Single and combined effects of Bacillus spp. and brown seaweed (Sargassum vulgare) extracts as biostimulants of eggplant (Solanum melongena L.) growth

Bacillus subtilis SV41, B. amyloliquefaciens subsp. plantarum SV65 and Sargassum vulgare extracts were evaluated for their plant growth­promot­ ing potential on eggplant (Solanum melongena L.) plants. Bio­treatments applied singly and/or in combination were further compared to a compost tea and to a commercial bio­fertilizer (AcadianTM). Results clearly showed that the combined treatments based on the two Bacillus spp. strains and the aqueous algal extract and the last one mixed with B. amyloliquefaciens subsp. plan‐ tarum SV65 induced the highest enhancements in the plant height and the maximum root length which were estimated at 32.4­33.9%, 23.9­25.5% and 23.4­25% and at 36.8­41%, 32.9­37.4% and 36.3­40.5% compared to water, compost tea and AcadianTM based treatments, respectively. Furthermore, the combined treatment based on the aqueous algal extract and B. amyloliquefa‐ ciens subsp. plantarum SV65 had significantly improved eggplant growth where the recorded increments in the stem diameter, the aerial part fresh weight, and the root fresh weight varied from 17.5 to 24.6%, 38.4 to 46.1%, and 32.3 to 50% as compared to the three controls, respectively. As for single treatments test­ ed, the aqueous extract had induced a significant improvement in the major growth parameters measured. Developed bio­stimulant was found to be more effective than compost tea and commercial bio­fertilizer based treatments.


Introduction
The eggplant (Solanum melongena L.) contributes to the diversification of market gardening products and constitutes a new product requested by foreign markets. In Tunisia, the exported quantities over the past five years were estimated at 187 tons. The export rate remains low compared to 56 thousand tons recorded in 2013/2014 agricultural campaign concentrated in tomato, watermelon, potato, and salad crops. To meet the requirements of consumers and increase the competitiveness of our exports at the interna tional markets level, significant efforts have been made in terms of improving quality and productivity of this crop (GIL, 2020).
The increasing demand for eggplants has gone along with the rapid population growth (Maghfoer et al., 2014). Eggplants contain low calories and high nutrient potential (Sowinska and Krygier, 2013). According to Gandhi and Sundari (2012), eggplant is widely used for medicinal features to reduce blood cholesterol and to regulate hypertension. Thus, due to these benefits, the demand of eggplant and its production is expected to increase (Sowinska and Krygier, 2013).
Long term use of inorganic fertilizer has altered soil fertility leading to decreased efficiency of nutri ent absorption and productivity and adverse effects on environment and human health (Jagatheeswari, 2013;Waseem et al., 2013). Therefore, research efforts are concentrated on alternative nutrients to improve soil physical, chemical, and biological traits through the application of chimerical organic fertiliz ers (Maghfoer et al., 2014) and/or various organic soil amendments such as compost (D'Hose et al., 2012), plant extracts (Bijarniya, 2011), algae (Eyras et al., 2008), and microbial inoculants (Arora et al., 2020). Application of chemical fertilizers with inocu lants has been also explored (CarvajalMuñoz and CarmonaGarcia, 2012). Application of microbial inoc ulants has gained an increased interest in the last three decades (Babalola and Glick, 2012).
Microbial inoculants are the formulations of bene ficial living microorganisms that, when added to the soil, they can improve the availability of nutrients to host plant either directly or indirectly, thereby lead ing to improved plant growth (Gaind, 2011). Various microorganisms are explored for the production of microbial inoculants such as Azotobacter, Azospirillum, Bradyrhizobium, mycorrhizae, phospho rus solubilizing bacteria, and Rhizobium. These bio inoculants can colonize the soil and perform various biophysical and biochemical soil activities that facili tate the availability and the uptake of nutrients to plants (Alori et al., 2017). Microbial inoculants could be grouped into nitrogen fixers i.e. Rhizobium and Bradyrhizobium, phosphate solubilizers i.e.
Recent demands of organic farming enhanced the application of organic treatments such as seaweed extracts in agriculture. Seaweeds are aquatic plants belonging to the plant kingdom of Thallophyta (Arioli et al., 2015). At least 59 species of seaweeds can stimulate germination, growth, and yields of some horticultural plants (Sunarpi et al., 2010). Seaweed application in the agricultural field has numerous benefits such as stimulation of seed germination, promoting plant growth, improvement of water and nutrient uptake, enhancement of frost and saline resistance, biocontrol and resistance towards phy topathogenic agents, and remediation of pollutants of contaminated soil (Nabti et al., 2016). Fresh and dry seaweed or its derived products i.e extracts, com posts, and soil conditioners, have been long used in agriculture to enhance plant growth and productivity (Eyras et al., 2008). Seaweeds applied, singly or in combination with other macroalgae and/or bacteria, enhance crop productivity. Sridhar and Rengasamy (2010) successfully applied a brown marine alga S. wightii combined with a green seaweed Ulva lactuca to enhance peanut growth. Additionally, a mixture of two bacteria Azotobacter chrocoocum and Bacillus megaterium var. phosphaticum combined with sea weed extracts increased growth of bitter orange plants (Ismail et al., 2011).
In view of previous studies, aqueous and methanolic extracts from a brown macroalgae (S. vulgare) were assessed singly and in combination with two endophytic bacteria i.e B. subtlis SV14 and B. amyloliquefaciens subsp. plantarum SV65 for egg plant growth. Both Bacillus spp. used in this study showed growth and health biostimulating effects on tomato plants through their capacity to produce indole3acetic acid, organic acids siderophore and their ability to solubilize phosphate and to biocontrol Fusarium wilt disease in tomato , 2018. Furthermore, Ammar et al. (2017) demonstrated S. vulgare aqueous and methanolic extracts' ability to efficiently control Fusarium dry rot disease in potato. Phenolic acids and flavonoids are the major components in the methanolic extract of S. vulgare using HPLCDAD analysis (Ammar et al., 2017).
The main objective of this study was to evaluate the ability of two Bacillus spp. strains applied singly and/or combined with S. vulgare aqueous or methanolic extracts on eggplant growth and produc tivity.

Materials and Methods
Bacillus spp. Culture B. subtilis SV41 (Accession number KR818071) and B. amylolequifaciens subsp. plantarum SV65 (Accession number KR818073) isolated from two wild Solanaceous species Datura metel and Solanum nigrum, respectively, were used in this study. Their isolation protocol, characterization and identification analysis were mentionned in Aydi Ben  and (2018) studies. They were previously selected based on their growth biostimulating effect and ability to control tomato Fusarium wilt disease when tested in pot experiment or under field condi tions . The plant growthpromoting traits of both Bacillus strains are detailed in Table 1.
Stock cultures of both bacterial strains, were con served at 20°C in Nutrient Broth (NB) medium amended with 40% glycerol. Bacillus spp. colonies of a 1dayold culture on Nutrient Agar (NA) medium were transferred to LuriaBertani broth (LB) and incu bated at 28 ± 2°C for 48 h and under continuous shaking at 150 rpm. The bacterial strains were tested at the exponential stage of growth (data unpub lished). The concentration of Bacillus spp. was adjust ed at 10 8 cells/ml using spectrophotometre at DO 600 nm.

Preparation of aqueous and methanolic extracts from Sargassum vulgare
Brown seaweed was sampled during February 2014 from Monastir, Tunisia (N 35°46'47.754"; E 10°47'9.312"). The alga sampling and processing are detailed in a previous study (Ammar et al., 2017). Grounded samples were packed and stored at 4°C until use.
For aqueous extraction, 1 kg of powder sample of S. vulgare was soaked in 20 l of sterile distilled water (SDW) and boiled at 100 ± 2°C for 1 h. After cooling, extracts were filtered twice through Whatman N°1 sterile filter paper and further sterilized by filtration through sterile microfilter (0.22 μm pore size). The collected aqueous extracts, prepared at the concen tration 50 g/l, were stored at 4°C until further use within a week to avoid any chemical alteration (Ammar et al., 2017).
For methanolic extraction, samples of the brown seaweed (1 kg each) were subjected to a series of maceration in methanol (3 l) for three days under ambient room conditions. After filtration, the solvent was evaporated using a rotary evaporator under reduced pressure (at 60°C). One gram of the methanolic dry residue was separately dissolved into 10 ml of methanol. Methanolic extracts used at the concentration 1 g/l were stored at 4°C until further use (Ammar et al., 2017).

Eggplant seedling preparation
The cultivar Bonica, the most used by agricultures in the Tunisian CentreEast regions, was used in this study.
Eggplant cv. Bonica seeds were disinfected by immersion into 0.2% sodium hypochlorite for 3 min. They were washed several times with SDW. Disinfected seeds were subsequently treated with bacterial suspensions (~10 8 cells/ml) and/or aqueous and methanolic S. vulgare extracts using 20 μl per seed for 1 h. The same volume of SDW was used for treatment of control seeds.
Eggplant treated and untreated seeds were sown in alveolar plastic trays (7×7 cm) filled with sterilized peat ™ (Floragard VertriebsGmbH für gartenbau, Oldenburg). Seeds were further treated at trays with 5 ml of bacterial suspensions (~10 8 cells/ml) and/or aqueous and methanolic extracts from the brown seaweed. Control seeds were treated with the same volume of SDW. During all the growing period, trays were watered regularly to avoid drought stress and seedlings were kept under greenhouse conditions (2030°C with a 16 h light and 8 h dark cycle, and 60 IAA production z + + Phosphatase activity y + Organic acids x + + Siderophore production w + + 70% relative humidity) until reaching the twotrue leaf growth stage.

Screening of the effects of Bacillus spp. and Sargassum vulgare extracts on eggplant growth Effect of single bio-treatments
Each Bacillus spp. strain (B. subtilis SV41 or B. amyloliquefaciens subsp. plantarum SV65) was singly inoculated to eggplant seedlings by dipping roots for 30 min in a bacterial suspension (10 8 cells/ml) pre pared as described above . Control seedlings were dipped in SDW only and LB medium. Treated and control seedlings were transplanted into individual pots (12.5 cm × 14.5 cm) containing sterilized peat. Treated seedlings were re treated as substrate drenching with 50 ml of each bacterial cell suspension or with 50 ml of S. vulgare aqueous and methanolic extracts prepared as described above. Four weeks after transplanting, eggplant seedlings were retreated with 50 ml of each bacterial suspension and/or tested aqueous and methanolic extracts.
Seven replicates of one seedling each were used for each individual treatment and the whole experi ment was conducted twice. Treated and control seedlings were grown for 60 days under greenhouse conditions as described above (Botta et al., 2013). After 60 days of growth, the plant height, the stem diameter, the aerial part fresh and dry weights, the maximum root length, the root fresh and dry weights, the flower number, the fruit number, and the fruit fresh and dry weights were noted.

Effect of combined bio-treatments
For combined biotreatments, equal volumes of cell suspensions of each bacterial strain from 2 dold LB cultures were mixed and adjusted to 10 8 cells/ml with SDW. Equal volumes of each aqueous and/or methanolic extract from the brown seaweed were mixed with an equal volume of bacterial suspension of B. subtilis SV41and B. amyloliquefaciens subsp. plantarum SV65 adjusted at 10 8 cells/ml or their com bination. Seven combined biotreatments were test ed and detailed in Table 2.
Eggplant cv. Bonica seedlings were treated by dip ping roots for 30 min in each combined biotreat ment prepared as described above. Control seedlings were dipped in SDW only and LB medium. Treated and control seedlings were potted in commercialized sterile peat. Treated seedlings were retreated as substrate drenching with 50 ml of each tested com bined biotreatment. Four weeks after transplanting, eggplant seedlings were retreated with 50 ml of each combined biotreatment as described above.
Seven replicates of one seedling each were used for each individual treatment and the whole experi ment was conducted twice. After 60 days of growth under the same greenhouse conditions, the same growth parameters detailed above were measured.

Comparative efficacy of tested bio-treatments (Bacillus spp. and Sargassum vulgare extracts) and organic amendments
Bacillus spp. strains and S. vulgare extracts even applied singly or in combination were compared to a compost tea and to a commercial biofertilizer for their growthpromoting potential on eggplant seedlings.

Comparative efficacy of tested bio-treatments and a compost tea
The compost used in this study contained 70% of bovine manure, 25% of sheep manure and 5% of olivemill solid waste. The characterization of com post and the preparation procedure of compost tea (1:5 w/v) were described in a previous study . The physicochemical and microbial char acterization of compost are listed in Table 3. The compost used in this study had significantly improved the plant height, the leaf number, the aerial part dry Eggplant seedlings were treated by dipping roots for 30 min in compost tea (CT). Control seedlings were dipped in SDW only. Treated and control seedlings were transplanted into individual pots (12.5 × 14.5 cm) containing sterilized peat. Treated seedlings were retreated as substrate drenching with 50 ml of compost tea. Four weeks after trans planting, eggplant seedlings were retreated with 50 ml of compost tea.
After 60 days of growth under greenhouse condi tions, the growth parameters were measured as described above.

Comparative efficacy of tested bio-treatments and a commercial bio-fertilizer
The commercial biofertilizer used in this study was the Acadian ™ seaweed extract powder used at 2 g/l. The procedure of seedling treatment, the green house conditions and the noted growth parameters were the same as described above.

Statistical analysis
A oneway analysis of variance (ANOVA) was used for data analysis. The software used is the Statistical Package for the Social Sciences (SPSS) for Windows version 16.0. Each Experiment was conducted twice yielding similar results. No significant interactions between treatment and experiment were noted. Therefore, one representative trial of each experi ment is reported. Experiments were undertaken according to a completely randomized design. Means were compared using Multiple Range Duncan test at P≤0.05.

Results
Growth-promoting potential of tested single biotreatments B. subtilis SV41 and B. amyloliquefaciens subsp. plantarum SV65 based treatments and the aqueous and methanolic S. vulgare extracts were screened singly for their plant growthpromoting (PGP) ability on eggplant plants. As shown in Figs. 1, 2 and 3, the plant growth parameters (plant height, stem diame ter, fresh and dry weight of the aerial part, maximum root length, root fresh weight, flower and fruit num ber, and fruit fresh weight), noted 60 days posttreat ment, varied significantly (at P≤0.05) depending on tested bacterial and/or algal extracts.
Plants treated separately with the whole bacterial cells of both Bacillus strains and the aqueous extracts from S. vulgare were significantly 19 to 29.2% taller than the untreated control plants (Fig. 1a). The treat ments based on B. amyloliquefaciens subsp. plantarum SV65 cells, the aqueous and the methanolic extracts from the brown seaweed led to a significant increase by 14.3 to 20.9% in the stem diameter as compared to water control (Fig. 1b). Treatments with the methanolic and the aqueous algal extracts had stimulated by 33.8 and 43.4% the aerial part fresh over the untreated control (Fig. 1c). Only the aque ous extract had significantly enhanced the aerial part dry weight by 32.3% compared to control (Fig. 1d). It should be highlighted that eggplant aerial part devel opment was similar for LB medium treated plants and water control ones (Figs. 1a,1b,1c,1d).
As for their effects on the root development, all tested biotreatments induced a significant (at P ≤ 0.05) increment in the maximum root length and the root fresh weight when compared to control (Figs. 2a,2b). The maximum root length was significantly increased by 16.6 to 27.7% with both Bacillus spp. strains and S. vulgare aqueous extract when applied separately as compared to control (Fig. 2a). The root z, y Bacteria and fungal counts from compost during the matura tion phase of composting after 72 h of incubation at 35°C onto PCA and PDA, respectively. 3.92 Fungal count y (10 4 CFU/g of compost) 6.6 fresh weight was enhanced by 29.9 and 38.2% over control following treatments with B. amyloliquefaciens subsp. plantarum SV65 (B2) and S. vulgare methanolic extract, respectively (Fig. 2b). It should be highlighted that eggplant root development parame ters were comparable on plants treated with LB medium as well as water control plants (Figs. 2a,2b,2c). Data illustrated in figure 3a indicated a significant increase by 65.5 to 78.7% over the untreated control in the flower number following the individual applica tion of all tested biotreatments where the highest increment, of about 78.7% over control, was achieved using the algal aqueous extract. The fruit number was 30% higher than control in plants treat ed with the aqueous extract (Fig. 3b). Eggplant plants treated separately with S. vulgare extracts and the whole cell suspensions of B. subtilis SV41 showed 25.728.7% higher fruit fresh weight relative to con trol (Fig. 3c). It should be highlighted that plants treated with LB medium behaved similar than water control plants for eggplant flower and fruit produc tion (Figs. 3a, 3b, 3c, 3d).

Growth-promoting potential of tested combined biotreatments
Seven combinations of the tested biotreatments were evaluated for their effect on eggplant growth. Analysis of variance revealed a significant (at P ≤ 0.05) variation in the plant height, the stem diame ter, the fresh and dry weights of the aerial part, the maximum root length, the root fresh weight, the flower number, and the fruit fresh weight, depending on tested treatments. As shown in figure 1a,  the aqueous and the methanolic algal extracts com bined each one with both bacterial strains (EAq+B1+B2 and EMeth+B1+B2) and the methanolic extract mixed with B. subtilis SV41 (EMeth+B1). The highest increase of this parameter, of about 32.4 33.9% over control, was recorded following treat ments with S. vulagre aqueous extract combined with B. amyloliquefaciens subsp. plantarum SV65 (EAq+B2) and the algal aqueous extract mixed with the two Bacillus strains (EAq+B1+B2). A significant enhancement of the stem diameter of the treated plants, estimated at 13.6 to 24.7% over control, was also noted following the seven test ed combined biotreatments. The highest increment (24.7%) was induced by the aqueous extract com bined with B. amyloliquefaciens subsp. plantarum SV65 (EAq+B2) and at a lesser extent this extract when mixed with both Bacillus strains (EAq+B1+B2) (20.9%) (Fig. 1b).
The aerial part fresh weight was also significantly increased by 29.6 to 46.1% over control following treatments with the two bacterial strains (B1+B2), the aqueous extract combined with each bacterial strain separately (namely EAq+B1 and EAq+B2) or in combination (EAq+B1+B2) and the methanolic extract mixed with B. subtilis SV41 (EMeth+B1) (Fig.  1c). The highest increment (by 46.1% relative to the control) was induced by the aqueous extract com bined with B. amyloliquefaciens subsp. plantarum SV65 (EAq+B2). Eggplant plants treated with the algal aqueous extract combined with B. amyloliquefaciens subsp. plantarum SV65 cells (EAq+B2) or mixed with both Bacillus strains (EAq+B1+B2) showed a signifi cant enhancement in their aerial part dry weight by 25.426.5% compared to control (Fig. 1d).
As shown in figure 2a, the maximum root length increase over control ranged between and 18.4 to 41% following all tested combined biotreatments and the highest improvement, of about 36.841% over control, was induced by S. vulgare aqueous extract mixed with both Bacillus strains (EAq+B1+B2) or with B. amyloliquefaciens subsp. plantarum SV65 (EAq+B2). All tested biotreatments combined with the algal aqueous extract induced a significant enhancement in the root fresh weight of about 30.6 50% as compared to control (Fig. 2b). Furthermore, the combined treatment based on S. vulagre methanolic extract and B. subtilis SV41 (EMeth+B1) had also significantly improved this parameter by 41.4% over control. The highest increment in the root fresh weight, of about 50% relative to control, was noted on plants treated with the combined treat ment composed of the aqueous extract and B. amyloliquefaciens subsp. plantarum SV65 (EAq+B2).
When screened for their effects on the flower number, the seven combined biotreatments led to 54.573.7% increment in this parameter compared to control (Fig. 3a). Also, the fruit fresh weight was sig nificantly increased by 24.2% over control on egg plant plants treated with S. vulgare methanolic extract mixed with B. amyloliquefaciens subsp. plantarum SV65 (EMeth+B2) (Fig. 3c).

Comparative efficacy of tested bio-treatments with a compost tea and a commercial bio-fertilizer
Eleven tested biotreatments, applied singly or in combination, were evaluated for their growthpro moting potential on eggplant seedlings as compared to a compost tea and to a commercial biofertilizer (Acadian™).

Aerial part development
Analyses of variance of all growth parameters measured (plant height, stem diameter, aerial part fresh and dry weights) showed a significant variation (at P ≤ 0.05) between tested biotreatments as com pared to compost tea and Acadian ™ based treat ments.
Data showed a significant enhancement by 18.3 to 25.5% over compost tea based treatment in plant height of eggplant plants treated with S. vulgare aqueous extract applied either singly or in combina tion with both Bacillus spp. strains and/or singly with each tested bacterial strain (Fig. 1a). Furthermore, plants treated with the methanolic extract combined with B. subtilis SV41 and B. amyloliquefaciens subsp. plantarum SV65 (EMeth+B1+B2) were 19.3% taller than those treated with the compost tea. Similarly, compared with the tested commercial biofertilizer, the recorded increment varied from 17.7 to 25% depending on treatments. The highest increase of plant height, of about 23.425.5% and 23.325% over the commercial biofertilizer (i.e. Acadian™ treat ment), were induced by the aqueous extract mixed with B. amyloliquefaciens subsp. plantarum SV65 (EAq+B2) or with both bacterial strains (EAq+B1+B2), respectively.
All treatments with S. vulgare aqueous extract, applied either singly or in combination with each Bacillus strains separately or both strains combined, showed significant improvement in eggplant stem diameter by 9.917.5% and 11.218.7% over compost tea and the commercial biofertilizer treatments, respectively (Fig. 1b). B. amyloliquefaciens subsp. plantarum SV65 applied singly (B2) induced a signifi cant increment in this parameter by 12.2% versus compost teabased treatment. S. vulgare methanolic extract mixed with B. subtilis SV41 (EMeth+B1) led to a significant improvement in the stem diameter by 9.2% when compared to Acadian™ based treatment (Fig. 1b). As compared to both tested organic amend ments, eggplant plants treated with S. vulgare aque ous extract combined with B. amyloliquefaciens subsp. plantarum SV65 (EAq+B2) showed an incre ment by 17.518.7% in this growth parameter.
As shown in Fig. 1c, the aerial part fresh weight was significantly improved by 24.4 to 38.5% over the compost tea treatment in plants treated separately with the aqueous (EAq) and the methanolic (EMeth) S. vulgare extracts, the aqueous one combined with B. amyloliquefaciens subsp. plantarum SV65 (EAq+B2) or mixed with both Bacillus strains (EAq+B1+B2), and the methanolic extract associated with B. subtilis SV41 cells (EMeth+B1). Compared to the commercial biofertilizer, tested biotreatments had also significantly improved this parameter by 24.9 to 38.9%. The highest increments in the fresh weight of the aerial part, of about 38.5 and 38.9% compared to compost tea and Acadian™ based treatments, were induced by the aqueous extract combined with B. amyloliquefaciens subsp. plantarum SV65 (EAq+B2), respectively.
The aerial part dry weight was enhanced by 26.7 33.4% and 31.237.5% over the compost tea and the Acadian™ controls, using S. vulgare aqueous extracts either singly or in combination with B. amyloliquefaciens subsp. plantarum SV65 and both selected bac terial strains (Fig. 1d). Plants treated singly with the aqueous extract showed the highest increment of this parameter of about 33.4 and 37.5% compared to those amended with compost tea and Acadian ™ , respectively.

Root development
The maximum root length and the root fresh weight varied significantly (at P ≤ 0.05) depending on tested biotreatments. Plants treated with seven bio treatments (the aqueous and methanolic extracts combined with each bacterial strain and/or with both strains and the aqueous extract applied singly) showed significant improvement in the maximum root length of about 15.9 to 37.4% compared to ones treated with the compost tea (Fig. 2a). As compared to Acadian ™ treatment, nine biotreatments (same as previously, combined Bacillus spp. strains and B. amyloliquefaciens subsp. plantarum SV65 applied singly) induced a significant enhancement by 17.7 to 40.5% in this parameter. The highest increments of the maximum root length, of about 37.4 and 40.5% over the compost tea and the commercial biofertiliz er controls, were induced by S. vulgare aqueous extract combined with B. amyloliquefaciens subsp. plantarum SV65 (EAq+B2) or with both Bacillus strains (EAq+B1+B2), respectively, (Fig. 2a). The root fresh weight was significantly improved by 32.3% compared to compost tea using the aqueous extract mixed with B. amyloliquefaciens subsp. plantarum SV65 and by 36.1 and 25% versus Acadian ™ treat ment using the last biotreatment and the methano lic extract combined with B. subtilis SV41, respective ly (Fig. 2b). The highest increments on this parame ter, by 32.3 and 36.1% compared to compost tea and Acadian ™ based treatments, were induced by the aqueous extract from S. vulgare mixed with B. amyloliquefaciens subsp. plantarum SV65, respectively (Fig. 2b).

Fruit production
ANOVA analyses performed for the flower num ber, fruit number and fruit fresh weight showed a sig nificant variation (at P ≤ 0.05) between the eleven biotreatments tested and compost tea and Acadian ™ based treatments.
As compared to compost tea control, the seven tested biotreatments had significantly improved the flower number by 40.6 to 59.6%. The highest increase (59.6%) was noted on plants treated singly with the aqueous S. vulgare extract. Compared to the commercial biofertilizer (Acadian ™ ), only the treat ment with the algal aqueous extract had significantly enhanced this parameter by 38.3% (Fig. 3a). This aqueous extract when applied singly had also induced a significant improvement of the fruit num ber by 40% compared to compost tea and Acadian ™ based treatments (Fig. 3b). As shown in Fig. 3c, the average fruit fresh weight was significantly enhanced by 15.8 to 20.8% over the compost tea control using separately B. subtilis SV41, the aqueous and the methanlic S. vulgare extracts and the last one com bined with B. amyloliquefaciens subsp. plantarum SV65. These biotreatments had significantly improved this production parameter by 35.8 to 39.6% relative to the commercial biofertilizer.

Discussion and Conclusions
The use of ecofriendly resources has been a major focus of attention in the past three decades. Although reports on the benefits of using microbial inoculants for the promotion of plant growth and health in agricultural soil have been inconsistent, there is a promising trend for microbial inoculants to meet the sustainable agricultural production needs (Alori et al., 2017). The use of seaweeds as biofertil izers in horticulture and agriculture has increased in the recent years Basmal et al. (2019).
This study was aimed to evaluate the efficacy of combining Bacillus spp. strains and S. vulagre extracts (aqueous and methanolic extracts) in order to select the best combination for the biostimulation of eggplant growth. Furthermore, biotreatments (bacteria and algae extracts) tested singly and/or in combination were compared against two organic amendments i.e compost tea and Acadian™ (a com mercial biofertilizer) to select the most effective bio stimulant among the tested treatments.
Biotreatments (bacteria and/or algae extract) could be applied either singly as seed priming prior sowing, seedlings root dipping prior transplanting, soil drenching and foliar spraying or combination of two or more methods of application (Papenfus et al., 2013). In this study, biotreatments either used singly or in combination were applied as seed priming, then seedlings root dipping and finally as substrate drenching. The recommended method, timing and the rate of applications were greatly different accord ing to plant variety and growth stages (LolaLuz et al., 2013). According to Matysiak et al. (2011) study, the stimulatory potential is more efficient at the early stage of plant growth. In this study, all biotreat ments were applied early at presowing, the first application occurred at the twotrue leaf stage and the second one four weeks postplanting.
As single application, the aqueous extract from S. vulgare used at 50 g/l showed higher growthpro moting potential based on major growth parameters of eggplant than B. subtilis SV41, B. amyloliquefaciens subsp. plantarum SV65 and the methanolic extract compared to the untreated control, and to compost and Acadian ™ based treatments. As demon strated by Michalak and Chojnacka (2015), water extraction was found the most effective for better release of micro and macroelements from seaweed biomass even used as fertilizer and biostimulant. The application of seaweed extracts exhibit stimulat ing activities of plant growth, yield and fruit quality in a variety of horticultural crops (Battacharyya et al., 2015;Kocira et al., 2018;Mahmoud et al., 2019). Indeed, the use of water extract from algae as plant growth biostimulant was described in several crops such as wheat, tomato, Arabidopsis, spinach, and Vigna sinensis and this under normal and stressed environments (Nabti et al., 2010;Craigie, 2011;Kavipriya et al., 2011). In this study, the boiling aque ous extract from S. vulgare at 100°C for 1 h did not affect its growthpromoting potential and the con tents of polyphenol and flavonoids (Ammar et al. 2017). Water extracts prepared by autoclaving or heating previously washed marine alga in distilled water are found to have beneficial growth stimulat ing effects in cereal and flowering plants (Nabti et al., 2010;Craigie, 2011).
The aqueous S. vulgare extract applied singly had significantly improved the majority of growth para meters as compared to the untreated control and to the two tested organic amendments. Some seaweeds have been successfully used as soil conditioners and fertilizers in agriculture (Duarte et al., 2018). Commercially, extracts from brown algae such as Acadian are good sources of fertilizer (Hurtado et al., 2008). Fertilizers derived from seaweeds such as Fucus, Laminaria, Ascophyllum, Sargassum etc. are known to be biodegradable, nonpolluting and non hazardous to human and environment (Dhargalkar and Pereira, 2005). Mathur et al. (2015) study demonstrated the benefical effects of seaweed liquid fertilizer from Sargassum wightii, Ulva lactuca and Enteromorpha intestinalis on stimulation of seed ger mination and growth, and enhancement of biochemi cal traits of Glycine max plants. Seaweeds extracts were found to be more active than chemical fertiliz ers in enhancing seed germination and growth para meters (Godlewska et al., 2016). Vasantharaja et al. (2019) found that foliar spraying of cowpea plants with the brown seaweed extract at 3% significantly improves the shoot length, the number of leaves per plant, yield, the total phenolic and flavonoid contents and the antioxidant activity as compared to control plants. Foliar spray of liquid fertilizer based on S wightii extract has successfully enhanced the chloro phyll content, the internodes and the shoot length of tomato and chilli pepper plants compared to seed soaking (Murugan et al., 2020). The mechanisms of stimulation of plant growth by the marine algal extracts may be due to the diverse compounds observed in their extracts. Indeed, macronutrients, organic substances such as amino acids and plant growth regulators substances are presents in the sea weed liquid fertilizer of Sargassum species (Zodape et al., 2008;Nabti et al., 2016;Murugan et al., 2020). Furthermore, seaweed based treatments are able to increase the level of nutrient in soil such as nitrogen, phosphorus and potassium and other compounds as polysaccharides wich are necessary for plant growth that are highly diverse and constitute the major com pounds of algae cell walls (Heltan et al., 2015;Mirparsa et al., 2016;Nabti et al., 2016).
To improve the plant growthpromoting ability of both selected Bacillus spp. used in the current study, they were combined either single or in combination with the aqueous and/or the methanolic S. vulgare extracts. Microbial inoculants, applied singly or in combination, are able to improve nutrient availability and uptake, and to strengthen plant health (Alori et al., 2017).
As compared to untreated control and to the two tested organic amendments (compost tea and Acadian™), eggplants treated with combined formu lations of B. amyloliquefaciens subsp. plantarum SV65 and aqueous S. vulgare extract showed the highest enhancements in plant height, stem diame ter, aerial part fresh weight, maximum root length, and root fresh weight. Furthermore, the combination of B. subtilis SV41, B. amyloliquefaciens subsp. plantarum SV65 and the aqueous extract had significantly increased the plant height, the stem diameter and the maximum root length as compared to water, compost tea and Acadian™ based treatments. When applied on seeds, plant surfaces or soil, microbial inoculants are shown able to enhance root exuda tion, increase the availability and supply essential nutrients to host plants, and thereby promoting their growth (Trabelsi and Mhamdi, 2013). The phytohor mones synthetized by microbial inoculants can result in development of the root system, expansion and elongation of the root hairs and lateral roots, leading to improved uptake of water and nutrients (Halpern et al. 2015). Fixation of atmospheric nitrogen, solubi lization of minerals such as phosphorus by microbial inoculants are also involved in plant growth promo tion (Babalola, 2010). Indirectly, microbial inoculants also affect the status of plants by eliciting the induced systemic resistance (ISR) or the systemic acquired resistance (SAR) thus improving their health. These acts prevent soilborne pathogens from inhibiting plant growth (Yang et al., 2009). The ability to trigger a salicylic acid (SA)independent pathway controlling systemic resistance is a common trait of ISRinducing biocontrol bacteria. Both Bacillus spp. used in this study, have been demonstrated as promising biostimulants when challenged to tomato plants and their ability to produce the indole3acetic acid, organic acids and/or siderophores, to solubilize phosphate, and to control Fusarium wilt disease was evidenced , 2018.
Plant growthpromoting rhizobacteria (PGPR) applied singly and/or in combination reduced appli cation rates of chemical fertilizers. As demonstrated by Adesemoye et al. (2009), a mixture of PGPR strains B. amyloliquefaciens IN937a and Bacillus pumilus T4, and the arbuscular mycorrhizae (AM) Glomus intraradices added to 75% fertilizer success fully enhance growth, yield, and nutrient (nitrogen and phosphorus) uptake of tomato plants compared to the 100% fertilizer control. In the same way, three biostimulants consisting of a mix of rhizospheric microorganisms i.e. Pseudomonas sp. 19Fv1T, P. fluorescens C7 and AM fungi, tested in conditions of reduced fertilization, induced an increment in the yield, the fruit quality and the nutritional value of tomato fruits (Bona et al., 2018). ElYazeid et al. (2007) demonstrated that the double inoculation with Paenibacillus polymyxa and Bacillus megaterium associated with a foliar spray of boron led to an enhancement of growthpromoting hormone levels including gibberellic acid, 3indole acetic acid and cytokinines associated with a decrease in the abscisic acid inhibitor. Double inoculation especially with the mycorrhizal fungus G. intraradices and boron spray improved sex ratio and early production of fruits accompanied with high yield of squash.
Several investigations support different aspects of potential macro algal applications in agriculture. Currently, seaweed extracts are the new type of prod ucts used in plant cultivation (Elsharkawy et al., 2019). It should be highlighted that the improvement of growth parameters in eggplant plants treated with combined Bacillus spp. and aqueous extract from S. vulgare, recorded in the current study, is higher than that induced following the single application of aque ous extract. Hence, the combinations of biotreat ments enhance either the efficacy of bacteria and algal aqueous extract more than when applied singly. However, the combinations of the methanolic extract with tested Bacillus spp. strains did not induce signifi cant increments in the major growth parameters. The synergism occurring between both tested bacterial strains and the aqueous S. vulgare extract was con firmed based on various growth parameters. Crocker (2018) investigation clearly demonstrated the in vitro ability of seaweed extract to enhance PGPR growth which may explain the synergism noted. Also, Basmal et al. (2019) found that the biological fertilizer formu lation based on Sargassum sp. extract enhance the growth rate of beneficial Pseudomonas fluorescens. Through the in planta experiments, combined PGPR inocula and seaweed extract enhanced significantly the root growth parameters of treated soybean plants compared to the untreated ones (Crocker, 2018). The addition of biofertilizer containing multistrains of Bacillus acting as phosphorusfixing agents and Azotobacter, Azospirllum and Rhizobium as nitrogen fixing inoculants combined with a foliar spay with mixed seaweed extracts from Ulva lactuca, Ulva faciata and Peterocladia caplicia at 10 ml/l led to incre ment of growth characters and to enhancement of the total yield of pea plants (Elsharkawy et al., 2019).
As conclusions, the use of plantgrowth promoting bacteria especially Bacillus strains and the brown seaweed extracts (aqueous and methanolic extracts) as biostimulants on eggplant plants was emphasized as compared to untreated ones. The combined treat ment based on Bacillus spp. strains and the aqueous S. vulgare extract was found to be the most efficient biostimulant as compared to a compost tea and a commercial biofertilizer tested i.e. Acadian™. The beneficial roles of the above combined biotreat ments on growth parameters were higher than their single applications. The influence of the combined biostimulant developed based on the two tested Bacillus spp. strains and the brown seaweed aqueous extract on the soil microbial community need to be explored in the future to find out ways to more effec tively apply this combined biotreatment and to elu cidate its effects on soil microbiome including phy topathogenic and beneficial microorganisms.