Effect of biostimulants on soybean seedlings

www.afc.kg.ac.rs  Effect of biostimulants on soybean seedlings Gorica Cvijanović1, Ninoslav Čolić2, Nenad Đurić2, Gordana Dozet2, Abduladim Eltreki2, Marija Cvijanović3, Milena Žuža2  1University of Kragujevac, Institute for Information Technologies Jovana Cvijića bb, Kragujevac, Serbia 2Megatrend University, Faculty of Biofarming Maršala Tita 39, 24300 Bačka Topola, Serbia 3University of Bijeljina, Faculty of Agriculture Pavlovića put bb, 76300 Bijeljina, BiH *Corresponding author: cvijagor@yahoo.com Received 8 April 2020; Accepted 9 September 2020  A B S T R A C T The aim of this study was to analyze the effect of biostimulants on the morphological characteristics of soybean seedlings. The testing was conducted in the laboratory of the Faculty of Biofarming in Bačka Topola. The experimental material included three soybean varieties (‘Galina’, ‘Sava’ and ‘Rubin’) selected at the Institute of Field and Vegetable Crops in Novi Sad. The study lasted for two years, 2015–2016, and identical biostimulant treatments were applied in both years. In order to determine the effect of biostimulants on soybean seedling root, hypocotyl and weight, the following commercial biostimulants were applied: EM Aktiv, Terra Green Hobby, Slavol and Bioplant Flora. In addition to the single application of biostimulants, two combinations of Slavol + Bioplant Flora and Slavol + Bioplant Flora + Epin Extra + Slavol S were used as treatments. EM Aktiv showed the greatest effect on root growth. The root was on average 12% longer than the control. Slavol S had the greatest influence on seedling hypocotyl and weight. The increase was 8.24% and 5.15%, respectively, compared with the control.


Introduction
Soybean (Glycine max (L.) Merr.) is a major crop, not only in agricultural production but also in industrial processing. It ranks fourth in terms of production area in the world (Balesević and Miladinović, 2014). Worldwide, it is grown on an area of about 123.5 million hectares (http://faostat.fao.org, 2017). Using high quality seeds is a basic factor that determines high crop yields (Milošević et al., 1996). However, since soybeans are sown in different agroecological conditions, seed germination and vigor are influenced by various adverse environmental factors such as drought and extreme temperatures (Casenave and Toselli, 2007). Therefore, different methods are used to reduce the negative impact of environmental factors (Djukić et al., 2017;Miladinov et al., 2019). Biostimulants can be used to improve seed quality (Yildirim et al., 2007). Biostimulants are neither plant nutrients nor pesticides; they are organic materials that, when used in small amounts, improve plant growth and development, but to a degree different from the use of traditional plant nutrients (Yakhin et al., 2017).
Biostimulants can be divided into humic acidcontaining biostimulants, hormone-containing biostimulants and amino acid-containing biostimulants, or biostimulants of plant growth and development depending on the type of biostimulant (Tkalec, 2010). Also, biostimulants must be able to penetrate the plant tissue. This is of great importance, especially in field conditions, where treated plants are exposed to different agroenvironmental conditions (Kolomazik et al., 2012). However, in addition to their positive effect on seed germination, biostimulants in some cases have the ability to reduce seed quality (Miladinov et al., 2014a) as well as to inhibit seedling growth (Miladinov et al., 2015).
There has not been much research on the effect of biostimulants on seedlings. Therefore, the aim of this paper was to investigate the influence of different biostimulants and their combinations on the morphological parameters of soybean seedlings.

Material and Methods
The research was carried out in the laboratory of the Faculty of Biofarming in Bačka Topola. Testing was performed on seeds of three soybean varieties: 'Galina', 'Sava' and 'Rubin' (Factor A The seeds treated with distilled water (C) were used as controls.
During the preparation of the solutions, given the negative effect of light on biostimulants containing active microorganisms, the whole process (from dosage to treatment of the seed and its comparison with the substrate) took place in a slightly darkened part (away from the light source) of the laboratory. The seeds were sprayed with biostimulant solutions. After 30 minutes, the seeds were spread on filter paper. They were evenly applied to filter paper measuring 580 x 580 mm (58 cm in length), and 580 x 290 mm (Factor D). Seed germination was performed under laboratory conditions using a standard laboratory test. Standard laboratory germination was tested for 4 × 100 seeds. The incubation period was eight days at 25 °C and 95% relative humidity (ISTA, 2008). After eight days of incubation, 4 × 10 average fresh soybean seedlings were taken from each treatment for the analysis of seedling root, hypocotyl, and weight.
The statistical significance of differences between treatment means was tested by four-way ANOVA with LSD test at two levels of significance (5% and 1%). Pearson's correlations between the tested properties were assessed for significance by the t-test.

Results and Discussion
The analysis of the effect of variety (A), treatment (B), year (C) and type of filter paper (D) on soybean seedlings showed that these factors had a very significant effect on root growth. All factors except year significantly influenced the hypocotyl growth and weight of seedlings (Table 1). Also, for all parameters, a very significant interaction was established between the factors, AxB, AxD, AxBxC and AxBxCxD.

Effect of the factors under consideration on the parameters examined Factors
Seedling root

Effect of biostimulants on soybean seedling root growth
The results showed that the application of a regulator can have a positive effect on soybean seedling root growth. The greatest effect is achieved by EM Activ treatment. Eight days after seed treatment, soybean seedlings had significantly longer roots relative to other treatments and controls. Root length averaged 15.5 cm, which is 12% longer than the control. Effective microorganisms found in EM Activ, in addition to nitrogen fixation and mineralization of organic forms of phosphorus in the soil, synthesize active substances such as enzymes, amino acids, vitamins, and fungicidal substances, thus directly or indirectly affecting the growth and development of plants (Cvijanović, 2017). Szymanski et al. (2003) reported that EM Activ led to an increase in seed germination and root development, enhanced flowering and fruit formation, and improved soil fertility. EM Activ was also found to have a positive effect on soybean yield. In two-year studies, the yield increased by an average of 10.84 % i.e. by 6.86 % and 14.81 % per year, respectively (Dozet et al., 2014). The effect of the other treatments on root growth was not significant, but they gave longer roots than the control. Compared with the control, Slavol S promoted root elongation by 7.27 %, Terra Green Hobby by 6.77%, S + B + E + SS by 5.61%, and Bioplant Flora by 5.28 %, whereas the combination of S + B increased root length by 4.82% (Figure 1 The beneficial effect of Slavol S can be attributed to the active ingredient, which is indole-3-acetic acid. Indole-3-acetic acid is considered the most important natural auxin in higher plants. Auxins are phytohormones that affect plant growth by participating in stretching and cell division, inducing root growth (Normanly et al., 1995). Slavol S was also found to have a positive influence on sunflower seeds (Miladinov et al., 2014b;Miklić et al., 2016).
However, when combined with other biostimulants, its effect is reduced, and therefore the root is shorter than when treated with Slavol S alone, which is in agreement with the results conducted by Miladinov et al. (2015). Miladinov et al. (2014b) have also indicated that the effect of biostimulants is also significantly dependent on genotype, which is consistent with the research on soybeans. The results showed that the use of biostimulants had the greatest effect on root growth in 'Rubin'. 'Rubin' had a significantly longer root system than the other varieties. The root length of 'Rubin' was 15.61 cm, which is 2.97% and 18.19% longer than that of 'Galina' and 'Sava', respectively. The rapid development of the root system is of great importance to the plant, especially under water deficit conditions during germination, because it allows plants to quickly reach soil moisture (Gupta et al., 2008). Using alonger strip of filter paper, a significantly higher root length (14.57 cm) was observed, which is a 7.62 % increase. This is understandable, because the root had no physical barriers, and could therefore unfold vertically downward, which is of great importance for the initial growth of the seedling.

Effect of biostimulants on soybean seedling hypocotyl growth
The biostimulants Slavol S, Terra Green Hobby and EM Aktiv significantly influenced hypocotyl growth. Compared with the control, Slavol S increased hypocotyl length by 8.24 %, Terra Green Hobby by 6.52 %, and EM Aktiv by 4.97 %. By contrast, the combination of S + B + E + SS reduced the length of the hypocotyl by 4.67% (Figure 2 Figure 2. Influence of seed treatment on hypocotyl length Similar results were reported on sunflower by Miladinov et al. (2014b). They found that the combined effect of Slavol S and Bioplant Flora resulted in an inhibitory effect on sunflower seedling hypocotyl, which was reduced by as much as 9.09% over control. Although many studies have indicated the positive effect of humic acids -the active substance of Bioplant Flora -on germination, plant growth and development (Russo and Berlyn, 1990;Parađiković, 2008;Zeljković, 2013), their effect on hypocotyl length has not been established. The results of the present study showed that the effect of biostimulants is significantly dependent on genotype, which is in line with the results on soybean (Cindrić, 2019) and sunflower (Miladinov et al., 2014b). The results showed that the use of biostimulants had a significantly better effect in 'Galina' and 'Rubin'. The hypocotyls of 'Galina' and 'Rubin' were, respectively, 33.41% and 33.10% longer than those of 'Sava'.

Effect of biostimulant on soybean seedling weight
As regards the influence of biostimulants on soybean seedling weight, Slavol S gave the greatest increase. In comparison with the control, seedling weight was increased by 5.15 %. The results are consistent with the research conducted by Jelacic et al. (2006). Kaludjerovic and Mirecki (2013) determined that Slavol S increased the weight of lettuce seedlings by an average of 35.74 %. The weight was increased regardless of the substrate used for seedling growth Bioplant Flora reduced seedling weight by 2.17 % over control, which is statistically insignificant. However, the results contradict the research conducted by Türkmen et al. (2004) on tomatoes. They found that humic acid based products, such as Bioplant Flora, increased the content of macro and microelements, resulting in improved growth. However, a positive effect was only present when the product was used at certain concentrations. At high concentrations, its effect was negative. The combination of two biostimulants, S + B, did not have a significant effect on seedling weight relative to the control. However, the combination of three biostimulants, S + B + E + SS, gave a significantly lower seedling weight, compared with the control of 8.70% (Figure 3

Correlation between tested parameters
The application of Pearson's correlation coefficient revealed the highest positive correlation between hypocotyl length and seedling weight (r = 0.703**). The smallest positive correlation was found between root length and seedling weight (r = 0.314 **), which is understandable, since most nutrients and water are taken up for building aboveground vegetative biomass and creating favorable conditions for the start of autotrophic feeding of a new young plant (Table 2).

Conclusion
Soybean varieties responded differently to biostimulant treatments. The most significant difference was found in the influence of biostimulants on root growth. The greatest effect was found in 'Rubin'.
Pre-sowing seed treatment with EM Aktiv showed the greatest effect on root growth. The root was on average 12% longer than the control. Slavol S had the greatest influence on seedling hypocotyl and weight. The increase was 8.24% and 5.15%, respectively, compared with the control.
The biostimulants were more effective on largesized filter paper, which was an expected finding because the seedling had no physical barriers and could develop smoothly.
The year of production had an effect only on root growth, whereas it did not have a significant effect on the other morphological parameters.
However, unlike the positive effects of certain biostimulants when used alone, their improper combination can inhibit the morphological properties of soybean.