Background
Phytopathogens such as fungi and bacteria can cause serious diseases to humans and lead to large losses in agricultural crops production [
1‐
3]. Recent studies show that natural plant products are safer alternatives to antibiotics and commercial pesticides that are commonly used to control these pests [
4,
5]. Enhancing the plant’s natural photochemistry might therefore serve as a useful technique to increase the production of natural defensive compounds and safely control diseases. In this respect, seaweed extracts (SWE) which are obtained from algal species growing along coastal regions around the world, might be used as natural plant biostimulants [
6]. These extracts are usually associated with enhanced plant growth following foliar or drench application [
7,
8]. However, horticultural crops exhibit a range of diverse responses following different application methods and doses of SWE [
7,
9]. Few reports on vegetable crops indicate that SWE treatment may affect the crop nutritional composition by means of increasing phenolics, flavonoids, and antioxidant capacity [
10,
11]. Antioxidant activities are commonly associated with phenolic and flavonoid content in medicinal shrubs [
12] and in plants in general [
13,
14]. Furthermore, little attention has been given to ornamental medicinal plants [
15,
16].
In addition, the antifungal and antibacterial activities of SWEs are widely demonstrated and these activities are mostly linked to the presence of thermo-stable lipophilic compounds [
17‐
20]. For example, some SWEs have bactericidal effects [
18] associated with the presence of terpenes and phenolic compounds [
17,
19]. Other reports indicate that seaweeds might stimulate the production of plant defense elicitors (e.g. oligosaccharides) against pathogens [
21]. SWE foliar spray may also stimulate microbes antagonistic to
Pythium ultimum [
22]. Further, SWE foliar sprays on turfgrasses may enhance resistance to dollar spot [
6,
17]. Many investigations also indicate that plant extracts have useful value in controlling microorganisms [
4,
5] which may allow for a reduction in the application of fungicides and bactericides, and consequently minimize their potential side effects on human health and the environment. Nevertheless, how to use SWE as a biostimulant of natural phytochemistry production to enhance the medicinal values of plants is a poorly investigated question. To our knowledge, the effects of SWE on plant extract activities against microorganisms had not been investigated in vitro.
Solanaceae is a large plant family that contains many horticultural crops that are of economic and medicinal importance such as the
Petunia and the recently delimited genus
Calibrachoa [
23‐
25].
Calibrachoa cultivars are widely grown by seed in North and South America from hybrid cultivars [
26]. Some
Calibrachoa cultivars vary in their content of phenolic, flavonoid and tannin compounds and their leaf extracts may exhibit antifungal and antibacterial activities against a wide spectrum of pathogenic microorganisms [
27], further, close genera to
Calibrachoa such as
Petunia had shown obvious flavonoid composition [
28] and antimicrobial activities [
29], however little is known about Solanaceae members such as
Calibrachoa responses to SWE from the point of chemical composition.
In the current study, we investigate the effects of SWE of Ascophyllum nodosum on enhancing Calibrachoa plants medicinal value by way of increasing the phenolic, flavonoid and tannin contents as well as their antioxidant capacity. Further, we examined the use of SWE technologies to enhance antifungal and antibacterial activities of plants against a selected spectrum of microorganisms. This research may represent an important methodology towards enhancing the quantity and quality of natural products obtained from promising sources of natural products such as Calibrachoa; it could also lead to reducing the use of antibiotics and commercial reagents in agricultural crop disease and pest control programs, thereby minimizing their impact on human health and our environment.
Methods
Chemicals and cell cultures
Analytical/HPLC grade chemicals were obtained from Sigma Aldrich, Canada. Fungi and bacteria were obtained from Henry farms Laboratories, Guelph, Canada.
Plant material and SWE treatments
Uniform 2 weeks old plants of Calibrachoa x hybrida of ʻSuperbells® Dreamsicleʼ (CHSD) and ʻSuperbells® Frost Fireʼ (CHSF) were obtained from local nurseries, identified by Dr. Hosam Elansary, University of Guelph, and vouchered at the Biodiversity Institute of Ontario, University of Guelph, Guelph, Canada. The plants were subjected to a weekly treatment of the commercially available liquid extract of the marine plant Ascophyllum nodosum (Stella MarisTM, Acadian Seaplants, Canada) at 5 and 7 ml/L per plant as a soil drench or foliar spray. Untreated plants were considered as controls. SWE treatments continued for 8 weeks before sampling plant leaves. Ascophyllum nodosum was harvested from the intertidal zone along the North Atlantic coastline of Canada and then hydrolyzed under atmospheric pressure using KOH. The liquid extract was evaporated under vacuum to a concentration of 29 % solids.
Plant material, growing conditions and morphological parameters
The plants were transplanted into a simulated green roof pot system in commercial 4-in. pots measuring 13 cm in height and equipped with a filter (fabricated non-woven geotextile of polypropylene) and 4 cm drainage layers of gravel (0.5 ml). Pots were arranged during April 2015 in a controlled greenhouse environment located in Guelph, Ontario, Canada (43° 30′ 18.24″ N 80° 22′ 15.86″ W). The substrate used for the green roof system was black peat and perlite (3:1 w/w) and was supplemented with Osmocote Plus® (14:13:13 N, P, K + microelements) (2 g/l media). The experiment was conducted under the following conditions: Temperatures ranged between 19.6 °C and 27.4 °C; relative humidity ranged between 55 and 62 %; photosynthetically active radiation was maintained at 1000 μmol/m2/s at 10.00 am, and plants were maintained at 12 h light conditions for 1 week before starting treatments. A daily watering (25–40 ml) was applied to allow for 100 % evapotranspiration rate (ET). ET was determined for 5 plants per cultivar by watering with enough water and leaving them to dry for 1 h, then weighing each representative, reweighing every 24 h, and the daily changes in weight represent the daily ET. The volumetric water content was calculated for 5 pots before and during the experiment by determining the weight before and after irrigation by allowing free draining for 1 h. The difference between the fresh weight and oven-dry weight (at 70 °C until constant weight at the end of the experiment) was calculated to provide the volumetric water content. Plants were grouped into three blocks/repetitions (n = 3) containing 4 replicates per treatment in the experiment and totaling 120 plants per cultivar distributed on three plots. After 8 weeks of treatments, the plants were harvested and plant height and leaf number were calculated. A WinDIAS Leaf Area Measurement System (Delta-T Devices Ltd., Cambridge, UK) was used to calculate the leaf area. Total dry weight was determined by drying cleaned plants in an oven at 70 °C to reach constant weight.
Leaf samples of
Calibrachoa cultivars were collected in June 2015 and leaves’ extracts were obtained using methanol as per Pérez-Tortosa et al. [
30] with some modifications [
31]. Dried leaves (0.25 g) were ground then dissolved in 3 ml methanol (99 %). The solution was shaken on a magnetic agitator at minimal speed under darkness (to maintain the activity of bioactive compounds), for 24 h at room temperature. The solution was centrifuged for 5 min (4°C) at 10000 RPM (7000 ×
g) and the supernatant (~2.6 ml) was stored in sealed vials at −20 °C. The preparation was evaporated in a rotary evaporator to produce a semisolid extract that was kept for further analyses.
Antioxidant capacity
The 2,2′-diphenypicrylhydrazyl (DPPH) method was employed to determine the free radical scavenging activity of the samples [
32]. The absorbance was measured at 517 nm and experiments were repeated twice in triplicate. The
β-carotene-linoleic acid assay was conducted using the method described by Tepe et al. [
32] with modifications [
27]. The mixture of
β-carotene-linoleic acid was prepared by dissolving 0.5 mg
β-carotene in 1 ml of chloroform, 25 μl linoleic acid and 200 mg Tween 40. Chloroform was removed by vacuum evaporation then 100 ml of distilled water saturated with oxygen were added and shaken vigorously. 2.5 mL of the mixture was mixed with 350 μl of the liquid extracts, incubated for 48 h at room temperature and the absorbance was measured at 470 nm. The BHT was used as positive control and a blank was prepared in the same manner and the antioxidant capacities of each sample were compared to the BHT and the blank. Antioxidant activity was expressed as the concentration of the sample required to inhibit 50 % of DPPH or β-carotene-linoleic acid (IC50).
Calibrachoa total phenolic, tannin and major flavonols contents quantification
The Folin-Ciocalteau colorimetric method using gallic acid as the reference was employed to determine the phenolic contents of leaf extracts with results expressed as gallic acid equivalents (mg GAE/g ext.) [
33,
34]. Flavonoids were determined in plant leaves by extracting 100 mg ground leaf tissues in 2.1 ml methanol:acetic acid:water (70:4:29, v/v) for 72 h at 4 °C, then the supernatant was removed and the pellet was re-extractred in 2 ml methanol:acetic acid:water (90:1:9, v/v) for 24 h at 4 °C. The combined supernatants were dried (under vacuum) and completed to 0.5 ml with a mixture of methanol:acetic acid:water (80:2:17 v/v) [
35]. The samples were subjected to HPLC analysis of flavonoids. The HPLC analysis was performed using liquid chromatographic system equipped with a Waters Alliance 2695 separations module (Waters, Milford, MA, USA). A LiChrosphera RP-18 reversed-phase column (119 mm 4 mm) supplied by Merck (Quebec, Canada) was employed. A gradient solvent system of A [HOAc:CH3CN:H3PO4:H2O (20:24:1.5:54.5)] and B [1.5 % H3PO4] was used starting with 80 % A, decreasing to 30 % A at 30 min, 15 % A at 34 min and 0 % A at 40 min. The flavonoids were determined as quercetin-3-O-rutinoside equivalents by integrating areas and the absorbance was monitored at 352 nm. Tannins were determined following the gravimetric method [
36] with modifications [
37]. All experiments were repeated twice in triplicate.
Chemical composition of SWE
Inductively Coupled Plasma Spectroscopic Analysis (ICPSA) was used for SWE to determine the mineral compositions in Optima 4300DV (Perkin-Elmer, USA). The nitrogen content (N) was determined using AOAC method No. 990.03 in the LECO FP-528 analyzer. Phosphorous pentoxide (P2O5) was determined using the ammonium citrate in AOAC method No. 960.08 by ICP-OES. Potassium oxide (K2O) was determined using the ammonium oxalate in AOAC method No. 960.08 by ICP-OES. Heavy metals quantification followed the AOAC method No. 6020A using the Atomic Absorption -Hydride Generation. Experiments were repeated twice in triplicates.
Antifungal activities
Four fungi were used:
Aspergillus flavus (ATCC (American type culture collection) 9643),
Candida albicans (ATCC 26555),
Penicillium funiculosum (ATCC 56755), and
Penicillium ochrochloron (ATCC 48663). Cultures were kept on malt agar at 4 °C then sub-cultured monthly. Spore suspension concentration of (1.0 × 10
5) was maintained and the minimum inhibitory (MIC) and minimum fungicidal (MFC) concentration assays were performed using the microdilution method [
38]. Leaf extracts were diluted in 5 % DMSO (1 mg/ml and 10 mg/ml), then added (2 μl) to microplates containing broth Malt medium with inoculum and incubated for 72 h at 28 °C on a rotary shaker. MIC was determined by serial sub-cultivations of 2 μl of leaf extract and isolated compounds dissolved in a medium. The sub-cultivations were incubated for 72 h in microtiter plates that contain 100 μl broth and inoculum, then incubated for 72 h at 28 °C. MIC was defined as the lowest concentration that inhibits fungi growth at the bionocular microscope level. To determine the MFC a serial dilutions of the inoculum was prepared in 96-well microtiter plates and the MFC was defined as the lowest concentration with no visible growth indicating 99.5 % killing of the original inoculum. Triplicate samples were used in all experiments and each experiment was repeated twice. The fungicides fluconazole (FLZ) and ketoconazole (KLZ) were used as positive controls (1–3500 μg/ml).
Antibacterial activities
Experiments were performed using Gram + Bacillus
cereus (clinical isolate),
Staphylococcus aureus (ATCC 6538) and
Micrococcus flavus (ATCC 10240) and Gram-bacteria
Enterobacter cloacae (ATCC 35030). The microdilution method [
38] was used to determine the minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations. Bacterial suspensions were adjusted with sterile saline to a concentration of 1.0 × 10
5 CFU/ml and stored at 4 °C. Inocula were screened for contamination by culturing on a solid medium. Leaf extract were added (1 and 10 mg/ml) to 100 μl Triptic Soy broth (TSB) with a bacteria inoculum (1.0 × 10
4 CFU/well), reaching the desired concentration in a microtitre plate to measure the MICs and MBCs. The mixtures in microplates were incubated for 24 h at 37 °C in a rotary shaker. After incubation of the microplates, the lowest concentration that completely inhibited bacterial growth (at the binocular microscope) was defined as the MIC. To determine the MBC, a serial sub-cultivations of 2 μL into microtitre plates containing 100 μL of TSB for each well and incubated for 24 h. The MBC was defined as the lowest concentration indicating killing of 99.5 % of the original inoculum. To determine the optical density a microplate manager was used at 655 nm and experiments were in triplicates and repeated three times. The results was compared to positive controls (streptomycin and ampicillin, 1 mg/ml), and negative control (5 % DMSO).
Statistical analysis
The results were expressed as means ± standard deviation (SD). Further, the data was subjected to the Least significant differences (LSD) one-way analysis of variance (ANOVA) implemented in SPSS (PASW Ver. 21) at a level of significance of P ≤ 0.05.
Discussion
The improved growth in
Calibrachoa plants following SWE treatments concord previous results on other plants [
8,
9,
11]. In both cultivars, increases found in phenolic and flavonols contents following SWE treatments were associated with concurrent increases in antioxidant activity. These results are in agreement with previous studies on vegetable crops [
8,
10,
11]. The chemical analyses of SWE showed some important major and minor nutrients available in SWE for the treated plants that might enhance their growth and secondary metabolite compositions. Several reports indicated that organic fertilizers [
39], NPK mineral fertilizers [
40], higher supplementation of potassium [
41] enhance overall plant growth, the phenolic and flavonoid composition as well as the antioxidant activities. The increased phenolic and flavonoid content associated with increased antioxidant activities following SWE treatment of
Sargassum johnstonii Setchell & Gardner [
8] and
Ascophyllum nodosum [
10,
11]. In this study, SWE significantly enhanced plant leaf total phenolic, flavonols and tannin content that consequently increased the antioxidant activity of leaf extracts. Further, increases in the phenolic content of the leaves of the cuttings of
Pelargonium were observed following SWE treatment [
42]. However, one report found that the phenolic and flavonoid content might not increase following SWE treatment [
43]. It is well documented that phenolic compounds are the main secondary metabolites in plants that are considered as the major antioxidant resource in horticultural crops [
44]. In addition, the flavonoids as polyphenolic compounds are secondary metabolites that exhibit strong antioxidant activities [
45‐
47]. The increased antioxidant activities associated with increased tannins in leaf extracts is in agreement with previous reports that highlighted the role of tannins in enhancing the overall antioxidant values of plant extracts [
48,
49]. The drench applications of SWE enhanced the secondary metabolite composition of both
Calibrachoa cultivars compared to foliar application and control plants. This finding is in agreement with previous reports showing that foliar and drench applications of SWE may result in diverse effects on crops [
7‐
9]. Positive differences were found in this study in almost all parameters among plants treated with different doses of SWE. Mattner et al. [
50] found that soil drench application of SWE enhanced the vegetative growth of broccoli; also, it was found that SWE soil drench doses increased the leaf area in one orange cultivar but had no effect on dry weight and stem dry weight compared to foliar applications [
7].
We found that the increase in the phenolic, flavonols and tannin content was associated with enhanced antifungal and antibacterial activities. The enhanced antifungal activities against
A. flavus,
C albicans,
P. funiculosum and
P. ochrochloron were observed following treatment with extracts of plants sprayed or soil drenched with SWE; this may be attributed to the increased contents of phenols [
51,
52]. Yazdani et al. [
53] reported that certain phenolic compounds isolated from the methanolic extracts of
Piper betle L. (Piperaceae) could inhibit the growth of
A. flavus. Phenolics isolated from the root bark of
Lycium chinense Miller (Solanaceae) [
54], the leaves of
Baseonema acuminatum (Apocynaceae) [
55], and the leaves of
Hyssopus officinalis [
56] have been associated with the antifungal and antioxidant activities. The bioactivity of phenolic compounds might be attributed to the interferece with proteins and forming non-covalent bonds leading to conformation changes and protein inactivation in microbes [
57]. Hussin et al. [
58] reported that
Barringtonia racemosa L. (Lecythidaceae) leaf extracts have strong antifungal activities against
Aspergillus sp. and
Penicillium sp. as well as other fungi which they explained by the presence of four different flavonoids and two phenolic acids. In the current study, increased specific flavonols associated with increased antifungal activities which may agree with previous reports that specific phenolic compounds were responsible for antifungal activity [
59]. Some reports even indicate that tannins might be responsible for the antifungal activity of plant extract such as that reported from the fruit peels of
Punica granatum which mainly contains tannins and was efficient against
Aspergillus niger and
Penicillium citrinum [
60]. The tannins in the current study significantly increased in SWE treated plants, which support previous investigations.
Observed antibacterial activities following SWE treatments might be attributed to increases in phenolic compounds which are commonly reported in antibacterial plant extracts [
61,
62]. A wide range of studies have provided support for this. For example, Stanković et al. [
63] found that phenolic and flavonoids compounds found in leaf extracts of
Teucrium sp. have strong antibacterial activities against
Staphyllococcus aureus,
Pseudomonas aeroginosa and
E. coli, with
S. aureus being the most sensitive. Similarly, Nitiema et al. [
64] found that specific phenolics such as coumarins have antibacterial activities against a wide spectrum of organisms such as
Enterobacter aerogens. Also Vaquero et al. [
65] reported that the antimicrobial property of different wines depends on the presence of pure phenolic compounds and polyphenols, and that clarified wines were inactive against all bacteria. What’s more, Edziri et al. [
59] found that two flavonoids were responsible for antibacterial activity against
Pseudomonas aeruginosa and
Escherichia coli (7.81–15.62 μg/ml). Furtheremore, Dahham et al. [
60] reported that pomegranate fruit peelings, which mainly contain tannins, have strong antibacterial activity against
S. aureus and moderate antibacterial activity against
Bacillus cereus. Finally, Saravanakumar et al. [
66] reported that
Thespesia populnea flower extracts showed strong antibacterial activity against wide spectrum of species including
Micrococcus flavus due to the presence of flavonoids and phenols in the extracts.
In the present study, SWE as biostimulant that contain important nutrient composition might boost the vegetative growth and secondary metabolite composition of
Calibrachoa plants that might enhance their respective bioactivity against microorganisms. Diverse responses were found among fungi and bacteria. The most sensitive fungus was
C. albicans and the most sensitive bacterium was
E. cloacae while the most resistant fungus was
P. ochrochloron and the most resistant bacterium was
S. aureus. CHSD showed higher antioxidant activities than CHSF due to higher phenols, flavonols and tannins content. The antioxidant activities found in this study matches those found in
Petunia [
67,
68] and also are in agreement with recent studies as response to SWE [
69,
70]. The cultivars CHSD and CHSF showed enhanced antibacterial and antifungal activities following SWE treatments, and this implies that SWE treatments might be used to enhance the medicinal values of these plants and their use as potential alternatives for antibiotics and commercial reagents to protect human health and the environment.
Acknowledgments
The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at king Saud University for its funding this Research Group NO. (RG 1435-011).