The individual two microbial strains out of five constituting the biostimulant consortium from EVL Inc., stored in glycerol stocks, were used for this study. The stocks were stored at −80°C; a loopful was added into 50 mL fresh sterile M13 or MRS medium broth, contained in 250 mL flasks. The cultures were then incubated for 24–48 h at 30 or 37°C in an orbital shaker at 120 rpm (except for the Lactobacillus stain which does not require agitation) after which suitable dilution was carried out to obtain ~10⁸ CFU at the required turbidity, measured as optical density (OD) at 600 nm. The pH tolerance screening for growth was conducted prior to the start of plant effect experiments (Table 1). Based on initial screening three pHs (5, 7, & 8) were selected for testing on seed germination and seedling growth. Microbial strains were then grown at these pHs after which cells were removed by centrifugation at 12,000 g and the cell free aliquots were used at various concentration as treatments in seed germination and seedling growth bioassays.

Vigorous sorted seeds of corn (Zea mays L. var 25M75) and tomato (Solanum lycopersicum L. var. Beefsteak) were used in the study. The germination was 95 and 98% for corn and tomato, respectively. Seeds were surface sterilized to avoid fungal contamination using 70% ethyl alcohol for 2 min, then washed with 3.5% NaOCl for 5 min. Seeds were then thoroughly rinsed 5 times with deionized water. Surface sterilized seeds were germinated inside petri dishes (sterile 100 ×15 mm polystyrene Petri dishes) lined with filter paper (Fisherbrand™ - P8 Grade, Pittsburgh, US). Each petri dish received 10 seeds for corn and 20 for tomato. The treatment solutions were then prepared using deionized water and cell-free supernatant (CFS) with concentrations of 1, 0.4, 0.2, and 0.1% (v/v). The respective broth medium concentrations were used as a positive control while deionized water was used as a negative control. The filter paper of each Petri dish was then wetted with 5 and 4 mL of each concentration of the CFSs of corn and tomato, respectively.

Petri dishes were then sealed using parafilm to avoid water loss, which would hinder the normal process of germination. Petri dishes were arranged in a completely randomized design (CRD) with three replicates of each treatment in a germination cabinet set at 25°C with a relative humidity of 70% and 24 h darkness. Only two ^*microbial strains (Table 1) were selected as showed effect on seed germination assay with corn and tomato after screening (data not shown). In all experiments seeds were considered germinated when their radicle was about 2 mm long; data were collected at 24, 30, 42, and 54 h for corn and 48, 60, 72, and 84 h for tomato. The experiments were each repeated twice.

To detect the differences between treatments for seedling growth experimental data were subjected to analysis of variance (ANOVA) using SAS^® OnDemand for Academics. Where the program found treatment means to be different, they were separated using the Fisher least significant difference (LSD) at p ≤ 0.05.

The tested CFS from pH 5, at all evaluated concentrations, slowed tomato seed germination both for EB2004S and EL2006H (Figures 2, 3B). The CFSs obtained from EB2004S had no effect on germination of tomato seeds. The same results were obtained for EL2006H CFS obtained from pH 7.

The screening of the treatment concentrations was conducted so that the best was chosen, and other concentrations were not retained for further experimentation. To achieve this, data analysis of seed germination was used as the basis to further confirm the effect of the CFS on early seedling growth. Visual effects on roots development (number and length), seedling vigor and leaves appearance and size as seen in some of the selected concentrations used in the seedling growth experiments are provided in Figure 3.

Seed germination is a critical step in the progression of plant development, and particularly so for any plant that solely depends on seeds for propagation. Seeds that germinate robustly lead to early establishment of seedlings; as a result final production/yield is heavily impacted by these early plant growth stages (Tian et al., 2014). The present study has demonstrated that the use of CFS from EB2004S and EL2006H increased corn and tomato seed germination. Prior to evaluation of the CFSs, seeds were first germinated under normal conditions, to get the average germination percentages, to confirm the quality of the seed used (corn 95% and tomato 98%). Our study is one of several efforts to increase early plant establishment, particularly at high levels of abiotic stresses such as more extreme soil pHs. Previous studies have used a range of methods to improve seed germination including physical, physiological and biologicals (Afzal et al., 2016); the current study focused on the use of CFSs a derivative of biological seed enhancement applied at various concentrations obtained from pH 5, 7, and 8 to explore the potential of microbes in improving germination.

PGPM suspension are known to enhance tomato seed germination (Widnyana, 2018). Similar studies have been reported for corn such enhancement of seed germination in most plant species. Results from this study (Figures 1, 2) show that CFS increases the rate of seed germination, congruent with a study on CFSs to increase seed germination which was reported for Burkholderia seminalis on tomato (Tallapragada et al., 2015).

The CFS from EB2004S cultured at pH 5 positively effected corn seed germination at 42 h (Figure 1). The CFS obtained at pH 5 meaningfully enhanced corn germination, even at the lowest concentration of 0.1% (v/v) (Figure 1). The lowest concentration of CFS applied to corn seeds enhanced germination significantly, in contrast to an 8% (v/v) CFS solution used to affect rice seed germination (El-Khawas and Adachi, 1999).

The study also observed effects, although smaller of the CFSs obtained from pH 7 and 8 on corn and tomato seed germination. This was not expected for CFS from pH 7, but provided insight regarding culturing microbes below or above their optimum pH, or perhaps at stressful levels of some other abiotic stress, to increase potential for benefiting plants, as was the case for seed germination. The most probable explanation for this situation could be that when a microbe is dealing with stress it excretes more material related to adaptation mechanisms (Decho and Gutierrez, 2017), perhaps in the form of beneficial microbe-to-plant signal compounds that increase seed germination rate.

Seedling growth, the earliest stage of actual plant establishment, needs to withstand the initial stresses the plant confronts as it grows. The use of products that enhance seedling growth are of great importance as they exert an ultimate effect on final crop performance/production. PGPM have shown to promote seedling growth from both their suspensions (Almaghrabi et al., 2014; Widnyana, 2018) and CFSs (Pellegrini et al., 2020) for both controlled and open field conditions. In the reported work we scanned seedling roots to determine their total length, volume, and average diameter (Tables 3–6) among seedling growth variables. This is the first report of the L. helveticus enhancing seedling growth directly, as opposed to the report by Rodríguez-Morgado et al. (2017), which stimulation of microbial activity in the soil and hence increased soluble phosphates, which increased plant root development. Furthermore, Lactobacillus rhamnosus CFS reported was analyzed whereby LLA was a metabolite responsible for improved soil properties (Rodríguez-Morgado et al., 2017) and LLA, peptides, and AA were responsible for increased microbial growth in the soil (Caballero et al., 2020). Whether the same metabolites are responsible for reported results in this study or not remains to be found out and further test under field conditions.

Visual inspection of the corn seedlings (Figure 4) indicated that 1% (v/v) CFS from B.s subtilis significantly increased root volume; these findings are consistent with a study on rice seedlings (El-Khawas and Adachi, 1999) which observed increased root length and volume following treatment with PGPM CFS. Moreover, Azospirillum brasilense Ab-V5 and Ab-V6 CFSs application yielded positive results in improving Glycine max root morphology and nodulation (Rondina et al., 2020).

Despite, the positive effects of the CFS on treated corn seedlings (Table 3), there was no statistically significant effect on WSDW, as compared to the positive controls. For tomato, the same treatment provided little to no effect. Most of the B. subtilis CFS seedling treatments did not significantly improve seedling variables regardless of pH level, the exception being root diameter which was increased at pH 8.

These interesting and in some cases apparently contradictory results provide a reason for investigating this hypothesis further under greenhouse conditions and even further under open field conditions. Currently, studies of CFS as biostimulants remain scarce for both greenhouse and open-field condition. Grain yield from Z. mays L. and G. max L. was enhanced by rhizobial CFS metabolites (LCOs, phytohormone and exopolysaccharides) by combined inoculation with Azospirillum sp. and Bradyrhizobium sp. (Marks et al., 2013). Similarly, one recent study by Tewari et al. (2020) has shown more interesting results under field condition that a combined formulation of Bradyrhizobium sp., its CFS and exopolysaccharides, which resulted in increased productivity and nodulation of pigeon peas as opposed to CFS or bacterium inoculum applied alone.