Biocontrol Activity of Actinomycetes Strains against Fungal and Bacterial Pathogens of Solanum lycopersicum L. and Daucus carota L.: In Vitro and In Planta Antagonistic Activity †

Plants are affected by various biotic and abiotic stresses due to climate change. Tomato and carrots are important crops that are attacked by various pathogens. Fourteen plant growth promoting bacteria (PGPB) belonging to the genera Streptomyces sp. and Nocardiopsis sp. were selected for the biocontrol of several common fungal and bacterial pathogen. Antifungal activity was assessed against Fusarium oxysporum f. sp. radicis-lycopersici (FORL) and Rhizoctonia solani (RHS). Antibacterial activity was evaluated against Pseudomonas syringae, Pseudomonas corrugata, Pseudomonas syringae pv. actinidiae, Pectobacterium carotovorum subsp. Carotovorum. In vitro antifungal and antibacterial antagonistic activities were evaluated by dual culture method. Fungal-bacterial interaction areas were analysed by scanning electron microscopy (SEM). Cell-free culture filtrates (CF) from strains showing good biocontrol potential, were produced and investigated for their in vitro antifungal and antibacterial activity. Two most effective strains were also combined in consortium and utilized for in planta pre-emergence biocontrol assays on both S. lycopersicum and D. carota. For each pathogenic strain, four experimental conditions were compared: CNT (no bacterial inoculation/no infection), PGPB (with bacteria/no infection), PGPB+INF (with bacteria/and infection), INF (with infection/no bacteria). The PGPB strains Streptomyces albidoflavus H12 and Nocardiopsis aegyptica H14 showed good in vitro antifungal (inhibition > 50%) and antibacterial (inhibition halo > 10 mm) activity. The SEM micrographs showed deterioration of fungal filaments and modification of hyphal structures. The CFs of both strains were also able to inhibit FORL and RHS in in vitro growth (minimum inhibitory concentration of 0.2–0.8%). In planta biocontrol assessments showed that, the consortium was effective in reducing the infection effects of both fungal and bacterial pathogens. Dual onsortium allowed normal plant development compared to the control. These results confirm the usefulness of actinomycetes strains as a bio-control agent and can therefore be an alternative to chemicals used in agriculture.


Introduction
Plants are affected by various biotic and abiotic stresses due to climate change. Areas affected by temperature increase are more susceptible to pathogens attack [1]. The use of microorganisms in agriculture is a sustainable strategy to control phytopathogens. These bacteria can improve plant health and growth, providing a long-term protection [2,3]. Several rhizospheric microorganisms act as biostimulants, and show antagonistic properties against several pathogens [4]. Among them, actinomycetes have the ability to produce a wide range of secondary metabolites (e.g. antibiotics and extracellular enzymes) [5], that inhibit the growth of several fungal and bacterial pathogens [6]. Moreover, biocontrol activity is obtained through the induction of systemic resistance [5]. The present study is aimed at evaluating the biocontrol capability of actinomycetes isolates against several fungal and bacterial pathogens of Solanum lycopersicum and Daucus carota. In vitro antifungal and antibacterial antagonistic activities were evaluated by dual culture method. Fungal-PGPB interaction areas were also analysed by scanning electron microscopy (SEM). From strains with good biocontrol potential, cell-free supernatant (CFS) were produced and investigated for their in vitro antifungal and antibacterial activity. The most effective strains were also combined in consortium and utilized for the seed treatment for in planta pre-emergence biocontrol assays on both S. lycopersicum and D. carota. For each pathogenic strain, four experimental conditions were compared: CNT (without PGPB/infection), PGPB (with PGPB/no infection), PGPB+INF (with PGPB/infection), INF (with infection/no PGPB). The induced protection was assessed by estimation of plant survival, morphobiochemical parameters, damages and chlorophyll contents.

Experiments
In vitro antagonistic activity by diffusible and volatile compounds was carried out by dual culture method on PDA culture medium using fourteen actinomycetes strains of the genus Streptomyces sp, and Nocardiopsis sp. The fungal pathogens tested were Fusarium oxysporum f. sp. radicis-lycopersici (FORL), and Rhizoctonia solani (RHS). The morphological deterioration of PGPBfungus interaction areas were analyzed by SEM microscopy. The pathogenic bacteria tested were: Pseudomonas syringae, Pseudomonas corrugata, Pseudomonas syringae pv. actinidiae, Pectobacterium carotovorum subsp. carotovorum. The inhibition percentages of fungi were calculated after incubation until the complete growth of the control plate [7]. The bacterial inhibition halos were assessed after 48 hours. Cell-free supernatant (CFS) of Streptomyces albidoflavus H12 and Nocardiopsis aegyptica H14, which showed good in vitro biocontrol, were investigated for Minimal Inhibitory Concentration (MIC), and for Minimum Bactericidal Concentration (MBC) as described by CLSI guidelines [8]. Using polystyrene microplate, 100 μL of media were introduced in each well, then 100μL of CFS of single strains, and consortium were inserted in the first wells and the dilutions were maintained. Finally, the fungi and test-bacteria were inoculated.
In planta antagonistic activity of H12 and H14 consortium-the most actives strains against the different tested pathogens -was assessed on S. lycopersicum, and D. carota against the abovementioned pathogens in pre-emergence [7]. The experiment was organized as follows: (i) CNT (without PGPB/infection), PGPB (with PGPB/no infection), PGPB+INF (with PGPB/infection), INF (with infection/no PGPB). Each experimental unit was realized in 25 pots with two seeds per pot under natural light conditions until the disease's symptoms showed up. The induced protection was assessed by estimation of plant survival, morpho-biochemical parameters, damages and chlorophyll contents. The plants were analyzed for the morpho-physiological characters; damages, and total chlorophyll contents [7].

Results
Almost all 14 strains (64%) showed good in vitro antagonistic activity by producing diffusible and volatiles compounds against fungal pathogens (inhibition percentage up to 85%). Most of the tested strains (70%) exhbit at least one activity against pathogenic bacteria (inhibition halo up to 25mm) ( Table 1).    A, B, respectively). In the presence of H12 and H14 consortium, the hyphal structures change in the interaction zones of both FORL and RHS with the PGPB (C, D, respectively).
Concerning the in planta experiment, the inoculation with the consortium (PGPB) improved development and growth of both tomato and carrot plants compared to the control (better germination rates, morpho-physiological characters, and chlorophyll content) (Figure 2,3).

Discussion
The different abilities to promote plant-growth by actinomycetes strains, such as nutrient solubilization, nitrogen fixation, and phytohormones production act indirectly in the control of plant diseases [5]. The use of combined bacteria is a strategy for plants protection against pathogens attack [7]. Actinobacteria are well known for their ability to produce various bioactive compounds [9]. They are biological agents for their antagonistic activities and plants protection against several soil borne pathogens [10]. The bio-control activity of actinomycetes is linked to antibiosis, lysis mechanisms, and host defenses induction [5]. The actinomycetes strains investigated in this study also have different plant growth-promoting traits [11]. These bacteria enhance plant physiological status and offer an additional advantage to their use as biological control agents for sustainable agriculture.

Conclusion
Nowadays, bacterial and fungal plant diseases are controlled almost exclusively by agrochemicals. These chemical products entail serious consequences for human's and ecosystems' health. The use of biocontrol agents should be encouraged to counteract this problem. Our findings show that actinomycetes could be considered a valid biocontrol agents. Further experiments are needed to determine their effectiveness on other plants, against other pathogens and under different cultivation conditions. However, these preliminary results underline that actinomycetes, and in particular Streptomyces and Nocardiopsis genera, can be biological alternatives for plants disease management.

Conflicts of Interest:
The authors declare no conflict of interest.