Cyanobacterium strain and preparation of the extract
The freeze-dried biomass of A. minutissima (BEA 0300B), originally isolated from a biofilm sample collected on the Fuerteventura coast, Canary Islands, was provided by the Banco Español de Algas, University of Las Palmas, Spain. The strain was cultivated in photobioreactors and the biomass was dehydrated as previously described (Righini et al. 2021a). This strain does not produce cyanotoxins, as previously assessed (Roberti et al. 2015).
For the preparation of the A. minutissima extract (AME), the biomass was suspended in sterile distilled water at 20 mg mL− 1 concentration, through an autoclave-assisted method (100°C, 1 bar, 20 min), at the University of Bologna. The obtained extract was centrifuged twice for 20 min at 5000 rpm with a Beckman Coulter Allegra (21R Centrifuge, Inc., Krefeld, Germany). The supernatant (extract) was collected under a sterile flow cabinet and frozen until use.
Chemical and spectroscopic analyses of the aqueous extract of Anabaena minutissima
Total C and nitrogen contents of AME were determined by a CHN elemental analyzer (CHN Elemental Analyser 1110, Thermo Scientific GmbH, Dreieich, DE) on 10 mL samples after freeze-drying, in duplicates. The analyses were performed in triplicate. The total solid content of AME was measured gravimetrically. Moisture and ash were analyzed with the TG-DTA92B thermogravimetric instrument (SETARAM, France). A quantity of 6 mg of lyophilized extract was placed in an alumina crucible and heated in a temperature range of 25°C to 750°C at a rate of 10°C min− 1. The oven atmosphere consisted of ultra-zero grade air at a flow rate of 130 mL min− 1. The analysis was performed in triplicate.
The total carbohydrate content was determined by the anthrone method which is suitable for carbohydrate determination in the presence of proteins (Fagen et al. 1954). The assay was performed at a microscale (Laurentin & Edwards, 2003) and modified as follows: anthrone reagent was prepared daily by dissolving anthrone (ACS reagent 97%, N° 319899, Sigma-Aldrich) 2 mg mL− 1 in ice-chilled 98% H2SO4 (84727, Sigma-Aldrich). The assay was performed in a rigid 96-well Polypropylene PCR Microplate (Corning® Thermowell® 96-well Product Number 6551, Corning, NY, USA). Samples and standards (50 µL/well) were cooled by maintaining the plate in a 0°C water bath (containing approx. 30% ice by volume), ice cold anthrone reagent (200 µL) was then carefully added and mixed. As standards several dilutions (0; 0.1; 0.2; 0.3; 0.4; 1.0 mg/mL) of pure glucose were used. The plate was covered with adhesive aluminum foil for microplates (VWR 60941-112, VWR International, Radnor, Pennsylvania, USA), and then it was incubated for 10 min at 95°C in a PCR thermal cycler (T3 DNA thermal-cycler, Biometra GmbH, Gottingen, Germany). Finally, 200 µL aliquots of each sample were transferred to a 96-well flat-bottom polystyrene microplate (Costar 3595, Corning, NY, USA) and the absorbance at 620 nm was measured by a spectrophotometer (Infinite 200 PRO series, Tecan, Männedorf, Switzerland). Please note that the adhesive aluminum cover was also heated at 95°C in the thermal cycler to avoid acid vapor condensation. Visible alterations of the aluminum cover were not observed probably thanks to the protection offered by the adhesive layer on the acid-exposed face. The analysis was performed in triplicates on multiple samples (n = 4).
Total soluble reducing sugars were quantified via the 3,5-dinitrosalicylic acid (DNS) method (Miller 1959) adapted for 96-well microplates (Cianchetta et al. 2010). Pure glucose dilutions were included as standards. The assay was performed in citrate buffer 50 mM, pH 4.8, for 5 min at 95 ◦C. The microplates were analyzed using a spectrophotometer (Infinite 200 PRO series, Tecan, Männedorf, Switzerland) at a 660 nm wavelength. The analysis was performed in triplicates on multiple samples (n = 4).
Glucose in the aqueous extract was quantified by an enzymatic colorimetric assay (D-Glucose HK Assay Kit, Megazyme, Astori Tecnica, Poncarale, Italy) according to the producer’s instructions. The analysis was performed in triplicates on multiple samples (n = 4).
Phycobiliproteins were determined according to Righini et al. (Righini et al. 2023) by resuspending the freeze-dried aqueous extract in phosphate buffer (0.2 M, pH 7) and then stirring the suspension at room temperature in the dark. After 4 h, the suspension was centrifuged for 20 min at 13°C, 5000 rpm, and the phycocyanin, allophycocyanin, and phycoerythrin in the supernatant were quantified at 652, 615, and 562 nm spectrophotometrically (Bennett and Bogorad 1973; Bryant 1982) by using the following equations:
$$Phycocyanin \left(PC\right)\left(\frac{mg}{g}\right)=\left[{A}_{615}-\left(0.474\times {A}_{652}\right)\right]/5.34$$
$$Allophycocyanin \left(APC\right)\left(\frac{mg}{g}\right)=\left[{A}_{652}-\left(0.208\times {A}_{615}\right)\right]/5.09$$
$$Phycoerythrin \left(\frac{mg}{g}\right)=\left[{A}_{562}-\left(2.41\times PC\right)-(0.849\times APC)\right]/9.62$$
Values expressed as %TS of total carbohydrates, glucose, reducing sugars, phycoerythrin, phycocyanin, and allophycocyanin were calculated by dividing the measured concentration (in mg L− 1) by the concentration of total solids in the aqueous extract (in mg L− 1). Errors were calculated by adding errors in quadrature assuming errors in the measurements are governed by the normal distribution and that the measured quantities are independent from each other.
Lipids were extracted from 70–75 mg of freeze-dried extract in duplicates with hexane. Briefly, 40 mL of solvent g− 1 of dry material were utilized, the mixture was vortexed for 3 min with glass beads then the liquid fraction was recovered after centrifugation, for a total of three extractions on the same pellet. The recovered liquid fractions were pooled and warmed at 75°C in a water bath to eliminate most of the solvent and finally dried to a constant weight under a vacuum (centrifugal evaporator Jouan RC10-10, Thermo Electron Industries SAS, Château-Gontier, France). Finally, the lipid fraction was determined gravimetrically.
FT-IR spectra of lyophilized AME were recorded by using a Tensor FT-IR spectrometer (Bruker Optics, Ettlingen, Germany) equipped with an accessory for analysis in micro-ATR (Specac Quest ATR, Specac Ltd., Orpington, Kent, UK). The spectra were acquired (64 scans per sample or background) in the range of 4000–400 cm− 1 at a resolution of 4 cm− 1 and processed using the Grams/386 spectroscopic software (version 6.00, Galactic Industries Corporation, Salem, NH, USA). The analysis was performed three times.
Tomato seeds and treatment
Tomato (L. esculentum L.) seeds, ‘Marmande’ cultivar, were purchased from L’ortolano s.r.l. (Cesena, Italy). Before treatment, the seeds were sterilized in 70% ethanol for 2 min, then 2.5% sodium hypochlorite for 2 min, rinsed thrice in sterilized distilled water, and blotted on sterile filter paper.
For the seed treatment, AME was opportunely diluted to obtain 2.5, 5, and 10 mg mL− 1 doses, into which the sterilized seeds were immersed at room temperature in the dark for 12 h. Sterile distilled water was used as a control. Then the seeds were collected and rinsed in sterile distilled water to eliminate any extract residual and left to dry on filter paper under a laminar air flow hood for 10 min before use.
Pathogens and substrate inoculation
The strain of P. ultimum 22 (PU) was made available by CREA-Research Centre for Agriculture and Environment, Bologna, Italy, while the strain of R. solani DAFS3001 (RS) was made available by the Department of Agricultural and Food Sciences, Alma Mater Studiorum, University of Bologna, Italy.
Both pathogens were maintained as mycelium on potato dextrose agar 3.9% (PDA, Difco) slant at 4°C and transferred on PDA plates at room temperature for short-term use.
For the greenhouse assay, a growth substrate (7/3 weight/weight, sterile peat/sand mix) was inoculated with the single pathogens (PU or RS) as follows: mycelium disks from actively growing colonies were transferred on PDA plates and incubated at room temperature for 5 days, then the entire plate content (colony + PDA) was homogenized in sterile distilled water with a kitchen blender and mixed with the substrate (2% weight/weight, pathogen/substrate). After inoculation, the substrate was covered with a black plastic film and incubated at 24–26°C for 2 days. Not inoculated substrate (control) was prepared in the same way.
Direct activity of the extract toward the pathogens
The poisoned substrate technique was used to test the effect of the extract on the in vitro growth of the pathogens. PDA was amended with dilutions of AME to obtain the following final concentrations: 0, 1, 2.5, and 5 mg mL− 1, then 10 mL poisoned PDA was poured into Petri plates (9 cm diam.) which were inoculated with one mycelial disk from PU or RS actively growing colonies, then incubated at 22°C for 42 h, in triplicates. The averaged radial growth of the colonies was calculated based on three measurements per plate.
Seed treatment effect on the plantlet growth in microcosm and chemical changes
Tomato seeds were treated with dilutions of AME at 0, 2.5, 5, and 10 mg mL− 1 doses as described above and seeded in 400 mL glass jars containing 200 g perlite and 100 mL of diluted Hoagland’s solution, prepared according to (Maurer et al. 2021) (3 replicates × 2 jars × 6–10 seeds each). After 2 weeks of incubation in a growth chamber (Percival® AR-36LC8, Percival Scientific, Inc. Perry, IA, USA), at 24°C and 16h/8h light/darkness photoperiod, the plantlets were collected, and the fresh weight of the entire plantlets and rootlets was recorded (3 replicates), after exudate collection (see below).
The lyophilized root samples were ground with a ball mill before FT-IR analysis. The section on chemical and Spectroscopic Analyses describes the procedure. The spectra of the roots from seeds non-treated and treated with AME at 5 mL− 1 were shown. The region between 1800 and 1500 cm− 1 corresponding to the main functional groups was studied in more detail using curve-fitting analysis by Grams/386 spectroscopic software (version 6.00, Galactic Industries Corporation, Salem, NH, USA). The best-fit parameters were determined using the Gaussian model. This was achieved by minimizing the reduced Chi-square (χ2), resulting in coefficients of determination (R2) ranging from 0.999 to 0.988 and standard error (SE) ranging from 0.0005 to 0.001. The identified peaks were integrated to calculate the peak area.
Effect of root exudates from treated plantlets on pathogen growth
The root exudates were collected from the plantlets of the experiment described above. About 20 two-week-old plantlets per each tested AME dose (0, 2.5, 5, and 10 mg mL− 1) were collected and pooled. The root apparatus was washed in distilled water then immersed in 12 mL sterilized distilled water into 20 mL glass vials and incubated in the growth chamber at the above-described conditions for 20 h. After plantlet removal, the exudate solution was sterile filtered, normalized per seedling or root weight, obtaining 10 mL exudates per dose, then stored at -20°C until analysis. To assess the effect of the exudates on the mycelial growth of the pathogens, 96-well flat-bottom polystyrene microplates (Costar®3595, Corning, NY, USA) were prepared, each well containing 200 µL normalized root exudate, 15 µL potato dextrose broth, and 15 µL of diluted inoculum obtained from 24 h (PU) or 7 d (RS) old, colonized PDA plates, after milling and syringe extrusion, with 10 replicates. Microplates were incubated for 16 h at 24°C, then the mycelial growth was followed through spectrophotometric analysis (200 PRO series, Tecan, Kawasaki, Japan) at a 660 nm wavelength from 16 until 80 h after pathogen inoculation. Blanks without inoculum were included.
Greenhouse assay
The experiment was conducted at the fully air-conditioned and automated glass greenhouse complex of DISTAL, University of Bologna. Plastic pots (13.5 × 11.5 × 7.5 cm) containing the artificially inoculated substrate, as already described above, were seeded with a total of 50 seeds per pot, with 3 pots per treatment (0, 2.5, 5, and 10 mg mL− 1 AME doses). Pots with non-inoculated substrate served as healthy controls. Pots were then disposed over a bench according to a completely randomized design, at the following greenhouse conditions: 24–26°C (day), 20–22°C (night), with a 12 h/12 h photoperiod, and 70% relative humidity.
The percentage of standing plants was recorded 21 days after the sowing. Then, all plants were gently removed from the substrate, their height was measured, and the root apparatus was washed under tap water to record disease symptoms. Disease incidence was calculated as the percentage of plants showing disease symptoms over the total of examined plants.
For PU, the disease severity was evaluated on the whole plant using a visual disease assessment based on a 0–4 scale (Jabiri et al. 2021) with modifications, as follows: 0 = no visible disease symptoms; 1 = ≤ 20% moderate level of general decay; 2 = extensive general decay, and with an obvious reduction in overall plant development (but < 50% of root system missing); 3 = very severe levels of general decay associated with an extensive reduction of the root apparatus (> 50% root system missing); 4 = dead plant.
The disease severity caused by RS was evaluated by scoring the root apparatus based on a 0–5 scale (Righini et al. 2022b), where: 0 = absence of necrosis (0% of symptoms); 1 = very slight necrosis (up to 5% of root with symptoms); 2 = slight necrosis (6–20% of root with symptoms); 3 = moderate necrosis (20–50% of root with symptoms); 4 = severe necrosis (51–70% of root with symptoms); 5 = severe crown and root necrosis (> 70% of root with symptoms
The experiment was repeated three times (n = 3).
Biochemical assays
To verify the possible involvement of induced resistance mechanisms in the plantlets due to seed treatment with AME, some enzymatic activities considered markers were determined on 14-day-old seedlings. For this purpose, seeds were treated with AME at the dose of 5 mg mL− 1 in sterile water and were seeded on sterile filter paper in sterile polystyrene Petri plates (diam. 14 cm), 60 seeds per plate, with 3 replicates, including non-treated control. Plates were incubated in a growth chamber (Percival®AR-36LC8, Percival scientific, Inc. Perry, IA, USA) at 24°C, 16 h light, and the experiment was repeated thrice (n = 3). The dose of 5 mg mL− 1 was chosen because no significant differences among treatments were observed in the greenhouse experiment.
To determine enzymatic activities, total proteins were extracted from 0.5 g fresh roots randomly collected from each plate of each experiment. The samples were immediately snap-frozen in liquid nitrogen and then ground to a fine powder using a pre-chilled mortar and pestle, and total proteins were extracted by 20 mM sodium acetate buffer pH 5.2 (1 mL g− 1 of fresh weight) containing 1% polyvinylpolypyrrolidone (Sigma–Aldrich Co.) (Roberti et al. 2015). After incubation at 4°C for 90 min under continuous gentle stirring, the samples were centrifuged twice at 12,000 rpm for 20 min at 4°C and the supernatant was harvested and filtered using a GV Millex® Syringe Filter Unit (Millipore Corporation, USA) to remove solid particles. Protein concentrations were determined by the protein-dye binding method of (Bradford 1976), using bovine serum albumin (BioRad Laboratories, Inc.) as the standard.
The activity of three chitinases, β-N-acetylhexosaminidase (EC 3.2.1.52), chitin 1,4-β-chitobiosidase, and endochitinase (EC 3.2.1.14), was assayed in triplicate for each experiment (n = 3) following a modified procedure by (Tronsmo and Harman 1993) in 96-well microplates. Chitinase assays were based on colorimetric determination of p-nitrophenyl cleaved from the chitin-analogous substrates, p-nitrophenyl-β-D-N, N’, N’’-triacetylchitotriose, p-nitrophenyl-N-acetyl-β-D-glucosaminide and p-nitrophenyl-β-D-N, N’-diacetylchitobiose, respectively (all from Sigma–Aldrich Co). Fifty microliters of each substrate in 50 mM acetate buffer, pH 5.0 (2 mg mL− 1) were added to 90 µL of the protein extract (15 µg total protein) from each sample. After a 15-minute incubation in a water bath at 50°C, 50 µl of 0.2 M Na2Ca3 were added to stop the reaction, and the absorbance was measured at 405 nm. Each chitinase activity was calculated using the p-nitrophenyl absorption coefficient of 18.5 mM− 1 cm− 1.
The activity of β-1,3-glucanase (EC 3.2.1.39) was evaluated in triplicate for each experiment by measuring the production of reducing sugars, using laminarin (Sigma, USA) as the substrate and following a modified protocol of (Abeles and Forrence 1970). The reaction mixture consisted of 60 µL of the protein extract (15 µg proteins) and 60 µL laminarin (2%). After incubation for 2 h at 37°C, 70 µL l of 3.5 dinitrosalicylic acid were added and the reaction mixture was heated at 100°C for 20 min. After cooling in an ice bath, the absorbance was measured at 492 nm. Glucose was used as a standard. Enzyme activities were expressed as U (µmol min− 1) g− 1 protein.
In addition, the epicotyl and hypocotyl content of total phenols, the epicotyl content of chlorophyll a and b, and carotenoids were determined as follows: freeze-dried seedlings (for each determination, 5 seedlings per plate, in triplicates, for n = 3 experiment) were first ground with 0.1 mL of absolute methanol per mg of root and incubated for 30 min on ice. The Folin method (López Arnaldos et al. 2001) was used for phenol measurement. The samples were centrifuged for 30 minutes at 4°C and 12,000 rpm, and 50 µL of supernatant was added with 1 mL of Na2CO3 (2%) and 75 µL of Folin Ciocalteau reagent (Sigma Aldrich, Merck). After 15 min of incubation in the dark at 25°C, the absorbance was measured at λ = 725 nm. Gallic acid (Sigma Aldrich, Merck, Darmstadt, Germany) was used as the standard curve (Meenakshi et al. 2009). The total phenolic content was expressed as µg gallic acid equivalents per g of fresh roots.
The determination of chlorophylls was carried out in fresh epicotyls (FE). Twenty-five mg of samples were crushed with MeOH (100%, 1 mL), vortex mixed 3 times for 1 min each time and then incubated overnight in the dark at 20°C. The absorbance at 665, 652, and 470 nm was measured in supernatants after centrifugation at 12000 rpm for 30 min. The chlorophyll content was calculated using the following equations (Wellburn 1994; Lichtenthaler and Buschmann 2001):
$$Clorophyll a \left(\text{C}\text{l}\text{a}\right)\left(\frac{{\mu }\text{g}}{\text{m}\text{g}} FE\right) =16.72{\times A}_{665}-9.16\times {A}_{652}$$
$$Clorophyll b \left(\text{C}\text{l}\text{b}\right)\left(\frac{{\mu }\text{g}}{\text{m}\text{g}} FE\right) =34.09{\times A}_{652}-15.28\times {A}_{665}$$
$$Carotenoids\left(\frac{{\mu }\text{g}}{\text{m}\text{g}} FE\right) =\left(1000{\times A}_{470}-1.63\times Cla-104.9\times Clb\right)/221$$
Before performing the biochemical assays, the percentage of germinated seeds per plate was recorded, and the root length, seedling fresh, and dry weight (70°C for 3 days) were determined on 10 seedlings per plate, for each experiment.
Statistical analysis
All experiments were arranged according to a completely randomized design. All data were analyzed by ANOVA, after checking the homogeneity of variance, and, if the p-value was less than 0.05, the means were separated by Tukey’s test (p < 0.05). Percentage data were arcsin transformed before analysis. All analyses were performed with GraphPad Prism software, San Diego, CA, USA, version 5.01.