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Article

Biocontrol of Soil-Borne Pathogens of Solanum lycopersicum L. and Daucus carota L. by Plant Growth-Promoting Actinomycetes: In Vitro and In Planta Antagonistic Activity

by
Rihab Djebaili
1,2,
Marika Pellegrini
1,*,
Claudia Ercole
1,
Beatrice Farda
1,
Mahmoud Kitouni
2 and
Maddalena Del Gallo
1
1
Department of Life, Health and Environmental Sciences, University of L’Aquila, Coppito, 67100 L’Aquila, Italy
2
Laboratory of Microbiological Engineering and Applications, University of Brothers Mentouri Constantine 1, Ain El Bey Road, Constantine 25000, Algeria
*
Author to whom correspondence should be addressed.
Pathogens 2021, 10(10), 1305; https://doi.org/10.3390/pathogens10101305
Submission received: 22 September 2021 / Revised: 7 October 2021 / Accepted: 8 October 2021 / Published: 12 October 2021
(This article belongs to the Special Issue Biological Control of Phytopathogens: Mechanisms and Applications)
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Versions Notes

Abstract

:
Biotic stress caused by pathogenic microorganisms leads to damage in crops. Tomato and carrot are among the most important vegetables cultivated worldwide. These plants are attacked by several pathogens, affecting their growth and productivity. Fourteen plant growth-promoting actinomycetes (PGPA) were screened for their in vitro biocontrol activity against Solanum lycopersicum and Daucus carota microbial phytopathogens. Their antifungal activity was evaluated 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, and Pectobacterium carotovorum subsp. carotovorum. Strains that showed good in vitro results were further investigated in vitro (cell-free supernatants activity, scanning electron microscope observations of fungal inhibition). The consortium of the most active PGPA was then utilized as biocontrol agents in planta experiments on S. lycopersicum and D. carota. The Streptomyces albidoflavus H12 and Nocardiopsis aegyptica H14 strains showed the best in vitro biocontrol activities. The diffusible and volatile compounds and cell-free supernatants of these strains showed both antifungal (in vitro inhibition up to 85%, hyphal desegregation and fungicidal properties) and antibacterial activity (in vitro inhibition >25 mm and bactericidal properties). Their consortium was also able to counteract the infection symptoms of microbial phytopathogens during in planta experiments, improving plant status. The results obtained highlight the efficacy of the selected actinomycetes strains as biocontrol agents of S. lycopersicum and D. carota.

1. Introduction

Tomato (Solanum lycopersicum L.) and carrot (Daucus carota L.) are among the most important vegetables of the Solanaceae and Apiaceae families [1,2]. These plants are attacked by several soil-borne pathogens that induce severe diseases, which affect plant productivity. Fusarium oxysporum and Rhizoctonia solani are among the most important ubiquitous fungal pathogens of several plants, including tomato and carrot [3,4]. Several bacterial pathogens attacks can also affect tomato and carrot productivity. Among these, tomato spot disease caused by Pseudomonas syringae induces necrotic lesions on the different parts of the plant [5], and Pectobacterium carotovorum causes carrot soft rot [6]. Chemical treatments are available to counteract these diseases. However, these compounds are converted into toxic derivatives [7], and their application harms the environment and nontarget organisms [8]. Plants, through beneficial microorganism enlistment via root exudates, counteract the attacks of soil-borne pathogens [9,10]. Among these microorganisms, known as plant growth-promoting bacteria (PGPB), actinomycetes are an important source of bioactive and antimicrobial metabolites and control a wide range of phytopathogens [11]. Actinomycetes can be used as biostimulants, biopesticides, bioherbicides, and biological control agents [12,13], thanks to numerous agroactive compounds and through direct and indirect mechanisms [14]. The few effective formulations commercially available for agricultural use require an expansion of scientific knowledge on the subject. The present work aims to select a consortium of actinomycetes as a valid biocontrol agent for tomato and carrot. We screened the biocontrol capabilities of fourteen Streptomyces spp. and Nocardiopsis spp. actinomycetes that have already been demonstrated to be used as biostimulants [15] and salt stress-tolerance agents [16]. Given these capabilities, we hypothesized that these strains could be valid biocontrol agents against fungal and bacterial pathogens. To test our hypotheses, we screened the actinomycetes in vitro for antifungal and antibacterial antagonistic activities. We combined the most effective actinomycetes in a consortium and subjected them to further in vitro screening, evaluating the effects on fungal hyphae and the antibiofilm and antimicrobial activities of cell-free supernatants. Finally, we evaluated the efficacy of in planta biocontrol in tomato and carrot plants in greenhouse experiments.

2. Results

2.1. Antibacterial Activity

2.1.1. In Vitro Antibacterial Activity of Selected Actinomycetes

The in vitro biocontrol activity against pathogenic bacteria was evaluated by dual culture, investigating the activity of diffusible compounds (classic Petri dishes). An interesting inhibitory effect was observed for seven of the fourteen actinomycetes. The other strains showed no inhibitory effect or moderate single-strain-specific inhibition (<25 mm). Table 1 presents the results recorded for actinomycetes with efficacy against at least two pathogenic strains.
Streptomyces albidoflavus H12 and Nocardiopsis aegyptica H14 showed the highest antibacterial activity in vitro, inhibiting all bacterial strains tested with an inhibition diameter >25 mm. Moderate inhibition (15–25 mm) against all the pathogenic strains was observed for Streptomyces iakyrus G10, Streptomyces xantholiticus G33, and Streptomyces anulatus J13, except for P. syringae (low inhibition, 5–15 mm). For Streptomyces ambofaciens, J27 was observed with only low antagonist activity against Pc. carotovorum strains, while for N. aegyptica S2, moderate inhibitory activity was observed for one of the Pc. carotovorum strains (PC2) and P. corrugata.

2.1.2. Cell-Free Supernatants Minimum Inhibitory Concentration (MIC) and Maximum Bactericidal Concentration (MBC)

S. albidoflavus H12 and N. aegyptica H14 cell-free supernatants (CFSs) were investigated for their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The MIC and MBC values of the CFSs singularly and in combination are shown in Table 2.
The CFS of S. albidoflavus H12 was active at small concentrations only against the strain PC2 Pc. carotovorum (0.2–0.8%) with bactericidal activity (same values for MIC and MBC). Against P. syringae and P. corrugata, the MIC of this CFS was 25%. At this concentration, the activity was only bacteriostatic; bactericidal activities were obtained with 50% CFS. The observed activity for CFS of Nocardiopsis aegyptica H14 was slightly lower, with MIC values of 1.6% against Pc. carotovorum PC2 and 50% against P. syringae and P. corrugata. Bacteriostatic and bactericidal activity were observed at these concentrations, respectively. The combination of CFSs of the two strains did not affect the inhibitory and bactericidal activities observed for H12 and inhibitory and bacteriostatic activity against PC1 (MBC > 50%) was recorded.

2.1.3. In Planta Biocontrol Activity

The growth parameters recorded in in planta experiments of tomato and carrot are shown in Table 3. Tomato and carrot plants treated with only actinomycetes consortium (CONS) showed increased survival and growth with respect to the control (CTL). The presence of pathogenic bacteria P. syringae and P. corrugata in tomato and Pc. carotovorum strains in carrot induced different negative effects on plants.
In tomato, in the presence of the pathogenic P. syringae (PS) infection alone, there was a decrease in survival in the number of leaves, in the length of root and shoot, and in the presence of damage on plants (p < 0.05). The simultaneous presence of the actinomycetes consortium with the bacterial pathogen (CONS + PS) improved plant response toward pathogenic attack, decreasing the damage and improving plant development (p < 0.05). P. corrugata (PC) pathogenesis affected survival and aerial parts of plants, damaging leaves and reducing shoot length and chlorophyll content compared to the control (CTL, p < 0.05). The simultaneous presence of the actinomycetes consortium (CONS + PC) protected the plants from these negative effects, improving their survival and developmental status (p < 0.05).
In carrot, infection of plants with both Pc. carotovorum strains (PC1 and PC2) induced damage and lower survival and chlorophyll content than the control (CTL, p < 0.05). Cotreatment with the actinomycetes consortium (CON S +PC1/PC2) reduced pathogenic negative effects, improving plant development compared to control in terms of root and shoots lengths and chlorophyll content (p < 0.05).

2.2. Antifungal Activity

2.2.1. In Vitro Antifungal Activity of Selected Actinomycetes

The results of in vitro antifungal activity of actinomycetes with interesting inhibition percentages (>25%) are presented in Figure 1.
In vitro mycelium growth of F. oxysporum f. sp. radicis-lycopersici was inhibited at ~50% by diffusible compounds of S. albidoflavus H12 and N. aegyptica H14. For these two strains, the mycelial inhibition was also obtained from volatile compounds, with percentages of 25%. Among the other strains, an interesting inhibition was recorded for the diffusible compounds of S. iakyrus G10 (50%), S. anulatus J13 (25%), and N. dassonvillei subsp. dassonvillei T45 (35%). The in vitro growth of the mycelium of R. solani was inhibited by a greater number of actinomycetes and with higher rates of inhibition. Mycelium inhibitions greater than 70% were recorded by diffusible compounds of S. xantholiticus G33, S. albidoflavus H12, N. aegyptica H14, N. aegyptica S2, and N. dassonvillei subsp. dassonvillei T45. With the exception of N. aegyptica S2, the volatile compounds of all these strains, together with those of S. xantholiticus G22, were able to inhibit the growth of R. solani by 50%. Given the best performances, the antifungal abilities of S. albidoflavus H12 and N. aegyptica H14 were further investigated.

2.2.2. Actinomycetes Effects on the Hyphal Structure

The effects of Streptomyces albidoflavus H12 and N. aegyptica H14 on fungal mycelia were investigated by scanning electron microscopy (SEM). Micrographs of FORL and RHS mycelia in the absence and presence of actinomycetes are presented in Figure 2 and Figure 3, respectively.
Micrographs of FORL and RHS mycelia grown without actinomycetes (left panels of Figure 2 and Figure 3) showed branched and intertwined hyphae, with continuous structures. The presence of actinomycetes (right panels in Figure 2 and Figure 3) induced morphological deterioration of the hyphae and a change in structures, which appear discontinuous and distorted.

2.2.3. Cell-Free Supernatants Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC)

CFS of S. albidoflavus H12 and N. aegyptica H14 were assayed for MIC and MFC against F. oxysporum f. sp. radicis-lycopersici and Rhizoctonia solani. The results of CFS tested individually and in combination are presented in Table 4.
Active MICs against F. oxysporum f. sp. radicis-lycopersici ranged from 0.4 to 0.8%, with greater inhibition by CFS of N. aegyptica H14. All these concentrations had fungicidal properties (MIC = MFC). For R. solani, the MIC and MFC values of N. aegyptica H14 (0.2% and 0.4%) were lower than those of S. albidoflavus H12 (0.8%). However, for both fungal pathogens, when the CFSs of the two actinomycetes were pooled, fungicidal activities comparable to the lowest values observed for N. aegyptica H14 were observed (0.4%).

2.2.4. In Planta Biocontrol Activity

As found during the in planta antibacterial activity experiments (Table 5), the survival and growth of tomato and carrot plants treated with the actinomycetes consortium (CONS) alone were higher than the control (CTL). The presence of pathogenic bacteria P. syringae and P. corrugata in tomato and Pc. carotovorum strains in carrot induced several negative effects on plants.
In tomato, F. oxysporum f. sp. radicis-lycopersici infection (FORL) reduced survival, caused severe damage, and negatively affected plant shoot and root development. Cotreatment of plants with fungal infection and actinomycetes consortium (CONS + FORL) improved plant status, allowing normal growth and improving some development parameters. Compared to the control (CTL), plants with consortium inoculation and pathogenic infection (CONS + FORL) had lower survival and shoot length (p < 0.05). Nonetheless, a similar number of leaves (p > 0.05) and higher chlorophyll content and root length (p < 0.05) were shown. A similar situation was observed for R. solani. The pathogenic attack of this fungus (RHS) reduced the survival and growth of plants. In the copresence of actinomycetes consortium (CONS + RHS), the pathogenesis was still evident. Compared to control, decreases in growth and development were observed (p < 0.05). However, the attack was less offensive, and less damage and fewer negative effects on plant development than RHS were observed (p < 0.05).
The R. solani pathogenesis on carrot (RHS) strongly reduced plant survival and all developmental parameters. The simultaneous presence of the actinomycetes consortium (CONS + RHS), even if not completely, significantly reduced the negative effects on survival; compared to the control, similar root length, amount of chlorophyll (CTL, p > 0.05), and higher shoot length and number of leaves (p < 0.05) were observed.
To investigate the contribution of the in vitro antimicrobial properties of S. albidoflavus H12 and N. aegyptica H14 on bacterial and fungal biocontrol in planta, the results were included in a single dataset and processed by a principal component analysis. The biplot is presented in Figure 4.
The first principal component (F1) accounted for a total variance of 55% and the second (F2) of 24%; the total variance explained by these two components was 78%. The biplot grouping, based on data correlations, showed a correlation of in planta fungal biocontrol of tomato and carrot with the in vitro inhibitory activities shown for S. albidoflavus H12 and N. aegyptica H14. Experimental conditions in planta of carrot bacterial pathogens were included in this grouping, together with an increased survival rate in plants treated with actinomycetes consortium and infected with pathogens, compared to plants with the sole infection. The other parameters in planta had the same correlations of bacterial biocontrol, with the exception of leaves number, which had similar correlations to in planta experimental conditions of tomato bacterial pathogens and to the in vitro inhibitory and microbicidal concentrations found for CFS S. albidoflavus H12 and N. aegyptica H14. These correlations underline that, at a statistical level, the differences in planta shown between plants treated with actinomycetes consortium and infected with pathogens and plants with the sole infection can be ascribed to in vitro properties highlighted by different tests. The fungal biocontrol was more correlated (and thus ascribed) to the in vitro inhibitory activities shown for S. albidoflavus H12 and N. aegyptica H14. Bacterial biocontrol was more correlated/ascribed to the activity of the metabolites present in the CFS of S. albidoflavus H12 and N. aegyptica H14.

3. Discussion

Diseases caused by pathogenic bacteria and fungi cause significant damage to crops, resulting in decreased yields and increased costs. To address this problem, farmers usually use synthetic chemicals, with major negative consequences for the environment and human health. The present work focused on the evaluation of several beneficial actinomycetes as biocontrol agents against bacterial and fungal pathogenic strains of tomato and carrot. Our approach made it possible to select a valid consortium useful for combating the selected pathogens. The in vitro assays and in planta experiments highlighted the suitability of the consortium formed by Streptomyces albidoflavus H12 and Nocardiopsis aegyptica H14 as a biocontrol agent. The consortium was able to control in planta bacterial and fungal pathogenic attacks. The biocontrol activity could be ascribed to the different abilities of S. albidoflavus H12 and N. aegyptica H14. This study also highlighted a contribution of the antibacterial and antifungal metabolites present in cell-free supernatants of both strains with bactericidal and fungicidal properties.
The antibacterial and antifungal activities are common traits found among actinomycetes strains [11,17,18,19,20,21,22,23,24,25]. The biocontrol properties of Streptomyces and Nocardiopsis genera are mainly related to the release of extracellular metabolites, siderophores, and enzymes, with a broad antimicrobial spectrum [25,26,27]. The genus Streptomyces is the major producer of antibiotics. It plays an important role in the suppression of plant diseases induced by bacterial pathogens and has a great diversity of genes for the production of biocontrol secondary metabolites [28,29].
The biocontrol of bacterial plant pathogens by Streptomyces has been reported in many studies [17,30,31]. Diffusible and volatile antibacterial metabolites have direct activity on pathogenic bacterial cells and on their quorum sensing, disrupting the gene expression regulation in response to cell-population density and biofilm formation [32]. The ability of Streptomyces to control plant diseases caused by fungal pathogens is widely described for F. oxysporum [20,33,34,35,36,37] and R. solani [7,38,39,40,41,42,43]. The diffusible and volatile compounds produced by Streptomyces also act against the polymeric compounds of fungal pathogens, such as chitin [44]. Cell-free supernatants produced by Streptomyces are a valid source of biocontrol metabolites against F. oxysporum and R. solani [45,46,47,48,49]. Recent work by Lyu and collaborators demonstrated that reveromycin A and B—polyketides compounds with strong antifungal activity—extracted from Streptomyces sp. 3–10 CFS inhibit several phytopathogenic fungi, including R. solani [50].
The genus Nocardiopsis produces a large variety of bioactive compounds, including antimicrobial agents and enzymes [51]. The biocontrol compounds described for this genus are pendolmycin, a strong antifungal agent, and griseusins, thiopeptides, and naphthospironones, with antimicrobial power [51]. The activity against bacterial and fungal plant pathogens is less reported than in Streptomyces. However, metabolites of this genus have been reported to be effective antimicrobial and antifungal agents [52]. The recent work by Sabu and collaborators described a good biocontrol attitude of a Nocardiopsis sp. isolate, with broad antibacterial, antibiofilm, and antiphytopathogenic activity [53]. The CFS compounds of a halophilic Nocardiopsis gilva isolate have been reported to contain a novel p-terphenyl 1 compound with in vitro inhibitory activities against Fusarium spp. [54].
The in planta biocontrol was tested by combining the most efficient isolates; this strategy was in line with literature evidence supporting the use of microbial consortia for the control of microbial phytopathogens [8,55,56,57,58]. The efficacy of in planta biocontrol shown by the consortium of S. albidoflavus H12 and N. aegyptica H14 can be ascribed not only to the activities mentioned above, but also to other indirect mechanisms. Once associated with plant rhizosphere and tissues, actinomycetes counteract microbial phytopathogens by promoting plant growth and development through the solubilization of phosphates, indoleacetic acid, hydrogen cyanide, and ammonia production [59]. Actinomycetes also induce systemic resistance [60], promoting a faster and stronger response of plants to pathogenic attacks [61].

4. Materials and Methods

The following fourteen strains of actinomycetes, previously isolated and characterized [62], were used in this study:
  • Nocardiopsis aegyptica H14 (MG597543).
  • Nocardiopsis aegyptica S2 (MG597572).
  • Nocardiopsis alba J21 (MG597576).
  • Nocardiopsis dassonvillei subsp. dassonvillei D14 (MG597514).
  • Nocardiopsis dassonvillei subsp. dassonvillei T45 (MG597502).
  • Streptomyces albidoflavus H12 (MG597552).
  • Streptomyces ambofaciens J27 (MG597599).
  • Streptomyces anulatus J13 (MG597579).
  • Streptomyces iakyrus G10 (MG597593).
  • Streptomyces thinghirensis K23 (MG597560).
  • Streptomyces thinghirensis J4 (MG597590).
  • Streptomyces xantholiticus K12 (MG597545).
  • Streptomyces xantholiticus G22 (MG597582).
  • Streptomyces xantholiticus G33 (MG597585).
Cultures of the strains were grown on International Streptomyces Project No. 2 (ISP2) medium at 30 °C for 7 d.

4.1. Antibacterial Activity

The fourteen isolates listed above were screened for in vitro biocontrol activity against pathogenic bacterial strains by the dual-culture method. Cell-free supernatants of the most effective strains (MIC up to 0.8%) were combined and assayed by minimum inhibitory concentration (MIC) and minimum bactericidal activity (MBC) on 96-well plates. Broth cultures of the most effective strains were also combined in a consortium and tested for in planta biocontrol effectiveness under greenhouse experiments. Phytopathogenic bacteria Pseudomonas syringae pv. tomato (PS) and Pseudomonas corrugata pv. tomato (PC)—from tomato—and Pectobacterium carotovorum subsp. carotovorum strains PC1 and PC2—from carrot—were provided by Prof. Giuliano Bonanomi of the Dept. of Agriculture, University of Naples Federico II (Italy).

4.1.1. In Vitro Antibacterial Activity of Selected Actinomycetes

The in vitro antibacterial activity was evaluated by cocultivation of bacterial pathogenic strains with agar plugs of actinomycetes. A 100 µL volume of nutrient broth (NB) cultures of pathogenic strains, grown overnight at 37° under 150 rpm with constant shaking, was spread on the plate of ISP2/NA (nutrient agar) dishes (Ø 90 mm dishes, 50% ISP2 and 50% NA). Agar plugs of 5 d old selected actinomycetes strains grown on ISP2 solid medium were placed on streaked ISP2/NA plates, and dishes were incubated at 37 °C for 24 h. The inhibition zones formed around the agar plugs after cultivation were measured with a ruler.

4.1.2. Cell-Free Supernatants Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

Streptomyces albidoflavus H12 and Nocardiopsis aegyptica H14 cell-free supernatants (CFSs) were investigated for antibacterial activity following the CLSI guidelines [63]. CFSs were obtained from broth cultures of ISP2 strains grown at 30 °C for 7 d with constant agitation (150 rpm). Cultures were centrifuged at 10,000 g for 10 min and filtered through a 0.22 μm bacteriological filter. CFSs were tested singularly and in combination (50:50). Round-bottom 96-well plates and nutrient broth (NB) medium were used for the test, and the readings were carried out spectrophotometrically, after incubation at 37 °C for 24 h, by resazurin (Alamar blue) addition [64]. For each CFS, the minimum inhibitory concentration (MIC) was identified in the well without growth containing the lower CFS concentration. Minimum bactericidal concentration (MBC) was estimated by plating 100 µL of the wells in which there was no growth on NB medium. After incubation of the plates at 37 °C for 24 h, the corresponding concentration of CFS was considered bactericidal in the absence of colonies, while it was considered bacteriostatic in the presence of colonies.

4.1.3. In Planta Antibacterial Activity

The consortium’s in planta antimicrobial activity was evaluated on tomato, against PS and PC strains, and on carrot, against PC1 and PC2. The experimental conditions were arranged as follows:
  • CTL: control, untreated plants.
  • CONS: plants inoculated only with actinomycetes.
  • CONS + PS/PC/PC1/PC2: plants treated with actinomycetes and infected with a bacterial pathogen.
  • PS/PC/PC1/PC2: plants infected only with a bacterial pathogen.
Plant seeds were treated with 108 spore mL−1 from the actinomycetes consortium (equal amounts of S. albidoflavus H12 and N. aegyptica broth cultures) and allowed to dry overnight at room temperature. Each experimental condition comprised 25 pots with two seeds per pot (6/7 diameter) filled with common soil (pH = 8.2, electrical conductivity = 1 ds/m, total porosity = 81% (v/v)). Infections were realized after plant germination, using bacterial suspensions of 106 CFU mL−1. The experiments were carried out under natural light and environmental temperature until the symptoms of the disease manifested. Induced protection was evaluated by estimating plant survival, number of leaves, shoot and root lengths, degree of damage, and chlorophyll content [2].

4.2. Antifungal Activity

The fourteen actinomycetes were screened for in vitro biocontrol activity against two fungal pathogens of tomato and carrot, Rhizoctonia solani and Fusarium oxysporum f. sp. radicis-lycopersici (provided by Prof. Giuliano Bonanomi of the Dept. of Agriculture, University of Naples Federico II, Italy). The in vitro biocontrol activity against fungal strains was assessed by dual culture, studying the inhibitory activities of diffusible (classic Petri dishes) and volatile (biplate Petri dishes) compounds. The inhibition zones of the most active strains were also studied by SEM. The cell-free supernatants of the most effective strains were combined and assayed by minimum inhibitory concentration (MIC) and minimal fungicidal concentration (MFC). Broth cultures of the most effective strains were also combined in a consortium and tested for efficacy in planta biocontrol.

4.2.1. In Vitro Antifungal Activity of Selected Actinomycetes (Diffusible and Volatile Compounds)

The in vitro antagonistic activity was evaluated by cocultivation of fungal pathogens with actinomycetes strains. Inhibition of diffusible compounds was estimated by ISP2/potato dextrose agar (PDA) plates (50:50) using classic Petri dishes. Volatile compounds were estimated using biplate Petri dishes containing ISP2 for actinomycetes and PDA for fungi. Actinomycetes and fungi were transferred to plates using agar plugs (Ø 5 mm) obtained from 5 d cultures. For both plate types of fungi, positive controls were prepared without the addition of plugs. Dishes were incubated at 28 °C until the mycelium covered the entire plate of the positive fungi control (5–7 d). Each trial was repeated 3 times, and the inhibition percentages were calculated as follows:
I   %   =   ( mm   growth   control   −   mm   growth   dual   culture ) mm   growth   control   ×   100

4.2.2. Actinomycetes Effects on the Hyphal Structure

The effects of actinomycetes strains on F. oxysporum f. sp. radicis-lycopersici (FORL) and R. solani (RHS) were investigated by scanning electron microscopy (SEM), using a Gemini SEM 500 SEM (Zeiss, Oberkochen, Germany). Small pieces of agar from the actinomycete/fungus interaction zone and the fungus growth control were taken and mounted on adhesive tape. Freshly collected samples were observed using a specific stage for Peltier technology, with a working temperature of −1 °C. Micrographs acquisition was performed with a working distance of 8.1 mm, an acceleration voltage of 10 kV, and using a backscattered electrons detector (BSD4 signal).

4.2.3. Cell-Free Supernatants Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC)

Streptomyces albidoflavus H12 and Nocardiopsis aegyptica H14 cell-free supernatants (CFSs) were also investigated for antifungal activity following the CLSI guidelines [64]. The CFSs were obtained as described in Section 4.1.2. CFSs were tested singularly and in combination (50:50) using 96-well round-bottom plates and PDB medium. The readings were performed spectrophotometrically, after incubation at 28 °C for 5 d, by adding resazurin (Alamar blue, Bio-Rad, Hercules, CA, USA) [64]. For each CFS, the minimum inhibitory concentration (MIC) was identified in the well without growth containing the lower CFS concentration. The minimum fungicidal concentration (MFC) was estimated by plating 100 µL of the wells in which there was no growth on PDA medium. After incubation of the plates at 28 °C for 5 d, the corresponding concentration of CFS was considered fungicidal in the absence of spore germination, while it was considered fungistatic in the presence of growth.

4.2.4. In Planta Antifungal Activity

The consortium’s in planta antifungal activity was evaluated on tomato against FORL and RHS and on carrot against RHS. The experimental conditions were arranged as follows:
  • CTL: control, untreated plants.
  • CONS: plants inoculated only with actinomycetes.
  • CONS + FORL/RHS: plants treated with actinomycetes and infected with a fungal pathogen.
  • FORL/RHS: plants infected only with a fungal pathogen.
Treatment of seeds with actinomycetes was carried out as described in Section 4.1.3. Fungal infections were carried out by inoculating the plants with the fungal spore/mycelial suspensions as previously described [2]. Twenty-five pots with two seeds per pot (6/7 diameter) were arranged for each experimental condition. The pots were filled with common soil, and the experiments were carried out under natural light and environmental temperature. Once symptoms of the disease manifested, plant survival, number of leaves, shoot and root lengths, degree of damage, and chlorophyll content were estimated [2].

4.3. Statistical Analyses

Data were analyzed by one-way analysis of variance (ANOVA), utilizing Fisher’s least significant difference (LSD) post-hoc test to compare the mean values to a significance level of 5% (p < 0.05). The principal component analysis (PCA) algorithm was used to decompose the dataset of in vitro and in planta results (increase the percentages in plants treated with actinomycetes consortium and infected with pathogens respect to plants with only infection). The plant tested and fungal or bacterial biocontrol were considered as supplementary categorical variables (columns). In Figure 4: Ps, Pseudomonas syringae; Pc, Pseudomonas corrugata, PC1/PC2, Pectobacterium carotovorum strain 1 and strain 2; FORL, Fusarium oxysporum f. sp. radicis-lycopersici; RHS, Rhizoctonia solani. All statistical analyses were carried out using XLSTAT 2014 software (Addinsoft, Paris, France).

5. Conclusions

Currently, agriculture is largely dependent on agrochemicals, with a negative impact on ecosystems. Alternatives are needed such as the use of bioagents to fight plant diseases. In this study, several methods were used to determine the antagonistic activity of actinomycetes isolates against various pathogenic fungi and bacteria. The results obtained showed the in vitro power to inhibit pathogenic strains with the dual-culture method and with cell-free supernatants. The results demonstrated good in vitro potential against both bacterial and fungal pathogens. The most effective strains, Streptomyces albidoflavus H12 and Nocardiopsis aegyptica H14, were further investigated and grouped in a consortium for testing in in planta experiments. In general, for both carrot and tomato plants, treatment with the bacterial consortium counteracted microbial infections, improving growth and development.
The results of this work are encouraging. These strains have shown good biofertilization and salt stress control properties in previous studies. More in-depth studies are needed on the characterization of secondary metabolites, on plant association, and on the formulation of consortia. The suitability of applying these strains against other pathogens and in the open fields also requires further research. However, our results underline the possible role of these actinomycetes as biocontrol agents in the sustainable management of tomato and carrot crops.

Author Contributions

Conceptualization, M.D.G. and M.K.; methodology, C.E.; software, M.P.; validation, M.P., C.E. and M.D.G.; formal analysis, R.D. and B.F.; investigation, R.D. and B.F.; data curation, R.D.; writing—original draft preparation, R.D.; writing—review and editing, M.P. and M.D.G.; visualization, R.D. and B.F.; supervision, M.P.; project administration, M.P. and M.D.G.; funding acquisition, M.D.G. and M.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data that support the findings of this study are available upon request from the corresponding author.

Acknowledgments

We wish to thank Lorenzo Arrizza for the support in the scanning electron microscope analysis.

Conflicts of Interest

The authors declare no conflict of interest.

References

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Figure 1. In vitro antifungal activity with diffusible compounds (DFCs) and volatile compounds VOCs of the selected actinomycetes strains against Fusarium oxysporum f. sp. radicis-lycopersici (graph on the top) and Rhizoctonia solani (graph on the bottom). In the figure: SiG10, Streptomyces iakyrus G10; SaH12, Streptomyces albidoflavus H12; NaH14, Nocardiopsis aegyptica H14; SaJ13, Streptomyces anulatus J13; NddT45, Nocardiopsis dassonvillei subsp. dassonvillei T45; SxG22, Strepptomyces xantholiticus G22; SxG33, Strepptomyces xantholiticus G33; NaS2, N. aegyptica S2.
Figure 1. In vitro antifungal activity with diffusible compounds (DFCs) and volatile compounds VOCs of the selected actinomycetes strains against Fusarium oxysporum f. sp. radicis-lycopersici (graph on the top) and Rhizoctonia solani (graph on the bottom). In the figure: SiG10, Streptomyces iakyrus G10; SaH12, Streptomyces albidoflavus H12; NaH14, Nocardiopsis aegyptica H14; SaJ13, Streptomyces anulatus J13; NddT45, Nocardiopsis dassonvillei subsp. dassonvillei T45; SxG22, Strepptomyces xantholiticus G22; SxG33, Strepptomyces xantholiticus G33; NaS2, N. aegyptica S2.
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Figure 2. SEM micrographs of Fusarium oxysporum f. sp. radicis-lycopersici hyphae in the absence (on the left) and in the presence (on the right) of actinomycetes. The panel on the left shows the normal branching of hyphae. In the right panel, thinner and more distorted hyphal structures can be seen, with many disaggregation points. Scale bars (in red) 20 µm.
Figure 2. SEM micrographs of Fusarium oxysporum f. sp. radicis-lycopersici hyphae in the absence (on the left) and in the presence (on the right) of actinomycetes. The panel on the left shows the normal branching of hyphae. In the right panel, thinner and more distorted hyphal structures can be seen, with many disaggregation points. Scale bars (in red) 20 µm.
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Figure 3. SEM micrographs of Rhizoctonia solani hyphae in the absence (on the left) and in the presence (on the right) of actinomycetes. The panel on the left shows the normal hyphal structure and development. In the panel on the right, the inhibition of development by disruption of the hyphal structures can be observed. Scale bars (in red) 20 µm.
Figure 3. SEM micrographs of Rhizoctonia solani hyphae in the absence (on the left) and in the presence (on the right) of actinomycetes. The panel on the left shows the normal hyphal structure and development. In the panel on the right, the inhibition of development by disruption of the hyphal structures can be observed. Scale bars (in red) 20 µm.
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Figure 4. Biplot (scores and loadings) obtained from the principal component analysis on a dataset of in vitro and in planta results (increase in the percentages in plants treated with the actinomycetes consortium and infected with pathogens compared to plants with the only infection). The tested plant and fungal or bacterial biocontrol were considered as supplementary categorical variables. In the Figure: PS, Pseudomonas syringae; PC, Pseudomonas corrugata, PC1/PC2, Pectobacterium carotovorum strain 1 and strain 2; FORL, Fusarium oxysporum f. sp. radicis-lycopersici; RHS, Rhizoctonia solani. Chl, chlorophyll; H12/H14 Inhibition, in vitro bacterial and fungal inhibition by Streptomyces albidoflavus H12/Nocardiopsis aegyptica H14; MIC CFS H12/H14, minimum inhibitory concentrations of CFS of S. albidoflavus H12/N. aegyptica H14; MMC CFS H12/H14, minimum microbicidal concentrations of CFS of S. albidoflavus H12/N. aegyptica H14.
Figure 4. Biplot (scores and loadings) obtained from the principal component analysis on a dataset of in vitro and in planta results (increase in the percentages in plants treated with the actinomycetes consortium and infected with pathogens compared to plants with the only infection). The tested plant and fungal or bacterial biocontrol were considered as supplementary categorical variables. In the Figure: PS, Pseudomonas syringae; PC, Pseudomonas corrugata, PC1/PC2, Pectobacterium carotovorum strain 1 and strain 2; FORL, Fusarium oxysporum f. sp. radicis-lycopersici; RHS, Rhizoctonia solani. Chl, chlorophyll; H12/H14 Inhibition, in vitro bacterial and fungal inhibition by Streptomyces albidoflavus H12/Nocardiopsis aegyptica H14; MIC CFS H12/H14, minimum inhibitory concentrations of CFS of S. albidoflavus H12/N. aegyptica H14; MMC CFS H12/H14, minimum microbicidal concentrations of CFS of S. albidoflavus H12/N. aegyptica H14.
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Table
Table 1. In vitro antibacterial activity of the selected actinomycetes strains against Pseudomonas syringae, Pseudomonas corrugata, and Pectobacterium carotovorum subsp. carotovorum (PC1 and PC2).
Table 1. In vitro antibacterial activity of the selected actinomycetes strains against Pseudomonas syringae, Pseudomonas corrugata, and Pectobacterium carotovorum subsp. carotovorum (PC1 and PC2).
SiG10SxG33SaH12NaH14SaJ13SaJ27NaS2
P. syringae+++++++++−−
P. corrugata++++++++++++−++
Pc. carotovorum PC1+++++++++++++−
Pc. carotovorum PC2+++++++++++++++
+++, high inhibition, > 25 mm; ++, moderate inhibition, (15–25 mm); + low inhibition, (5–15 mm); −, no inhibition. SiG10, Streptomyces iakyrus G10; SxG33, Streptomyces xantholiticus G33; SaH12, Streptomyces albidoflavus H12; NaH14, Nocardiopsis aegyptica H14; SaJ13, Streptomyces anulatus J13; SaJ27, Streptomyces ambofaciens J27; NaS2, N. aegyptica S2.
Table
Table 2. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) percentages of Streptomyces albidoflavus H12 and Nocardiopsis aegyptica H14 cell-free supernatants (singularly and in combination) against Pseudomonas syringae, Pseudomonas corrugata, and Pectobacterium carotovorum subsp. carotovorum (PC1 and PC2).
Table 2. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) percentages of Streptomyces albidoflavus H12 and Nocardiopsis aegyptica H14 cell-free supernatants (singularly and in combination) against Pseudomonas syringae, Pseudomonas corrugata, and Pectobacterium carotovorum subsp. carotovorum (PC1 and PC2).
Pathogenic BacteriaSaH12 CFSNaH14 CFSSaH12 + NaH14 CFS
MICMBCMICMBCMICMBC
P. syringae25.550.050.050.025.050.0
P. corrugata25.550.050.050.025.050.0
Pc. carotovorum PC1----50.0-
Pc. carotovorum PC20.80.80.81.60.80.8
In the table: SaH12, Streptomyces albidoflavus H12; NaH14, Nocardiopsis aegyptica H14; CFS, cell-free supernatants.
Table
Table 3. In planta antibacterial activity of the consortium of Streptomyces albidoflavus H12 and Nocardiopsis aegyptica H14 against Pseudomonas syringae and Pseudomonas corrugata in tomato and Pectobacterium carotovorum strains in carrot.
Table 3. In planta antibacterial activity of the consortium of Streptomyces albidoflavus H12 and Nocardiopsis aegyptica H14 against Pseudomonas syringae and Pseudomonas corrugata in tomato and Pectobacterium carotovorum strains in carrot.
Survival (%)Leaves (n°)Damage (Grade)Root (cm)Shoot (cm)Chlorophyll (mg g FW−1)
Tomato
CTL85.90 b3.40 c-2.30 c7.04 c0.94 b
CONS98.80 a5.30 a-3.99 a11.09 a1.08 a
CONS + PS64.70 c4.80 a,b2.80 c2.96 b9.32 b1.08 a
CONS + PC87.00 b4.60 b3.50 b2.79 b8.89 b1.00 b
PS60.70 e2.70 d2.50 a2.02 d6.06 e0.62 c
PC50.40 d2.20 d4.70 c1.25 c4.15 d0.62 c
LSD1.240.630.470.370.880.06
Carrot
CTL88.00 B2.00 B-1.79 B2.60 D,E0.89 C
CONS98.60 A3.70 A-3.35 A4.90 A1.10 A
CONS + PC186.80 B2.00 B2.50 B3.08 A4.27 B1.08 A
CONS + PC240.80 C2.00 B2.50 B3.07 A3.78 C0.99 B
PC140.20 C2.00 B4.80 A1.40 B2.65 D0.61 D
PC227.40 D2.00 B4.50 A1.63 B2.16 E0.61 D
LSD1.400.180.370.400.480.07
In the table: CONS, consortium formed by Streptomyces albidoflavus H12 and Nocardiopsis aegyptica H14; CTL, control; PS, Pseudomonas syringae; PC, Pseudomonas corrugata; PC1, Pectobacterium carotovorum subsp. carotovorum strain 1; PC2, Pectobacterium carotovorum subsp. carotovorum strain 2; LSD, least significant difference. Results followed by the same case letter are not significantly different according to Fisher’s LSD post-hoc test. Lowercase letters (a–d) refer to the comparison of tomato experimental conditions. Uppercase letters (A–E) refer to the comparison of carrot experimental conditions.
Table
Table 4. Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) percentages of Streptomyces albidoflavus H12 and Nocardiopsis aegyptica H14 cell-free supernatants (singularly and in combination) against Rhizoctonia solani and Fusarium oxysporum f. sp. radicis-lycopersici.
Table 4. Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) percentages of Streptomyces albidoflavus H12 and Nocardiopsis aegyptica H14 cell-free supernatants (singularly and in combination) against Rhizoctonia solani and Fusarium oxysporum f. sp. radicis-lycopersici.
Pathogenic BacteriaSaH12 CFSNaH14 CFSSaH12 + NaH14 CFS
MICMFCMICMFCMICMFC
Fusarium oxysporum0.80.80.40.40.40.4
Rhizoctonia solani0.80.80.20.40.40.4
In the table: SaH12, Streptomyces albidoflavus H12; NeH14, Nocardiopsis aegyptica H14; CFS, cell-free supernatants.
Table
Table 5. In planta antifungal activity of Streptomyces albidoflavus H12 and Nocardiopsis aegyptica H14 consortium against Fusarium oxysporum f. sp. radicis-lycopersici and Rhizoctonia solani in tomato and R. solani in carrot.
Table 5. In planta antifungal activity of Streptomyces albidoflavus H12 and Nocardiopsis aegyptica H14 consortium against Fusarium oxysporum f. sp. radicis-lycopersici and Rhizoctonia solani in tomato and R. solani in carrot.
Survival (%)Leaves (n°)Damage (Grade)Root (cm)Shoot (cm)Chlorophyll (mg g FW−1)
Tomato
CONS98.40 a6.00 a-3.85 a10.71 a1.10 a
CTL86.40 b3.60 b,c-2.98 c7.71 b0.94 b
CONS + FORL78.50 c4.00 b2.60 b3.51 b6.15 c1.07 a
CONS + RHS60.30 d3.40 c2.60 b3.37 b5.90 c0.72 c
FORL53.60 e2.60 d4.60 a1.76 e4.69 d0.39 d
RHS49.60 f2.00 e4.60 a2.23 d3.93 e0.42 d
LSD1.300.560.380.250.510.09
Carrot
CONS98.70 A3.70 A-3.10 A4.89 A0.84 A
CTL88.00 B2.00 C-2.30 B3.20 B0.94 A
CONS + RHS44.30 C2.50 B2.70 B2.40 B4.80 A1.06 A
RHS20.20 D1.50 D4.80 A1.43 C2.60 C0.57 B
LSD2.130.400.360.280.520.24
In the table: CONS, consortium formed by Streptomyces albidoflavus H12 and Nocardiopsis aegyptica H14; CTL, control; FORL, Fusarium oxysporum f. sp. radicis-lycopersici; RHS, Rhizoctonia solani; LSD, least significant difference. Results followed by the same case letter are not significantly different according to Fisher’s LSD post-hoc test. Lowercase letters (a–e) refer to the comparison of tomato experimental conditions. Uppercase letters (A–D) refer to the comparison of carrot experimental conditions.
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Djebaili, R.; Pellegrini, M.; Ercole, C.; Farda, B.; Kitouni, M.; Del Gallo, M. Biocontrol of Soil-Borne Pathogens of Solanum lycopersicum L. and Daucus carota L. by Plant Growth-Promoting Actinomycetes: In Vitro and In Planta Antagonistic Activity. Pathogens 2021, 10, 1305. https://doi.org/10.3390/pathogens10101305

AMA Style

Djebaili R, Pellegrini M, Ercole C, Farda B, Kitouni M, Del Gallo M. Biocontrol of Soil-Borne Pathogens of Solanum lycopersicum L. and Daucus carota L. by Plant Growth-Promoting Actinomycetes: In Vitro and In Planta Antagonistic Activity. Pathogens. 2021; 10(10):1305. https://doi.org/10.3390/pathogens10101305

Chicago/Turabian Style

Djebaili, Rihab, Marika Pellegrini, Claudia Ercole, Beatrice Farda, Mahmoud Kitouni, and Maddalena Del Gallo. 2021. "Biocontrol of Soil-Borne Pathogens of Solanum lycopersicum L. and Daucus carota L. by Plant Growth-Promoting Actinomycetes: In Vitro and In Planta Antagonistic Activity" Pathogens 10, no. 10: 1305. https://doi.org/10.3390/pathogens10101305

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