Biocontrol of Agroathelia rolfsii associated with stem rot disease in tomato (Solanum lycopersicum L.) and growth promotion using compost-associated actinobacteria

Abstract

Stem rot disease (Agroathelia rolfsii) biocontrol is an environmentally safe alternative that could potentially decrease disease severity and limit plant and yield losses. In the present investigation, 11 actinomycetes isolates, recovered from disease-suppressive composts, were tested as whole cell suspensions and cell-free culture filtrates for their ability to suppress tomato stem rot and to stimulate plant growth. Five isolates (namely A5-3, A2-4, A3-4, A4-4 and A5-4), applied as cell suspensions, were found to be the most effective in suppressing disease severity by 37.5–56.2% compared to the untreated control and 25-56.2% using their cell-free culture filtrates. The in vitro antifungal activity of isolates tested and their filtrates were estimated at 58.8–88% and 59-91.3% decrease in fungus mycelial growth, respectively. As for their growth-promoting ability, tomato plants treated with A5-3, A2-4, A3-4, A4-4 and A5-4 isolates were 20-89.1% and 10.3–79% higher than A. rolfsii-inoculated and pathogen-free controls, respectively. Inoculated and uninoculated plants treated with filtrates showed significant increments in their growth parameters by 18.2–91.9% and 15.3–93.4% over control, respectively. The most bioactive isolates against target pathogen were affiliated, based on 16 S rDNA gene sequencing, to Streptomyces, Saccharomonospora and Micromonospora genera. All these isolates were shown able to produce indole-3-acetic acid. Streptomyces sp. (A5-3) and Streptomyces sp. (A5-4) displayed chitinase, protease and lipase activities together with phosphate solubilization and nitrogen-fixing abilities. Streptomyces sp. (A5-3) displayed the greatest amylolytic activity and ability to solubilize zinc and to produce siderophores and hydrogen cyanide. This investigation demonstrated that actinomycetes recovered from disease-suppressive composts can be explored as potential sources of bio-active compounds with antifungal and bio-fertilizing abilities useful for the improvement of tomato growth and health.

Introduction

Soilborne diseases are a serious threat to crop yield [1]. Southern blight or stem rot, caused by Agroathelia rolfsii (Cruzi) Tu &.

Kimbrough (syn. Sclerotium rolfsii Sacc.), is a devastating soilborne disease [2, 3]. The fungus has a wide host range exceeding 500 plant species including leguminous and vegetable crops [4, 5]. Infection process occurs at all growth stages where the fungus directly attacks healthy plant tissues using various pathogenicity factors such as cell-wall degrading enzymes and other secondary metabolites. A. rolfsii is able to produce oxalic acid [6] and various cellulolytic and pectinolytic enzymes to lower pH, sequester calcium from cell walls, suppress host defensive responses, and induce serious alterations in the infected tissues [7, 8]. Severely diseased plants may ultimately wilt thus leading to serious stand reductions and subsequently yield losses reaching 100% depending on growing conditions and host plant [5, 9, 10].

A. rolfsii is especially difficult to control due to its wide range of alternative hosts, the lack of resistant cultivars, and its ability to overwinter in soil for several years as sclerotia [4, 11]. In the world, this disease is frequently managed using chemical methods. However, the hazardous application of these agrochemicals induces deleterious effects on human health and environmental and food safety [12].

Among the most widely explored alternatives for achieving sustainable plant disease management, biocontrol using various beneficial microorganisms has gained increased attention [13–17]. Microorganisms are able to suppress soilborne pathogens either directly through antagonism or indirectly through spatial and/or trophic competition for available resources and by stimulating plant defensive mechanisms or both [18, 19].

Beneficial microorganisms were isolated from disease-suppressive soils with low disease incidence [20, 21] and/or composts [22–24]. Among the widely recovered compost-associated microbial agents, bacteria, fungi, and actinomycetes were the focal driving forces for plant disease bio-suppression and growth stimulation [25].

Actinomycetes are prokaryotic organisms and a distinct group of gram-positive bacteria with a significant ability to suppress various airborne and soilborne plant diseases [26–28], to enhance plant and soil health [29, 30], and to mitigate the adverse effects of abiotic stresses [31, 32]. The key mechanism of action displayed by actinomycetes is the biosynthesis of secondary metabolites with various biological properties [33–35]. These microbial agents can protect plants by inhibiting the development of fungal pathogens through the production of antifungal compounds or cell-wall degrading enzymes [29, 36]. They can also improve plant growth via the production of siderophores and indole-3-acetic acid (IAA) [37, 38] and/or by carrying out symbiotic fixation of nitrogen [39]. Successful biocontrol using actinomycetes has been reported against numerous soilborne pathogens including Fusarium oxysporum [40, 41], Rhizoctonia solani [40, 42], Verticillium dahliae [43, 44], Phytophthora sp. [45], and Pythium ultimum [46]. However, to the best of our knowledge, little information is available on the potential use of actinomycetes for the biocontrol of stem rot disease of tomato induced by A. rolfsii.

Therefore, given the importance of the tomato crop in Tunisia, the polyphagic nature of target pathogen and its ability to induce serious plant losses, the present study was undertaken to evaluate the ability of compost-associated actinomycetes to suppress stem rot disease and to promote growth of tomato plants. The most effective isolates were screened for their in vitro antifungal potential and plant growth-promoting features.

Materials and methods

Plant material

Tomato (Solanum lycopersicum L.) cv. Rio Grande seeds were used in the current study. They were disinfected by immersion into 70% ethanol for 2 min then into 0.2% sodium hypochlorite solution for 3 min. They were rinsed several times with sterile distilled water and allowed to dry under laminar flow. Seeds were sown in 77-alveolated trays filled with commercialized peat (Klasmann-Deilmann, Bio-Substrate, Germany), regularly watered to avoid water stress and kept under greenhouse conditions at 25 ± 5 °C air temperature, 60–70% relative humidity and 12 h photoperiod. Seedlings at the two-true-leaf growth stage and having relatively similar heights were used in all in vivo trials.

Pathogen isolation and culture

The three A. rolfsii isolates used in the current study were originally recovered from artichoke (Cynara scolymus L.) stems showing typical Southern Blight symptoms (Sr₁) and potato tubers exhibiting severe soft rots (Sr₂ and Sr₃). They were identified and characterized based on their pathogenic and biological traits in our previous investigations [47, 48]. The most virulent isolate (Sr₁) was selected for the in vitro bioassays and a consortium of the three isolates (at 1:1:1 ratio) was used for the in vivo trials [49]. Isolates were grown for 7 days on Potato Dextrose Agar (PDA) medium and incubated at 30 °C in the dark [48] before use. Pathogen stock cultures were maintained on PDA slants at 4 °C until use.

Pathogen inoculum was produced by growing each isolate on sand corn-meal medium composed of 1000 g of air-dried clean sharp sand, 200 g of yellow corn meal, and 300 ml of sterile distilled water [50]. Mycelial plugs (6 mm in diameter), removed from 7-day-old cultures, were suspended into 500 ml Erlenmeyer flasks (15 plugs per flask) containing an autoclaved mixture of 200 g of sand corn-meal medium. The inoculated medium was incubated at 30 °C in the dark for 20 days and shaken daily during the first 7 days to ensure uniform pathogen growth.

Isolation and growth conditions of compost-associated actinomycetes

Composts C₃ (derived from 70% cattle and 25% chicken manures mixed with 5% green waste; Chemical characteristics: pH: 7, EC: 5.22 ms/cm, OM: 43.4%, N: 1.43%, P: 0.05%, K: 1.29%, Ca: 2.76%, Na: 0.45%) and C₄ (issued from 70% cattle, 25% sheep manures and 5% olive-mill solid waste mixture; Chemical characteristics: pH: 7.1, EC: 3.6ms/cm, OM: 42.8%, N:1.82%, P: 0.06%, K: 1.2%, Ca: 3.39%, Na: 0.44%), selected in a previous study based on their interesting antifungal activity against A. rolfsii and their high plant growth-promoting ability [48], were used for actinomycetes isolation. A collection of 17 actinomycetes isolates, exhibiting various macro-morphological traits, was recovered on Actinomycetes Isolation Agar medium cultures and grown on Yeast Malt Agar (ISP-2). Eleven isolates were previously screened for their seedling growth-promoting and biocontrol potentialities against A. rolfsii at the nursery stage [51].

Cell suspensions and cell-free culture filtrates preparation

All actinomycetes isolates were separately transferred to 50 ml of ISP-2 liquid medium poured into 250 ml Erlenmeyer flasks. The actinomycetes cultures were incubated at 28 ± 2^°C for 3 days under continuous shaking at 200 rpm [52]. The optical density (OD) from obtained cell suspensions was determined using a spectrophotometer (JENWAY 7205^®) and converted to colonies count (CFU/ml) by a calibration curve. A curve was prepared by counting living cells number (CFU/ml) using the serial dilution method in function of OD of cell growth at 600 nm.

Actinomycetes suspensions were further centrifuged thrice at 10.000 rpm for 10 min. The cell-free culture filtrates were collected following supernatant filtration through a membrane with 0.22 μm pore size [53]. ISP-2 liquid filtrate was used as control treatment.

Hypersensitivity test

Hypersensitivity test was carried out on tobacco seedlings (Nicotiana tabacum). Cell suspension (~ 10⁸ cells/ml, 10 µl) was injected to apparently healthy leaves using a microsyringe. Negative control leaves were injected with an equal volume of sterile distilled water. Tobacco seedlings (uninoculated and inoculated) were maintained for 24 h under ambient room conditions (~ 26 °C). The formation of necrotic and/or chlorotic zones on the inoculated leaf areas indicated positive response and isolate used for inoculation was considered as phytopathogenic and excluded from further biocontrol investigations [54].

Screening for plant growth-promoting ability

Cell suspensions and cell-free culture filtrates of a set of 11 actinomycete isolates were assessed for their ability to promote tomato cv. Rio Grande growth. Treatments were carried out by soaking seedling (at two-true-leaf stage) roots for 30 min into cell suspensions (~ 10⁸ CFU/ml) or their cell-free filtrates before being transferred to plastic pots containing commercialized peat (12.5 cm × 14.5 cm). Supplementary treatments were performed by substrate drench with 25 ml of cell suspension or 10 ml of culture filtrate at the three time points i.e. immediately, at 15 and 30 days post-planting (DPP). Control seedlings were similarly treated using sterile distilled water only. All seedlings (treated and untreated) were maintained under the similar greenhouse conditions detailed above and watered regularly with tap water. Ten seedlings were used per individual treatment. Seedlings were arranged according to a completely randomized design and the whole experiment was repeated at least twice.

At 45 DPP, tomato plants were uprooted and gently washed under running water to remove the adhering peat before recording the different growth parameters (plant height, stem diameter, aerial part and root dry weights).

Screening for stem rot suppression ability

The ability of cell suspensions and cell-free culture filtrates to suppress A. rolfsii-induced disease was evaluated on tomato seedlings grown under the same conditions as indicated above. The consortium of the three A. rolfsii isolates, prepared as described above, was mixed thoroughly with additional clean sharp sand at 5% (w/w), and spread onto the top 2-cm depth of the growing substrate [49]. Tomato seedlings were carefully removed from the alveolated trays, soaked (roots only) for 30 min into cell suspensions (~ 10⁸ CFU/ml) or their cell-free filtrates, and further transplanted into plastic pots (12.5 × 14.5 cm) previously filled with commercialized peat. They were substrate-drenched with 25 ml of cell suspensions or 10 ml of cell-free filtrates at three time points as the abovementioned test. Uninoculated and inoculated controls were watered with the same volume of sterile distilled water, and watered later regularly with tap water. The bioassay was carried out using ten seedlings per individual treatment.

At 45 DPP, growth parameters (plant height, stem diameter, aerial part dry weight, and root dry weight) were measured. Disease severity was scored based on 1–5 rating scale where 1 = no stem lesion, 2 = lesions girdled ≤ 25% of the stem circumference, 3 = lesions girdled 26–50% of the stem circumference, 4 = lesions girdled > 51% of the stem circumference, and 5 = stem completely girdled [51]. The whole experiment was repeated at least twice.

Screening for the antifungal activity of the selected isolates of actinomycetes

Test of cell suspensions effects

Actinomycete isolates were evaluated for their antifungal potential toward A. rolfsii (Sr₁) using the dual culture technique [51]. Two agar plugs (6 mm in diameter), cut from 7-day-old actinomycete colonies growing on ISP-2 agar medium, were placed on opposite sides of Petri plates (90 mm in diameter), equidistantly 3 cm away from the centre, on PDA medium amended with streptomycin sulphate (300 mg/l w/v). After incubation at 28 ± 2 °C for 5 days, a fungal plug (6 mm in diameter) was deposed in the centre of the plate. PDA plates without actinomycete plugs served as control. All plates were incubated in the dark at 28 ± 2 °C until pathogen mycelium reached the edge of the plate in control cultures. Ten replicate plates were used per each treatment. Percentage growth inhibition of A. rolfsii radial growth was calculated according to the following formula:

Growth inhibition (%) = [(Dc– Dt) / Dc] x 100.

where Dc is the diameter of pathogen colony in control plates and Dt is the diameter of pathogen colony in treated plates.

Test of cell-free culture filtrates effects

The cell-free culture filtrates of each actinomycete isolate were aseptically injected at the concentration of 20% (v/v) into Petri plates containing molten PDA medium amended with streptomycin sulphate (300 mg/lw/v). This concentration was preliminarily chosen as being effective against A. rolfsii mycelial growth compared to other concentrations tested (Table S1). After medium solidification, a mycelial plug (6 mm in diameter), removed from the edge of 7-day-old A. rolfsii cultures, was deposited in the centre of each treated Petri plate. Ten replicate plates were used for each individual treatment. Fungal cultures were incubated in the dark at 30 °C until the pathogen covered the whole control plates [53]. The colony diameters were measured and the inhibition rate of pathogen growth was noted as described above. All the experiment was repeated twice.

Plant growth-promoting (PGP) features of the selected isolates of actinomycetes

Indole 3-acetic acid (IAA) production

IAA production ability was tested using the colorimetric method after growing each isolate in yeast malt broth medium amended with 0.2% L-tryptophan at 28 °C for 2 days under rotary shaking at 125 rpm [55]. After centrifugation at 11,000 rpm for 15 min, 2 ml of Salkowski reagent were added to 1 ml of culture supernatant. Treatments were performed in triplicate. Absorbance was read at 530 nm. IAA concentration was determined based on a standard curve performed from IAA dilution series at 100 µg/ml (w/v) in ethanol.

Phosphate and zinc solubilization abilities

Phosphate and zinc solubilization abilities were determined according to Anusree and Bhai [55] protocol onto Pikovskya agar and mineral salt agar medium added with 0.1% of insoluble zinc oxide, respectively. The test was performed in triplicate. After 15 days of incubation at 28 °C, the clearing zone formed around colonies was noted.

Nitrogen fixation

Nitrogen fixation was qualitatively evaluated according to Gayathri et al. [56] method. The positive nitrogen fixation ability expressed by the capacity of each isolate to grow onto nitrogen (N)-free agar medium such as Jensen’s agar medium after 15 days of incubation at 28 °C. The test was performed in triplicate.

Antifungal potential features of the selected isolates of actinomycetes

Production of siderophores

Production of siderophores was determined on Chromium AzurolS (CAS) agar medium according to Anusree and Bhai [55] protocol. The test was performed in triplicate. After 10 days of incubation at 28 °C, the clearing zone formed around colonies showing color change from dark blue to yellow-orange was noted.

Production of hydrogen cyanide (HCN)

Production of HCN was determined qualitatively onto Yeast Malt Agar (YMA) medium amended with glycin (4.4 g / l) where any change in color from yellow to light-reddish brown indicated a positive HCN production after 10 days of incubation at 28 °C [57]. Treatments were performed in triplicate.

Production of hydrolytic enzymes

Isolates of actinomyctes were qualitatively checked for their ability to produce proteases, chitinases, amylases, pectinases, and lipases when grown onto skim-milk (3% v/v), chitin- (0.5% w/v), starch- (0.5% w/v), pectin- (0.5% w/v) and Tween 80- (1% v/v) agar media, respectively [58]. Treatments were performed in triplicate.

Molecular identification of the most active isolates of actinomycetes

Molecular identification was undertaken after extraction of the genomic DNA of the selected actinomycetes isolates according to Errakhiet al. [59] protocol. Primers used to amplify the 16S rDNA gene were 1F (5’-TCACGGAGAGTTTGATCCTG-3’) and 1530R (5’-AAGGAGGTGWTCCARCC-3’) [60]. Amplification was performed by an automatic Thermocycler (GeneAmp PCR system 2700, Applied Biosystems) programmed as follows: denaturation at 94 °C for 4 min for one cycle, followed by 30 cycles at 94 °C for 1 min, annealing at 53 °C for 1 min, extension at 72 °C for 1 min and final extension at 72 °C for 10 min. A negative control without DNA was included in each PCR run. The amplified products were visualized in a 1% agarose gel, stained with Ethidium bromide. The PCR products were purified and sequenced in the Centre of Biotechnology of Sfax (Laboratory of Biopesticides).

The homology of the 16 S rDNA sequences of eight selected actinomycetes isolates was performed, using BLAST-N program from GenBank database (http://ncbi.nlm.gov/BLAST/).

Statistical analysis

Data were analyzed using the SPSS (Statistical Package for the Social Sciences) software for Windows version 20.0. All in vitro and in vivo experiments were repeated at least twice. As no significant interactions were noted between tested treatments and experiments, one representative trial of each experiment was reported in the current study. Data were arranged in a completely randomized design. Means were compared using Duncan’s Multiple Range tests at P ≤ 0.05. Correlations between stem rot severity and tomato growth parameters were determined based on the bivariate Pearson’s test at P ≤ 0.05.

Results

Stem rot suppression potential of compost-associated actinomycetes

Variance analysis revealed a significant (at P ≤ 0.05) decrease in stem rot severity, noted on 45 DPP, depending on actinomycetes treatments whether applied as whole cell suspensions or cell-free culture filtrates.

Tomato plants treated with A5-3, A2-4, A3-4, A4-4, and A5-4 isolates showed 37.5 to 56.2% decrease in disease severity as compared to pathogen-inoculated and untreated control, with A5-3 being the most effective isolate. Interestingly, plants treated with A5-3, A3-4, and A4-4 isolates were comparable to the untreated and uninoculated controls (Fig. 1a).

Fig. 1.

Effects of compost-associated actinomycetes isolates (a) and their cell-free culture filtrates (b) on stem rot severity noted on tomato cv. Rio Grande plants 45 days post-planting as compared to controls. NIC: Uninoculated control; IC: Inoculated with A. rolfsii. Results are presented as mean ± SE (n = 10, P ≤ 0.05). Disease severity rating scale: 1 = no stem lesion, 2 = lesions girdled ≤ 25% of the stem circumference, 3 = lesions girdled 26–50% of the stem circumference, 4 = lesions girdled > 51% of the stem circumference, and 5 = stem completely girdled. Bars sharing the same letter are not significantly different according to Duncan’s Multiple Range test (at P ≤ 0.05)

All actinomycetes cell-free filtrates had suppressed disease severity by 25 to 56.2% relative to pathogen-inoculated and untreated control with those from A5-3, A3-4, A4-4, and A5-4 isolates being the most effective ones. Furthermore, plants already inoculated with pathogen and treated with these four filtrates showed similar health status as the untreated and uninoculated control ones (Fig. 1b).

Antifungal activities of actinomycete cell suspensions and cell-free culture filtrates

The diameter of A. rolfsii colonies varied significantly (at P ≤ 0.05) depending on actinomycetes isolates and their cell-free filtrates tested at 20% (v/v). The pathogen mycelial growth was reduced by 58.8–88% relative to control using their cell suspensions formulation (Table 1). A. rolfsii mycelial growth was lowered by 59-91.3% versus control using the eleven actinomycete cell-free filtrates.

Table 1.

Comparative antifungal activity of compost-associated actinomycetes cell suspensions and their cell-free culture filtrates toward Agroathelia rolfsii

Actinomycetes treatments	A. rolfsii colony diameter (mm)
	Cell suspensions	Cell-free culture filtrates
IC	85 a	83 ± 2.0 a
A3-3	33 ± 3.0 (61.1) bc	27.67 ± 0.73 (66.6) c
A4-3	29 ± 2.0 (65.8) bcd	22.6 ± 1.4 (72.6) bcd
A5-3	14.6 ± 1.4 (82.7) fg	8.5 ± 0.5 (89.7) fg
A8-3	26.3 ± 0.8 (69.0) cd	25.1 ± 1.6 (69.6) cd
A10-3	29.3 ± 2.3 (65.5) bcd	21.3 ± 2.4 (74.3) bcd
A1-4	35.1 ± 1.8 (58.6) b	34 ± 1.7 (59.0) b
A2-4	18.6 ± 0.4 (78.0) ef	18.1 ± 0.8 (78.1) ef
A3-4	14.6 ± 0.3 (82.7) fg	9.6 ± 1.20 (88.3) fg
A4-4	12.3 ± 1.4 (85.5) fg	8 ± 1.0 (90.3) fg
A5-4	10.1 ± 0.4 (88.0) g	7.1 ± 0.6 (91.3) g
A6-4	23.8 ± 3.3 (71.9) de	22.3 ± 1.4 (73.1) de

IC: Inoculated with A. rolfsii. Results are presented as mean ± SE (n = 10, P ≤ 0.05). For each column, values followed by the same letter are not significantly different according to Duncan’s Multiple Range test (at P ≤ 0.05). Values in parentheses indicate the decrease (in %) of pathogen mycelial growth as compared to the untreated control

Treatments with A5-3, A2-4, A3-4, A4-4, and A5-4 cell suspensions or their cell-free filtrates were found to be the most effective treatments by inducing 78–88% and 78.1–91.3% decrease in pathogen growth, respectively, compared to control (Table 1).

Growth-promoting potential of compost-associated actinomycetes on A. rolfsii-inoculated tomato plants

ANOVA analysis revealed a significant variation (at P ≤ 0.05) in all tomato growth parameters measured (plant height, stem diameter, and aerial part and root dry weights) at 45 DPP depending on treatments tested whether as cell suspensions or cell-free culture filtrates.

All treatments, except A1-4 and A6-4 cell suspensions, had improved plant height by 12.9–32.7% over A. rolfsii-inoculated and untreated control, and exhibited similar height as disease-free and untreated controls. Moreover, A3-3, A5-3, A2-4, A3-4, A4-4, and A5-4 cell suspensions led to increments, by 12.6–33.0% over the untreated and inoculated control, in the stem diameter. The aerial part dry weight was also increased, by 16.4–89.0% over control, following treatments tested. The root dry weight was enhanced on treated tomato plants except those challenged with A6-4 cell suspension (Fig. 2).

Fig. 2.

Growth-promoting potential of compost-associated actinomycetes isolates and their cell-free culture filtrates on tomato cv. Rio Grande plants inoculated with Agroathelia rolfsii noted 45 days post-planting as compared to controls. (a): Plant height; (b): Stem diameter; (c): Aerial part dry weight; (d): Root dry weight; NIC: Uninoculated control; IC: Inoculated with A. rolfsii. Results are presented as mean ± SE (n = 10, P ≤ 0.05). For each experiment, bars sharing the same letter are not significantly different according to Duncan’s Multiple Range test (at P ≤ 0.05)

As for the effects of their cell-free filtrates, the plant height increase, relative to the untreated and inoculated control, ranged between 18.2 and 36.7% depending on tested treatments. The stem diameter was improved by 20.4–54.4% over control using the majority of cell-free filtrates tested, except those from A4-3, A10-3, A1-4 and A6-4 isolates. Plant treatments with the filtrates tested, except those of A6-4, led to 33.2–91.9% increase in the aerial dry weight over control. Root dry weight was also improved by 55.2 to 81.1% over control on tomato plants already infected with A. rolfsii and treated with A3-3, A4-3, A5-3, A2-4, A3-4, A4-4, and A5-4 filtrates (Fig. 2).

Overall, treatments with A5-3 and A5-4 exhibited the highest growth-promoting effects when applied as cell suspensions or as cell-free culture filtrates on pathogen-inoculated plants.

Pearson’s correlation analysis revealed that the decrease in stem rot severity led to an increment in plant growth parameters. In fact, the plant height, the stem diameter, and the aerial part and root dry weights were negatively correlated to stem rot severity either when applied as cell suspension (r = -0.410, P = 0.001; r = -0.599, P ≤ 0.01; r = -0.451, P ≤ 0.01; r = -0.509, P ≤ 0.01) or as cell-free culture filtrates (r = -0.347, P = 0.005; r = -0.425, P ≤ 0.01; r = -0.569, P ≤ 0.01; r = -0.583, P ≤ 0.01), respectively.

Growth-promoting potential of compost-associated actinomycetes on pathogen-free tomato plants

Analysis of variance revealed a significant variation (at P ≤ 0.05) in all tomato growth parameters noted 45 DPP depending on actinomycetes treatments tested either as cell suspensions or as cell-free culture filtrates.

The plant height was improved, by 12.9 and 13.2% over the untreated and pathogen-free control was achieved, respectively, following treatments with A2-4 and A5-4 cell suspensions. In the same way, the stem diameter was increased, by 10.3% over control, was recorded following treatment with A5-4 cells. Moreover, all actinomycetes treatments tested had induced increments in the aerial part dry weight estimated at 26.6–79.0% over control with the highest growth-promoting effects (64–79%) recorded on plants treated with A3-3, A5-3, A2-4, A3-4, A4-4, and A5-4 cell suspensions. These treatments had also increased the root dry weight by 32.7–48% over control (Fig. 3).

Fig. 3.

Growth-promoting potential of compost-associated actinomycetes isolates and their cell-free culture filtrates recorded on tomato cv. Rio Grande pathogen-free plants 45 days post-planting. (a): Plant height; (b): Stem diameter; (c): Aerial part dry weight; (d): Root dry weight; NIC: Uninoculated control. Results are presented as mean ± SE (n = 10, P ≤ 0.05). For each experiment, bars sharing the same letter are not significantly different according to Duncan’s Multiple Range test (at P ≤ 0.05)

As for the effects of the cell-free filtrates, only tomato plants treated with A2-4 and A5-4 filtrates showed increase in their height, by 19.6 and 15.3% over control, respectively. All filtrates tested had enhanced the aerial part dry weight of tomato plants by 26.3–93.3% over pathogen-free control. The root dry weight was also increased by 62.5 to 79.8% over control, following treatments with A3-3, A5-3, A2-4, A3-4, A4-4, and A5-4 cell-free filtrates. The highest growth-promoting effects were recorded on plants treated with A5-3, A3-4 and A4-4 cell-free filtrates (Fig. 3).

Identification of the most active actinomycetes isolates

The isolates tested were checked for their hypersensitivity reaction and were found to be negative for this test. The most effective isolates in reducing stem rot severity and/or promoting tomato growth were subjected to molecular identification. Blast-N analysis of six (06) sequenced 16 S rDNA gene identity of sequences indicated that 4 isolates (namely A5-3, A3-4, A4-4 and A5-4) were affiliated to Streptomyces genus, one isolate (namely A2-4) belonged to Saccharomonospora genus and one isolate (A3-3) was identified as a member of Micromonospora genus.

The isolates A3-4 and A4-4 were affiliated with 100% identical similarity to S. griseorubens K5, Streptomyces sp. NAr5, S. labedae AJR1, S. griseoflavus TY175-08, S. griseorubens QH12-10, S. variabilisDJ1, S. euryhalinus UAE1, S. althioticus NMR2, S. griseoincarnatusEPE48, S. viridodiastaticus sh15, S. tunisiensis AI, S. coelicolor NIOT_ MBCTS80 and to the two type strains Streptomyces euryhalinus MS 3/20 and Streptomyces chartreusis NBRC 12,753 sequences. Both isolates A3-4 and A4-4 were identified as Streptomyces sp. The isolate A5-4 shared 100% identity to the type strain S. palmae CMU-AB204, S. youssoufiensis SNSAA03, S. lydicus HBERC-44,441, S. noursei Z1-04-02, S. albulus NJZJSA2, Streptomyces sp. JCM9888 and S. philanthi bv. triangulum 23Af2 sequences. This isolate A5-4 was identified as Streptomyces sp. The isolate A5-3 belonged to Streptomyces sp. with 100% of identity to S. thermogriseus BRM043931, S. thermovulgaris KSI, Streptomyces sp. BCC23167 and to the type strain S. thermogriseus NBRC 100,772 (Table 2).

Table 2.

Identification of the most active isolates of actinomycetes based on their 16 S rDNA sequences

Isolate	Accession number	The size of sequenced region (bp)	Most related species	Sequence identity (%)	Isolate identification
A2-4	OK043812	1145	Saccharomonospora sp. TH33 Saccharomonospora cyanea NA134 (type strain)	100 99.91	Saccharomonospora sp.
A3-4 and A4-4	OK043813 and OK043814	983 987	Streptomyces sp. NAr5 S. griseorubens K5 S. labedae AJR1 S. griseoflavus TY175-08 S. variabilis DJ1 S. euryhalinus UAE1 S. griseoincarnatus S. viridodiastaticus sh15 S. althioticus NMR2 S. tunisiensis AI S. coelicolor NIOT_ MBCTS80 S. euryhalinus MS 3/20 (type strain) S.chartreusis NBRC 12,753 (type strain)	100	Streptomyces sp.
A5-4	OK043815	391	S. palmae CMU-AB204 (type strain) S. youssoufiensis SNSAA03 S. lydicus HBERC-44,441 S. noursei Z1-04-02 S. albulus NJZJSA2 Streptomyces sp. JCM9888 S. philanthi bv. triangulum 23Af2	100	Streptomyces sp.
A3-3	OK043816	373	Micromonospora rosaria S4BS6 Micromonospora sp. YIM 68,233 M. rosaria DSM 803 (type strain)	100	Micromonospora sp.
A5-3	OK043817	1161	S. thermogriseus BRM043931 S. thermovulgaris KSI Streptomyces sp. BCC23167 S. thermogriseus NBRC 100,772 (type strain)	100	Streptomyces sp.

The isolate A2-4 was identified as Saccharomonospora sp. with 100 and 99.91% of identity to Saccharomonospora sp. TH33 and the type strain Saccharomonospora cyanea NA134, respectively. The isolate A3-3 belonged to Micromonospora sp. with 100% of identity to Micromonospora rosaria S4BS6, Micromonospora sp. YIM 68,233 and the type strain M. rosaria DSM 803 (Table 2).

Sequences of A2-4, A3-4, A4-4, A5-4, A3-3 and A5-3 were submitted to GenBank and attributed the following accession numbers OK043812 to OK043817, respectively.

Plant growth-promoting traits of the selected actinomycetes isolates

Production of IAA

The 11 selected isolates of actinomycetes were found able to produce the IAA after 48 h of incubation. The IAA production varied significantly from 3.03 to 41.8 µg/ml. The highest IAA production, of about 41.8 µg/ml, was recorded on A8-3 culture, followed by Streptomyces sp. A3-4 (22.8 µg/ml), then Streptomyces sp. A5-3 and A1-4 (14.9–15.6 µg/ml), and Saccharomonospora sp. A2-4 (14.1 µg/ml). The remaining isolates were found able to produce IAA at 12.2 to 3.03 µg/ml (Table 3).

Table 3.

Plant growth-promoting traits and antifungal activity features of the selected isolates of actinomycetes

Isolates of actinomycetes	A3-3	A4-3	A5-3	A8-3	A10-3	A1-4	A2-4	A3-4	A4-4	A5-4	A6-4
Plant growth-promoting features
Production of IAA (µg/ml)	2.1 ± 0.6 e	6.6 ± 2.4 cde	14.9 ± 3.3 bc	41.8 ± 4.7 a	3.6 ± 1.2 de	15.6 ± 2.4 bc	14.1 ± 2.3 bcd	22.8 ± 1.5 b	3 ± 0.8 de	9.1 ± 1.2 cde	12.2 ± 0.7 bcde
Solubilization of phosphate (mm)	0	0	8.8 ± 0.8 c	0	0	0	0	0	26.8 ± 3.3 a	17.5 ± 0.1 b	0
Solubilization of zinc (mm)	6.3 ± 0.1 c	7.3 ± 0.3 c	7 ± 0.2 c	0	0	9.8 ± 1.2 b	0	6.8 ± 0.3 c	23.8 ± 0.8 a	0	6.6 ± 0.3 c
Fixation of nitrogen	+	-	-	-	-	-	-	+	-	+	-

Antifungal activity features
Siderophores activity (mm)	0	0	0	0	0	0	47.5 ± 1.9 a	0	50.5 ± 3 a	0	0
Production of HCN	-	+	+	-	-	+	-	-	+	-	+
Enzymatic activity (mm)
Chitinase activity	8.2 ± 0.1 d	17 ± 0.2 c	34.1 ± 0.2 a	0	18 ± 0.2 c	9.3 ± 0.1 d	0	23 ± 0.1 b	22 ± 0.2 b	35.6 ± 0.3 a	8.5 ± 0.1 d
Proteolytic activity	33.6 ± 0.1 c	33.8 ± 0.1 c	42.8 ± 0.2 a	0	34.6 ± 0.2 c	0	0	38.6 ± 0.1 b	38.6 ± 0.2 b	43.6 ± 0.2 a	10.6 ± 0.2 d
Lipase activity	8.1 ± 0.1	14.5 ± 0.1	31.3 ± 0.1	0	18 ± 0.2	9.6 ± 0.1	0	20.8 ± 0.2	23.8 ± 0.2	32.5 ± 0.2	0
Amylase activity	7.8 ± 0.1 cd	14.8 ± 0.1 bc	28.6 ± 0.3 a	0	16 ± 0.1 bc	21 ± 0.7 ab	5.5 ± 0.1 d	20.6 ± 0.2 ab	19 ± 0.1 abc	21.3 ± 0.3 ab	8.3 ± 0.1 cd
Pectinolytic activity	7.1 ± 0.1 d	18.8 ± 0.2 bc	32.8 ± 0.2 a	6.3 ± 0.1 d	16.6 ± 0.2 c	6.1 ± 0.1 d	6.8 ± 0.1 d	20.6 ± 0.3 bc	24.5 ± 0.2 bc	25 ± 0.5 b	7.5 ± 0.1 d

A3-3: Micromonospora sp. A3-3; A5-3: Streptomyces sp. A5-3; A2-4: Saccharomonospora sp. A2-4; A3-4: Streptomyces sp. A3-4; A4-4: Streptomyces sp. A4-4; A5-4: Streptomyces sp. A5-4; A4-3, A8-3, A10-3, A1-4 and A6-4: Unidentified isolates of actinomycetes. IAA: Indole 3-acetic acid. HCN: Hydrogen cyanide. +: Presence of activity. -: Absence of activity. Results are presented as mean ± SE (n = 10, P ≤ 0.05). For each column, values followed by the same letter are not significantly different according to Duncan’s Multiple Range test (at P ≤ 0.05)

Solubilization of phosphate

Three (03) isolates of actinomycetes namely Streptomyces sp. A5-3, Streptomyces sp. A5-4 and Streptomyces sp. A4-4 were shown able to solubilize the phosphate as determined by the formation of a clear zone of about 8.8, 17.5 and 26.8 mm in diameter around their colonies, respectively. The highest phosphate solubilization ability was noted on Streptomyces sp. A4-4 cultures (Table 3).

Solubilization of zinc

Seven (07) isolates (namely A3-3, A4-3, A5-3, A1-4,A3-4, A4-4, and A6-4) were found able to solubilize the zinc as determined by the formation of clear zones, of 6.3 to 23.8 mm in diameter, around their colonies. The highest zinc solubilization activity was noted on Streptomyces sp. A4-4 cultures (Table 3).

Fixation of nitrogen

Three isolates namely Streptomyces sp. A3-4, Streptomyces sp. A5-4 and Micromonospora sp. A3-3 were found able to fix nitrogen as expressed by their capacity to grow on nitrogen free medium (Table 3).

Antifungal activity features of the selected actinomycetes isolates

Production of siderophores

Only Saccharomonospora sp. A2-4 and Streptomyces sp. A4-4 strains were shown able to produce siderophores as determined by the formation of clearing yellow-orange zone of about 47.5 and 50.5 mm in diameter around their colonies (Table 3).

Production of HCN

Two isolates belonging to Streptomyces sp. (namely A4-4 and A5-3) and three unidentified isolates of actinomycetes (A1-4, A6-4 and A4-3) were shown able to modify the filter paper color from yellow to light-reddish color thus indicating their positive HCN production potential when grown on yeast malt agar medium amended with glycin (Table 3).

Enzymatic activity

As for chitinase production, 9 isolates out of the eleven tested (except Saccharomonospora sp. A2-4 and the isolate A8-3 of actinomycete), displayed positive activity. The greatest activity was displayed by Streptomyces sp. A5-3 and Streptomyces sp. A5-4 cultures, where the clear zone formed around their colonies reached 34.1 and 35.6 mm in diameter (Table 3). Eight (8) isolates displayed proteolytic activity when grown on skim milk-agar medium. The highest production of proteases was noted on Streptomyces sp. A5-3 and Streptomyces sp. A5-4 cultures where the clear zones formed around their colonies were around 42.8 mm and 43.6 mm in diameter, respectively (Table 3). As for lipase activity, 8 isolates showed clear zones around their colonies when cultured on tween 80-agar medium and were found to be lipase-producing agents. The highest lipase production was noted on Streptomyces sp. A5-3 and Streptomyces sp. A5-4 cultures (Table 3). Concerning amylase production, all isolates except the actinomycete isolate A8-3 displayed positive activity with the highest activity recorded on Streptomyces sp. A5-3 culture where clear zone formed around its colony was around 28 mm in diameter (Table 3). All isolates tested displayed pectinolytic activity when grown on pectin-agar medium with the greatest clear zone (of about 32.8 mm in diameter) noted on Streptomyces sp. A5-3 cultures (Table 3).

Discussion

Soilborne pathogens associated to various economically important crops including vegetables, fruit trees, and many others cause significant losses around the world [61]. To overcome these soil microorganisms, chemical pesticides have been widely used, but good agricultural practices were recently more focused on alternative control methods including soil solarization [62], biofumigation [63], biocontrol [19, 64], and organic amendments such as composts [24].

The disease-suppressive effect displayed by composts is mainly related to the biological activity of their associated microbiota colonizing their organic matter [22–25, 65]. Among the widely compost-associated microbial agents, actinomycetes were the main driving forces for plant diseases suppression [29]. In a previous study, we demonstrated the ability of different composts to control A. rolfsii growth. However, the present investigation was more focused on the valorization of actinomycetes agents derived from the most disease-suppressive composts as potential growth-promoting and biocontrol agents.

In fact, eleven actinomycetes isolates were recovered from the most active composts and screened for their abilities to control stem rot disease and to promote growth of tomato seedlings in preliminary investigation. In the present study, the actinomycetes isolates, found to be the most bioactive in suppressing stem rot disease and/or in promoting tomato plant growth, were identified by 16 S rDNA sequencing as Micromonospora sp. A3-3 (OK043816), Streptomyces sp. A5-3 (OK043817), Saccharomonospora sp. A2-4 (OK043812), Streptomyces sp. A3-4 (OK043813), Streptomyces sp. A4-4 (OK043814), and Streptomyces sp. A5-4 (OK043815).

Stem rot disease severity was reduced using the cell-free cultures filtrates of all tested isolates with the highest disease-suppressive effect obtained using Streptomyces sp. (A5-3 A3-4, A4-4 and A5-4). The application of whole cells of Streptomyces and its partially purified crude metabolites reduced stem rot incidence by 64–67% and 22–49%, respectively [66]. These results confirmed other studies reporting the antagonistic potential of actinomycetes against stem rot caused by A. rolfsii, foot rot induced by Phytophthora capsici [55], apple fruit associated to Botryosphaeria dothidea, tomato Fusarium wilt induced by F. Oxysporum [67], and Verticillium wilt due to V. dahliae [68]. Several other reports indicated the use of actinobacteria for the biological control of various fungal and bacterial plant pathogens [26, 67]. The antagonistic effects of actinomycetes have been attributed mainly to their antifungal compounds [69]. In fact, the antifungal ability of actinomycetes suspensions and their cell-free culture filtrates was undoubtedly demonstrated on pathogen mycelial growth which decreased by 58.8–88% and 59-91.3% over control, respectively, with A5-3, A3-4, A4-4, A5-4 (Streptomyces sp.) and A2-4 (Saccharomonospora sp.) being the most active agents. Similar findings were described for various actinomycetes isolates able to inhibit (A) rolfsii radial growth via the formation an inhibition zone [55]. Liotti et al. [70] demonstrated the potential of some actinomycetes isolates to inhibit various pathogens including (B) dothidea, Colletotrichum gloeosporioides, (C) guaranicola, F. decemcellulare, and F. oxysporum.

In the current study, the ability of cell suspensions and cell-free culture filtrates from the tested actinomycetes isolates to promote tomato growth was investigated on A. rolfsii-inoculated plants. Isolates A5-3, A3-4, A4-4, A5-4 (Streptomyces sp.) and A2-4 (Saccharomonospora sp.) and their cell-free filtrates were found to be the the most effective in enhancing plant growth over the untreated and inoculated control. An important effect was also noted on tomato plants treated with Micromonospora sp. A3-3 isolate and its filtrate. Pearson’s correlation analysis showed that the reduced stem rot severity was related to the significant increment in all plant growth parameters. Goudjal et al. [71] also selected various endophytic actinomycetes for their plant growth promotion features and biocontrol activity against F. oxysporum f. sp. lycopersici. These findings are in accordance with other reports mentioning the ability of actinomycetes to improve plant growth [67, 72] and to control several plant pathogens [38, 73]. Different mechanisms and interactions involved in the plant disease suppression by actinomycetes acting either individually or synergistically are (i) pathogen inhibition using antimicrobial substances [33, 73], (ii) competition for colonization nutrients and sites supplied by roots [18], (iii) iron competition through production of siderophores [38, 74], (iv) induction of defence mechanisms [19, 75], (vi) degradation of pathogenicity factors of the pathogen such as toxins and extracellular cell wall-degrading enzymes [76], (v) inactivation of pathogen germination factors in root exudates [77].

The enhancement of plant growth by the tested actinomycetes cell suspensions and their cell-free culture filtrates were also reported in the present work when they were applied to pathogen-free tomato plants. Isolates A5-3, A3-4, A4-4, A5-4 (Streptomyces sp.), A2-4 (Saccharomonospora sp.), and Micromonospora sp. A3-3 and their cell-free culture filtrates showed their significant ability to improve growth parameters compared to the uninoculated and untreated control. In several reports, plant growth-promoting potentialities of actinomycetes have been also demonstrated [78, 79]. Plant growth-promoting actinobacteria are also able to decrease the soilborne disease severity and to benefit the plant growth [80]. They directly favour the plant development by providing nutrients (P, N, Fe) and synthesizing hormones (ethylene and indole-3-acetic acid) [79], as well as indirectly by protecting plants from biotic and abiotic stresses [81].

It should be mentioned that the genus Streptomyces was predominant, and the potent isolates selected in the current study belonged to this genus. Streptomyces spp. are known to be one of the main sources of bioactive natural products [18]. The genus accounts for about 80% of the production of natural substances by actinobacteria [82], and 60% of agricultural importance compounds are originated from Streptomyces species [83] which have been recorded to inhibit the growth of various fungal plant pathogens [66, 70]. Furthermore, different studies highlight the capability of Micromonospora sp. to improve plant growth via phosphate-solubilization and N-fixation [84, 85] with an antipathogenic effect through the production of cell-wall-degrading enzymes [86]. Nevertheless, Saccharomonospora has been considered as a rare genus in the phylum of actinobacteria and sparsely studied [87].

Actinobacteria display direct and indirect mechanisms for the improvement of plant growth. As for their direct mechanisms, they can enhance the availability of nutrients and produce phytohormones [88]. Nevertheless, through the indirect pathway, they have the capacity to produce hydrogen cyanide acids (HCN), siderophore, protease, and cellulase [22]. As for their effect on plant defence against plant pathogens, actinomycetes are able to produce antifungal compounds and antibiotics [69], hydrolytic enzymes such as β-1,3-glucanase [89] and chitinase [90]. In the current study, all the actinomycetes isolates were shown able to produce IAA with a highest production for A8-3 isolate, followed by Streptomyces sp. A3-4, then Streptomyces sp. A1-4 and Streptomyces sp. A5-3, and Saccharomonospora sp. A2-4. This phytohormone regulates different basic cellular processes including cell division, differentiation and elongation [29]. Therefore, several Streptomyces species, such as S. rimosus, S. viridis, S. olivaceoviridis, have the ability to produce IAA and to improve plant growth by increasing seed germination, root elongation, and root dry weight [84, 91]. Moreover, only Streptomyces sp. A5-3, Streptomyces sp. A5-4 and Streptomyces sp. A4-4 were phosphate producers with a marked phosphatase activity for Streptomyces sp. A4-4. They increase the phosphorus accessibility to plants through many processes like chelation, mineralization of organic phosphorus, and redox changes [92]. Some actinomycetes species such as S. griseus and M. aurantiaca are shown able to improve the growth of wheat plants due to increased N and P contents of the tissues as a result of their phosphate solubilizing activities [93]. Furthermore, Streptomyces sp. A3-4, Streptomyces sp. A5-4 and Micromonospora sp. A3-3 were shown able to fix nitrogen. Other actinomycetes species exhibit a nitrogen-fixing ability including Frankia, Agromyces, and Mycobacterium [39, 94]. Moreover, seven isolates (A3-3, A4-3, A5-3, A1-4, A3-4, A4-4, and A6-4) were found able to solubilize the zinc with the highest zinc solubilization activity displayed by Streptomyces sp. A4-4. Poovarasan et al. [95] reported the role of actinomycetes especially Streptomyces sp. in zinc solubilization and their association with arbuscular mycorrhizal fungi.

Some actinobacteria species are able to produce low molecular weight compounds named siderophores which are capable of sequestering Fe³ and other metals and influence their availability to plants [96]. In the current study, only Saccharomonospora sp. A2-4 and Streptomyces sp. A4-4 were found able to produce siderophores. Moreover, siderophores help for antagonistic activity by depriving the iron availability to the pathogens [96]. Siderophores such as peucechelin were produced by S. peucetius, desferrioxamine B by S. pilosus, and desferrioxamine E by S. coelicolor [97]. Streptomyces sp. A4-4 and A5-3 isolates and A1-4, A6-4 and A4-3 isolates showed positive HCN production; this compound is known to act through the inhibition of electron transport and thus energy supply disruption to the cells [81]. The HCN activity for plant disease control was also reported in Boukaew et al. [98] and Wang et al. [99] studies.

Actinomycetes are of huge importance since they possess a capacity to secrete a variety of extracellular hydrolytic enzymes [100]. Multiple enzymatic activities were recorded for Streptomyces sp. A5-3 and Streptomyces sp. A5-4 cultures synthesizing the highest levels of chitinase, protease and lipase. Furthermore, the highest amylolytic activity was noted on Streptomyces sp. A5-3 cultures. The extracellular enzyme activities are not only helpful in plant growth-promoting and organic matter decomposition, but they also play a crucial role in plant disease suppression by antagonizing soilborne phytopathogens and facilitating development of endophytic and intracellular colonization [101]. The hydrolytic enzymes such as chitinase, protease, amylase, lipase, and pectinase are capable to control plant diseases by degrading the fungal cell walls, cell membranes, and cell membrane proteins, and by decreasing the activity of extracellular virulence factors [102].

In conclusion, the screening for a set of actinomycetes isolates recovered from two composts exhibiting antifungal potential against A. rolfsii led to the selection of six promising biocontrol agents useful for stem rot control and for the improvement of tomato growth. Thus, composts, having different proportions of raw materials, were found to be a potentially crucial source for the isolation of potent actinobacteria. In the current investigation, we identified various species such as Streptomyces sp. A5-3, Saccharomonospora sp. A2-4, Streptomyces sp. A3-4, Streptomyces sp. A4-4, Streptomyces sp. A5-4 and Micromonospora sp. A3-3 as potential biocontrol agents and plant growth-promoting actinobacteria. Their ability to produce hydrolytic enzymes, HCN, siderophores and IAA, to solubilize phosphate and zinc and to fix the nitrogen were demonstrated. Therefore, these efficient actinomycetes isolates, acting both as biocontrol agents and biostimulants, may be potentially valorized in an integrated disease management program for stem rot in tomato.

Author contributions

All authors contributed to the study conception and design. FA conducted the experiment and wrote the manuscript. RABA contributed in the isolation of compost-associated actinobacteria isolates, analysis of the secondary metabolites and of the 16 S rDNA gene sequences and the revision of the manuscript. SBK identified the selected actinomycetes isolates. HJK contributed in data collection and data analysis. MDR supervised the experiments and approved the final manuscript. All authors read and approved the final manuscript.

Funding

The research leading to these results received funding from the Ministry of Higher Education and Scientific Research in Tunisia through the budget assigned to LR21AGR03-Production and Protection for a Sustainable Horticulture (2PHD), The Regional Research Centre on Horticulture and Organic Agriculture, University of Sousse, 4042, Chott-Mariem, Tunisia.

Declarations

Ethical responsibility

All authors confirm that the manuscript is original and has not been submitted or published elsewhere in any form or language (partially or in full). This work is an expansion of previous work published in Journal of Phytopathology and Pest Management (http://ppmj.net/index.php/ppmj/article/view/202).

Financial interests

The authors have no financial or proprietary interests in any material discussed in this article.

Competing interests

The authors have no competing interests to declare that are relevant to the content of this article.