacylisin and fengycin genes, as previously described for other antagonists [
58]. Instead, iturin and bacyllomicin genes were not always present. Furthermore, we noticed that the isolate P11-5 held all the detected genes, even if it did not show significant activity in in vivo assays in both pathosystems
R. solani/rocket and
S. minor/rocket. It must be considered that even if a BCA holds a set of genes for a function, it does not mean that the function itself is strictly correlated to the biocontrol ability [
59]. In many cases of beneficial plant–microbe interactions, several mechanisms are involved at the same time [
60] so that the disease suppression and the PGP mechanisms are difficult to differentiate, as are their relative importance, which can be different depending on the pathosystems [
61].
In conclusion, seven spore-forming isolates obtained from aromatic plant waste composts,
B. amyloliquefaciens strains P9-4 and P11-5, and
B. subtilis strains P6-10, P8-10, P9-2, P10-5 and P10-6, could have the potential to exert biological control on soil-borne diseases.
B. subtilis P6-10, and
B. amyloliquefaciens P10-5 and P10-6 strains proved to be the most effective isolates. Findings confirm that compost is a suitable source and precious reservoir of beneficial microorganisms to be potentially advantageously applied not only for improving soil fertility, but also for increasing the sustainable management of plant diseases [
62]. The production of antibiotics, secondary metabolites, volatile compounds or any other mechanisms, might be related to the biocontrol activities of these
Bacillus isolates. However, other experiments and field evaluations of BCAs need to be done to elucidate the mechanisms elicited by the bacterial isolates that have protected rocket seedlings against
S. minor in our study.