The Lithothamnium sp. sample was obtained off the coast of Espírito Santo State, Brazil (20°19’10” S – 40°20’16” W). After micronization (mechanical breakage, caused by the friction between the particles to sizes between 1 to 10 µm), the sample was provided by Valeagro Comércio Importação & Exportação / NaturVita Bioagroindustria (Petrolina, State of Pernambuco, Brazil).
The cyanobacterium Arthrospira platensis strain was provided by the “Elizabeth Aidar” Microalgae Collection of the Fluminense Federal University (Niterói, State of Rio de Janeiro – Brazil). The Axenic autotrophic cultivation of A. platensis was carried out in a semi-continuous system in a photobioreactor (Reichert et al. 2006) using a culture medium (Zarrouk, 1966) at the Department of Crop Sciences of the Federal University of Paraná (Curitiba, Paraná State - Brazil). After 45-day cultivation, the biomass was separated from the culture medium by centrifugation, attaining 0.95 g L− 1 DW and was freeze-dried. The freeze-dried powder was added to spraying solutions, so allowing the cells disruption (Stirk et al. 2020).
The experiments were carried out in the Biofertilizers Lab. and Organic production Research Area of the Federal University of Paraná, located in the municipality of Pinhais, Paraná State, Brazil, at 25°23’30” S and 49°07’30” W, at an average altitude of 920 meters. The climate in the region is a Cfb-type temperate climate (Köppen).
For determination of the effect of foliar application of solutions containing Lithothamnium sp. micronized (Lm) separately and associated with Arthrospira platensis (Ap), in terms of growth, development, biochemical alterations, and yield, three experiments were carried out.
2.1 Experiment I
It was carried out in greenhouse using pots with a capacity of three liters, filled (in a proportion of 1:1) with commercial substrate based on composted pine bark (Tropstrato®) and organic compost (Provaso®), with: C = 30.3 g kg− 1; N = 30.3 g kg− 1; P = 8.5 g kg− 1; K = 6.6 g kg− 1; Ca = 8.1 g kg− 1; Mg = 4.1 g kg− 1.
At 60 days after sowing (DAS) in nursery beds, the seedlings were transplanted to the pots (experiment I) and simultaneously to the field (experiment II). The seedlings had four leaves, an average pseudostem diameter of approximately 4 mm, showing adequate development. The pots were arranged on benches, with four pots and two plants in each pot, for repetition.
A completely randomized design was used, with two onion (Alliun cepa) cultivars (Alvará ® and Perfecta F1 ®), each treatment with four replications (n = 4) under a double factorial arrangement (two cultivars x five treatments). A localized irrigation system with a drip tape was used, aiming to maintain the humidity at 80%, controlled with the aid of a tensiometer. The pots contained 2 cm of expanded vermiculite on the surface so that there was no contact of foliar sprayed solutions with the substrate.
Four concentrations of Lithothamnium sp. micronized (Lm) associated or not with Arthrospira platensis (Ap) were weekly applied, as well the control sprayed with distilled water. The applications (n = 8) of the treatments were carried out using a 10-liter Kawashima® electronic sprayer at constant pressure (40 psi). The volume of application varied as a function of plant growth, from 20 mL (first to fourth application) to 36 mL (fifth to eighth application) per pot.
The Lithothamnium sp. (micronized powder) sample was suspended in distilled water at a concentration of 1.5 g L− 1 (Amatussi et al. 2020) before being sprayed on leaves. The sample of Arthrospira platensis (freeze-dried powder) was suspended in distilled water at a concentration of 0.75 g L− 1 and 1.5 g L− 1 (Mógor et al. 2018) before being sprayed to the leaves of onion plants. Five treatments were established: Control, Lm (1.5 g L− 1 of Lm), LAp1 (1.5 g L− 1 of Lm + 0.75 g L− 1 of Ap), LAp2 (1.5 g L− 1 of Lm + 1.5 g L− 1 of Ap) and Ap (1.5 g L− 1 of Ap).
After eight applications with a weekly interval, at 90 days after transplanting (DAT) at the beginning of bulbification, leaf material was collected for biometric and biochemical analysis. Data on plant height (cm), number of leaves, pseudostem diameter (mm), fresh and dry mass (g) of the aerial part, and roots (g) were obtained. Mass was quantified on a precision scale. The dry mass was quantified after drying in a forced-air circulation oven at 65°C until reaching a constant value. The biochemical analyzes are described below.
2.2 Experiment II
To observe the influence of Lithothamnium sp. (Lm) associated or not with A. platensis (Ap) on the yield of onion bulbs, experiment II was implemented in the field under organic system in August 2018. A completely randomized design was used with two onion cultivars (Alvará® and Perfecta®) where each treatment had four replications (n = 4). The treatments, application frequency, and seedling preparation were the same as described in experiment I.
The chemical analysis of the soil in 0–20 cm layer showed the following mean values: pH (CaCl2) = 5.84; pH H2O = 6.71; Al+3 = 0; H + Al+ 3= 2.93 cmolc dm− 3; Ca2+= 5.28 cmolc dm− 3; Mg2+= 3.05 cmolc dm− 3; K+= 1.32 cmolc dm− 3; P (Mehlich) = 49.0 mg dm; S = 33.49 mg dm− 3; C = 26 g.dm− 3; %; V% = 76.7 and CEC = 12.58 cmolc dm− 3.
The treatments were distributed in beds previously fertilized and prepared seven days before seedling transplanting. Soil preparation was carried out with the incorporation of eight tons per hectare of organic compost with the following mean values: C = 30.3 g kg− 1; N = 30.3 g kg− 1; P = 8.5 g kg− 1; K = 6.6 g kg− 1; Ca = 8.1 g kg− 1; Mg = 4.1 g kg− 1.
The spacing adopted in the experiment was 10 cm between plants and 30 cm between rows, each plot with 40 plants. There were 10 foliar applications of the treatments, with an application volume corresponding to 400 L ha− 1.
At harvest, at 135 days after transplanting (DAT), the bulbs were classified according to their diameter (I < 35 mm; II = 35–50 mm; III = 50–70 mm, and IV = 70–90 mm) and their masses were quantified. Yield was extrapolated as a function of the spacing adopted in the experiment (230,000 plants per hectare).
2.3 Experiment III
To evaluate the application frequency (weekly and fortnightly) experiment III was implemented in the field, in August 2019. This experiment used the same design, the same population of plants, the same number of replications (n = 4), and the same cultivars used in experiment II.
Applications were performed at two frequencies (weekly and fortnightly), resulting in a triple factorial arrangement (two cultivars x two frequencies x three treatments). The two treatments of Lm and Ap associated (LAp1 = 1.5 g L− 1 of Lm + 0.75 g L− 1 of Ap and LAp2 = 1.5 g L− 1 of Lm + 1.5 g L− 1 of Ap), in addition to the control, with water application were applied.
Plant material was collected at the beginning of bulbing, at 120 DAT. Plant height (cm), number of leaves, leaf area (cm2), pseudostem volume (cm3), fresh and dry mass (g) of leaves, pseudostem, and bulbs were evaluated. Leaf area and pseudostem volume were obtained using WinRhizo Pro® software (Regent Instr®Canada). At 135 DAT, the final collection was performed to evaluate yield, as described in experiment II.
2.4 Biochemical analyses
Subsequent to the collection of biometric (Experiment I) and yield (Experiment II and III) data, leaves and commercial bulbs (type III and type IV) were collected, macerated in liquid nitrogen and stored (-20° C) for biochemical analyses. Leaf collections were performed between 9 and 10 am.
Analysis of chlorophyll-a (Ch-a), chlorophyll-b (Ch-b) and carotenoids (C), extraction of pigments in the leaves was performed according to Lichtenthaler (1987). The absorbance readings were taken at 663, 647 and 470 nm. The formulas described by Lichtenthaler and Buschmann (2001), were applied, the values were expressed as µg of chlorophyll per g of fresh plant material.
Total sugars were quantified according to Maldonade et al. (2013), preceded by acid hydrolysis of the sample using 3,5-dinitro salicylic acid (DNS). The standard curve for reducing and total sugars was made with glucose (5.5 mM). The values were expressed as µg of sugars per g fresh plant material.
For the analysis of total free amino acids, the amino acids were extracted according to Winters et al. (2002) and the colorimetric reaction with absorbance reading done at 570 nm, according to Magné and Larher (1992). The standard curve was made with glutamine (2 mM). The values were expressed in µg g fresh plant material.
The analysis of phenolic compounds was by the Prussian Blue method (Price and Butler 1977). The absorbance reading was performed at 700 nm, the standard curve performed with gallic acid (0.01 M) in methanol. The values were expressed in µg g fresh plant material.
Nitrate Reductase enzyme activity of leaves was done according to (Jaworski 1971). Reading was performed in a spectrophotometer at 540 nm and values expressed as µ mol of NO2 h− 1 g− 1 plant material.
All data were tested for homogeneity and then submitted to analysis of variance, when significant (p < 0.5) the data were submitted to Tukey's test. Assistat® 7.7 Beta software (Silva and Azevedo 2016) was used.