1 Introduction

There is an ongoing worldwide search for new ways to promote sustainable agriculture by improving plant nutrition efficiency. In addition to improving plant yield, growth, and development, it is also important to reduce agrochemical usage. In recent studies, biostimulants have been used to enhance plant growth and to increase tolerance of unfavorable soil and environmental conditions as well as to improve the efficiency of resource utilization (Colla and Rouphael 2015). The European Biostimulant Industry Council (EBIC) describes plant biostimulants as substances and/or microorganisms whose function when applied to plants or the rhizosphere is to stimulate natural processes that enhance or benefit nutrient uptake, nutrient efficiency, tolerance to abiotic stress, and crop quality (du Jardin 2015). In recent years, there has been increased research interest in the application of biostimulants to many agricultural processes throughout the life cycle of plants, from seed germination to maturity. This field includes increasing plant growth and development efficiency, e.g., increasing yield, enhancing crop quality, increasing plant tolerance to and recovery from abiotic stresses, facilitating nutrient assimilation, translocation and use, enhancing the quality of produce, enabling more efficient water utilization, and enhancing certain physicochemical properties of the soil (Calvo et al. 2014). However, the mechanisms activated by biostimulants are often difficult to identify and are still under investigation (Rouphael et al. 2015; Nardi et al. 2016; Ertani et al. 2019; Paul et al. 2019).

Because of the special requirements of their sessile life style, plants have their own unique defense system. Abiotic environmental conditions like water deficit, salinity, and excessive light alter cell homeostasis. This leads to increase in the production of reactive oxygen species (ROS) such as the superoxide (O2·−), singlet oxygen (1O2), hydrogen peroxide (H2O2), and hydroxyl radicals (OH.) (Miller et al. 2010). To scavenge and detoxify high levels of ROS, enzymatic defense systems operate during both normal and stress metabolism. These include superoxide dismutase (SOD), peroxidase (POX), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR). Non-enzymatic defense systems that also operate under these conditions include ascorbic acid, alkaloids, carotenoids, glutathione, phenols, and tocopherols (Apel and Hirt 2004; Mittler et al. 2004). Moreover, ROS, in addition to being toxic by-products of stress metabolism, are also important signal transduction molecules in plant cells. Under optimal growth conditions, they are mainly produced at a low level in organelles such as chloroplasts, mitochondria, and peroxisomes during photosynthesis or respiration (Miller et al. 2010; Mittler et al. 2004). On the other hand, the endogenous defense system of plants is sometimes inadequate during normal or stress conditions, and in this case, biostimulants can be used exogenously. As an increasingly intensive area of research, the role of the antioxidant defense system alone or in combination with biostimulants is still only in the very early stages of investigation, and very limited information is currently available.

Arbuscular mycorrhizal (AMF) and humic substances are two of the seven main categories of biostimulants which have been used separately in previous studies to improve plant nutrient uptake, growth, and development, as well as to enhance tolerance and resistance to abiotic stress and to promote soil structural stability (Aalipour et al. 2020; Berruti et al. 2016; du Jardin 2015; Ertani et al. 2019; Ozfidan-Konakci et al. 2018; Yang et al. 2015). These fungi are among the most important of the soil microorganisms that develop mutual symbiotic association with the roots of most plants (Yang et al. 2016). In recent years, there has been an increasing interest in the use of AMF as a biostimulant to promote sustainable agriculture. In the literature, AMF studies have focused on the cultivation conditions, mycorrhizal host-plant family, origin of AMF inoculants, method of AMF propagation, type of AMF propagule, method of application, inoculated AMF species, and abiotic stress types (Berruti et al. 2016; Rouphael et al. 2015). One study was conducted on the plant growth parameters, photosynthesis, and antioxidant enzymes of Robinia pseudoacacia under lead stress. Results showed that AMF symbiosis had improved the physiological and biochemical properties of this woody plant via increased ROS scavenging capacity (Yang et al. 2015). Furthermore, plant growth, photosynthesis, and nutrient uptake were also improved with AMF inoculation in another woody plant, Zelkova serrata, grown under salt stress (Wang et al. 2019). Santander et al. (2019) found that AMF inoculation improved ionic balance and prevented the entry of toxic Na+ ions into the roots of lettuce under saline conditions. Further, the application of mycorrhizal fungi showed increased plant biomass and phosphorus concentration in turfgrass species under water stress conditions (Aalipour et al. 2020). Similarly, many studies dealing with abiotic stress treatments have focused on AMF application. Thus, investigations into AMF-mediated pathogen protection under non-stressed normal conditions, especially with respect to antioxidant metabolism, remain limited, a fact also mentioned in the study of Berruti et al. (2016). Hence, few works have been conducted that are concerned with the antioxidant enzyme defense system and the radical scavenging capacity of plants under these circumstances.

In addition to using AMF as a biostimulant, researchers in agronomy and the biological sciences have shown great interest in the use of humic substances. Humic substances represent a major pool of organic carbon on the Earth’s surface. They are formed by chemical and biological transformations of plant and animal matter via microbial metabolism and comprise more than 60% of the soil organic matter (Stevenson 1994). Several studies in the literature support the idea that humic substances play a major role in regulating plant growth and development under both normal and stress conditions (Bulgari et al. 2019). Humic acids (HA) are humus materials that are soluble in aqueous alkaline solutions but precipitate under conditions of pH 1–2 (Hayes 2006). Previous studies investigating the effects of humic substances on plants have focused mainly on their effects on plant growth processes. Treating plants with HA has led to improvements in root growth (increasing weight and length) (Akinci et al. 2009); seed germination (Fathy et al. 2010); adventitious rooting (Baldotto and Baldotto 2014); shoot fresh weight, crown breadth, and number of leaves (Zhang et al. 2014); and nutrient and water uptake (Canellas and Olivares 2014; Canellas et al. 2015), and nitrogen and potassium concentrations and catalase activity (Aalipour et al. 2020). More specifically, potassium humate (KH) is a potassium salt of HA and a complex of humic substances. It is manufactured commercially by alkaline extraction of brown coal (lignite/leonardite) and is used mainly as a soil conditioner. The extraction is performed in water with the addition of potassium hydroxide, sequestering agents, and hydrotropic surfactants. In terms of the KH effects on plants, Valdrighi et al. (1996) reported that KH from compost increased shoot fresh weight in chicory, and Ibrahim and Ramadan (2015) noted that KH combined with micronutrients and chitosan increased yield in common beans. Although these studies represent an intense area of research, the role of the antioxidant defense system has still not been fully elucidated.

Nearly 80% of the world’s terrestrial plant species have mutualistic relationships with arbuscular mycorrhizal symbiosis in their roots (Wang and Qiu 2006). Russian olive (Elaeagnus angustifolia L.) is among the nitrogen-fixing species of plants (DeCant 2008). It may also increase the availability of soil nitrogen resources and microbial diversity in the rhizosphere (Yildiz et al. 2017). Moreover, ecological studies conducted on this woody species have generally dealt with revegetation (Espeland et al. 2017), afforestation restoration (Yildiz et al. 2017), potential distribution and limiting climatic factors (Zhang et al. 2018), and its status as a bird habitat (Mahoney et al. 2019). However, no studies to date have been conducted on ROS formation, ROS detoxification, or enzymes and antioxidant capacity under the combined effects of biostimulants such as AMF and humic substances in Russian olive (Elaeagnus angustifolia L).

Therefore, in the present study, we examined the ROS capacity and antioxidant defense system of the woody plant Elaeagnus angustifolia L. inoculated with AMF and KH. To the best of our knowledge, evidence is lacking on how the combination of more than one biostimulant affects the antioxidant system and radical scavenging capacity in plants. Consequently, this became the main aim of this study and we hypothesized that the simultaneous application of AMF and KH could improve the growth and antioxidant defense system in Russian olive. Therefore, in this investigation, the physiological and biochemical characteristics of E. angustifolia were evaluated in terms of growth parameters, relative water content, chlorophyll fluorescence, osmotic potential, lipid peroxidation level, hydrogen peroxide content, SOD, POX, CAT, APX, and GR antioxidant enzyme activity, total phenolic compounds, total flavonoid content, and antioxidant capacity values.

2 Material and Methods

2.1 Plant Material and Experimental Design

The Russian olive (Elaeagnus angustifolia L.; Fam: Elaeagnaceae) seeds used for this study were collected from Ereğli District, in the province of Konya, Turkey. The seeds were refrigerated at 4 °C before use and were sown in pots filled with a soil medium for growing seedlings (70% soil + 20% peat + 10% perlite). The soil was first sterilized for 2 h at 120 °C in an autoclave. The non-autoclaved soil was analyzed, and the initial properties of the non-autoclaved soil are presented in Table 1. Samples of non-autoclaved soil were air-dried, sieved for the < 2-mm size fraction, and prepared for analysis. Soil texture was determined via the Bouyoucos hydrometer method (Gee and Bauder 1986). Acidity was measured by a microprocessor pH meter (Hanna-HI 221) and electrical conductivity by a WTW-Inolab (Cond Level1) electrical conductivity (EC) meter. The total carbonate content was determined using a Scheibler pressure calcimeter (Loeppert and Suarez 1996). All samples were analyzed for their total C and N concentrations by means of dry combustion using a LECO Truspec CN-2000 analyzer (LECO Corporation, St. Joseph, MI, USA). The available concentrations of P, K, Ca, Mg, Na, Fe, Cu, Zn, and Mn were determined by an ICP-OES spectrometer (PerkinElmer Optima 7000 DV). For the extraction, exchangeable cations (K, Ca, Mg, Na) in 5-g samples were extracted using a standard 1 N ammonium acetate solution (pH 7.0) at an extracting ratio of 1:5 (w/v; soil to solution). Exchangeable P (in 2-g samples) was extracted with a 0.5 M sodium bicarbonate solution (pH 8.5). Extraction of exchangeable Fe, Cu, Zn, and Mn (in 10-g samples) was carried out using 0.005 M diethylenetriaminepentaacetic acid solution (pH 7.3). After extraction, the samples were shaken for 30 min and then filtered. Cation exchange capacities (CECs) were determined via NH4OAc extraction (Sumner and Miller 1996). The prepared plants were placed in a climate-controlled greenhouse (temperature 25/20 °C; relative humidity 70/65%; light intensity 350 μmol m−2 s−1; day/night 16/8 h). Each pot contained one seedling, and 50 seedlings were used for each experimental group: C (control), AMF (arbuscular mycorrhizal fungi), KH (potassium humate), and AMF + KH (arbuscular mycorrhizal fungi + potassium humate). For native AMF inoculation, the seeds were inoculated with mixed indigenous mycorrhizal spores (Funneliformis, Claroideoglomus) collected from the Central Anatolian Region of Turkey. The spores (500 spores per seed) were placed 50 mm below the seed in each pot. For the KH application, K-humate (Gubretas, Istanbul, Turkey) was used at a ratio of 1.5 g per 1 kg of seeds during sowing. We conducted this research using a completely randomized design, with four factors (non-treated control plants, AMF inoculation alone, KH application alone, and a combined treatment of AMF and KH) with three replications. Plants that were not treated with AMF or KH served as control plants (50 pots, 1 plant per pot). Plants were randomly harvested after 4 months of treatment and frozen immediately using liquid nitrogen (− 196 °C). The leaves were then stored at − 86 °C until further analyses.

Table 1 Physicochemical properties and nutrient concentrations of the soil used in the seedling pots
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2.2 Determination of Growth Parameters

Before harvesting the leaves, the seedlings were photographed and directly measured for growth parameters (leaf, root, and shoot lengths, leaf fresh, and dry weights). Ten random plants from each group were used for the growth analyses and were separated into shoot and root fractions. The lengths were measured using digital calipers. The fresh weights (FW) of the leaves were measured first, and afterwards, the samples were dried in an oven at 70 °C for 72 h and the dry weights (DW) were then determined.

2.3 Determination of Leaf Relative Water Content, Chlorophyll Fluorescence, and Osmotic Potential

The leaves (7 leaf disks from 10 different plants from each group) were harvested and their FW was determined. Then, the leaves were floated on dI-H2O for at least 10 h and blotting the turgid tissue dry prior to determining the turgid weight (TW). The DW was measured after drying the leaves at 70 °C for 72 h. The leaf relative water content (RWC) was calculated using the following formula (Smart and Bingham 1974):

$$ \mathrm{RWC}\ \left(\%\right)=\left[\left(\mathrm{FW}-\mathrm{DW}\right)/\left(\mathrm{TW}-\mathrm{DW}\right)\right]\times 100 $$

The chlorophyll fluorescence, indicating the quantum efficiency of the photosystem II, was measured and expressed as the Fv/Fm ratio. Before harvest, ten fully expanded leaves from each treatment group were used for the measurements. After the clipped leaves (from 10 different plants from each group) had been placed in the dark for 20 min, a chlorophyll fluorimeter (Plant Efficiency Analyzer, Hansatech, UK) was used for the measurements.

For determining osmotic potential (Ψs), the leaves (from 10 different plants from each group) were crushed with a glass rod and then centrifuged for 5 min. An osmometer (Wescor Vapro Pressure Osmometer 5600) was used to measure the clear supernatant in order to determine the Ψs of the leaves. The results obtained were then converted to megapascal (Santa-Cruz et al. 2002).

2.4 Determination of Lipid Peroxidation and Hydrogen Peroxide Contents

Thiobarbituric acid reactive substances (TBARS) were used for determining lipid peroxidation levels (Heath and Packer 1968). Leaf samples (0.5 g) were homogenized with 0.1% trichloroacetic acid (TCA), and after centrifugation, the supernatant was mixed with 20% TCA containing 0.5% thiobarbituric acid (TBA). The amount of TBARS was calculated using an extinction coefficient of 155 mM−1 cm−1.

For determining the accumulation of hydrogen peroxide (H2O2), the method described by Liu et al. (2000) was used. Leaf samples (0.5 g) were homogenized with TCA and 1.5 mL of reaction mixture containing 0.1% (v/v) TiCl4, and the resulting extract was centrifuged. The H2O2 content was determined using a standard curve prepared on a UV-VIS spectrophotometer (UV-VIS 1800 Shimadzu Co., Kyoto, Japan) at 410 nm, and the results were defined as l mol of H2O2 per g of FW.

2.5 Enzyme Extractions and Determination of Antioxidant Enzyme Activity

All enzyme extractions were performed at 4 °C. Leaf samples (0.5 g) were ground with liquid nitrogen for the enzyme activity assay and then homogenized in ice-cold 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 1% polyvinylpyrrolidone (PVP). For the APX activity assays, 2 mM ascorbate was added to the homogenization buffer. The supernatants were used after centrifugation (12,000×g for 30 min) for the determination of the protein content and enzyme activity. The total soluble protein content in the enzyme extract was assayed using bovine serum albumin (BSA) as the standard (Bradford 1976).

The SOD (EC.1.15.1.1) activity was measured by the ability to inhibit the photochemical reduction of nitro blue tetrazolium (NBT) at 560 nm (Beauchamp and Fridovich 1971). The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 0.1 mM EDTA, 13 mM methionine, 0.075 mM NBT, and 0.002 mM riboflavin with the enzyme extract. The reaction was initiated by adding the riboflavin and exposing the solution to 300 μmol m−2 s−1 irradiance for 10 min. A unit of SOD was defined as the amount of enzyme that inhibited 50% of the photoreduction rate of NBT.

The POX (EC.1.11.1.7) activity was determined as an increase in absorbance activity at 470 nm for 2 min with the oxidation of guaiacol (Mika and Lüthje 2003). The reaction mixture contained 25 mM sodium acetate buffer (pH 5.0), 10 mM guaiacol, and 10 mM H2O2. A unit of POX activity was defined as the amount required to decompose 1 μmol H2O2 per min with an absorbance coefficient of 26.6 mM−1 cm−1.

The CAT (EC 1.11.1.6) activity was estimated as the decrease in absorbance activity at 240 nm for 3 min (Aebi 1984). The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0) and 10 mM H2O2. A unit of CAT activity was defined as the amount needed to decompose 1 μmol H2O2 per min with an absorbance coefficient of 39.4 mM−1 cm−1.

The APX (EC 1.11.1.11) activity was measured as the activity decrease in absorbance at 290 nm for 3 min (Nakano and Asada 1981). The reaction mixture contained 50 mM sodium phosphate buffer (pH 7.0), 250 μM ascorbate, and 5 mM H2O2. A unit of APX was defined as the amount needed to oxidize 1 μmol ascorbate per min with an absorbance coefficient of 2.8 mM−1 cm−1.

The GR (EC 1.6.4.2) activity was determined as the decrease in absorbance activity at 340 nm for 3 min (Foyer and Halliwell 1976). The reaction mixture contained 50 mM Tris-HCl buffer (pH 7.6), 5 mM NADPH, and 10 mM oxidized glutathione (GSSG). A unit of GR was defined as the amount required to reduce 1 μmol GSSG per min with an absorbance coefficient of 6.2 mM−1 cm−1.

2.6 Determination of Total Phenolic Compound, Total Flavonoid Content, and Antioxidant Capacity Values

Three replicates of 0.1-g leaf samples were extracted with 10 mL of 80% methanol over ice in a sonication bath for 1 h. The methanolic extracts were filtered under vacuum through Whatman No. 1 filter paper, and the filtrate was immediately used for the determination of the total phenolic, flavonoid, and antioxidant capacity (AC) values (Grúz et al. 2011).

The total phenolic compound (TPC) content of the leaves was determined by the Folin-Ciocalteu (FC) method (Singleton and Rossi 1965), and the total flavonoid (TF) content was determined according to the AlCl3 colorimetric assay (Huang et al. 2004). For the TPC content determination, 500 μL of the extract and the same amount of deionized water were mixed with 2% Na2CO3 (w/v) and 2 N FC reagent. After 30 min at room temperature (25 °C), a blue-purple color was formed as a result of the reaction and the absorbance was measured at 750 nm. The TPC content was expressed as micrograms of gallic acid equivalents (GAE) per milligram extract.

For the TF content determination, a total of 1 mL reaction mixture was prepared by mixing the aqueous extract and 2% (w/v) AlCl3 (1:1) in methanol. This was incubated at room temperature for 30 min and the absorbance of the reaction mixture was then measured at 415 nm. The TF content was expressed as micrograms of quercetin equivalent (QE) per milligram extract.

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was measured according to the colorimetric assay described by Blois (1958). For the DPPH determination, 1 mL of DPPH solution (0.033 mg mL−1) was freshly prepared and mixed with 100 μL of the extract. The mixture was then incubated for 30 min in the dark. Absorbance of the mixture was measured at 520 nm. The results of these three antioxidant capacity values were expressed as nanomoles of trolox equivalent (TE) per milligram extract.

The ferric reducing antioxidant power (FRAP) assay was conducted according to the procedure described by Benzie and Strain (1999). The FRAP reagent was freshly prepared (300 mM acetate buffer (pH 3.6), 10 mM TPTZ (2,4,6-tripyridyl-s-triazine), and 20 mM FeCl-6H2O (10:1:1, v/v)) and kept at 37 °C. After that, 100 μL of each sample was mixed with 2900 μL of the FRAP reagent and the reaction mixture was incubated at 37 °C in the dark for 30 min. The absorbance of the mixture was measured at 593 nm against a blank.

The cupric ion reducing antioxidant capacity (CUPRAC) was determined following the method of Apak et al. (2004). For the CUPRAC determination, 1 mL of extract was mixed with 1 mL of CuCl2 (10 mM), 1 mL of acetate buffer (1 mM, pH 7.0), and 1 mL neocuproine (7.5 mM). The total reaction mixture was gently shaken and then incubated in the dark at room temperature. After 30 min, the absorbance was measured at 450 nm.

2.7 Statistical Analysis

The experimental design of the greenhouse experiment was completely randomized and consisted of four treatments (C, AMF, KH, AMF + KH) with fifteen plants each. The experiments were repeated three times independently, and each data point was the mean of three replicates (except growth parameters, RWC, chlorophyll fluorescence, and osmotic potential). The results were expressed as means, and error bars were used to show the standard error of the mean (± SEM). All data obtained were analyzed using factorial analysis of variance (ANOVA), and Tukey’s post-hoc multiple range test was used to compare the differences among all the treatments, with P < 0.05 considered as significantly different. Further, correlations between variables were performed using Pearson’s coefficients. The SPSS 22.0 (IBM™) software was used for all the analyses.

3 Results

3.1 Growth Parameters

To show the interaction between the biostimulants, AMF and KH were applied to the soil and the morphological differences in the Elaeagnus angustifolia are shown in Fig. 1. The lengths of leaves, roots, and shoots were significantly (P < 0.05) increased with AMF and KH applications compared with the control plants, except for the root length of plants treated with KH (Table 2). However, extreme increases in length were exhibited with the AMF + KH application compared with the control. Under AMF + KH treatment, leaf, root, and shoot lengths of E. angustifolia showed 2.2-, 2-, and 2.7-fold increases, respectively. With AMF alone, these increases in leaf, root, and shoot lengths were recorded as 2.2-fold, 62.2%, and 92.7%, respectively. On the other hand, interestingly, with the treatments used in this study, there was a reduction trend in leaf FW and DW. The FW of E. angustifolia was more affected by AMF, KH, and AMF + KH than the DW. There was a reduction (by 25%) in the DW of the plants treated with AMF alone, while no significant (P < 0.05) differences were determined in plants treated with KH alone or with AMF + KH. However, the leaf FW was reduced by 34.8, 17.4, and 30.4% with treatments of AMF, KH, and AMF + KH, respectively, as compared with control plants (Table 2).

Fig. 1
figure 1

Combined effect of arbuscular mycorrhizal fungi (AMF) inoculation and K-humate (KH) on 4-month growth of Elaeagnus angustifolia. C, control; KH, potassium humate; AMF, arbuscular mycorrhizal fungi; AMF+KH, arbuscular mycorrhizal fungi + potassium humate. Scale bar, 5 cm

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Table 2 Growth parameters of Elaeagnus angustifolia as affected by arbuscular mycorrhizal fungi inoculation and K-humate
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3.2 Relative Water Content, Chlorophyll Fluorescence, and Osmotic Potential

There were no substantial differences in RWC between the E. angustifolia control and treatment groups (Fig. 2a). Likewise, in this species, the AMF, KH, and AMF + KH had no significant (P < 0.05) impact on Fv/Fm (Fig. 2b). On the other hand, significant changes in E. angustifolia leaf osmotic potential were detected, which showed increases of 17.4, 23.1, and 10.7% with the AMF, KH, and AMF + KH, respectively, compared with the control plants (Fig. 2c).

Fig. 2
figure 2

Elaeagnus angustifolia treatment results. Combined effects of arbuscular mycorrhizal fungi (AMF) inoculation and K-humate (KH) at 4 months on (a) leaf relative water content (RWC); (b) chlorophyll fluorescence (Fv/Fm); (c) leaf osmotic potential. C, control; KH, potassium humate; AMF, arbuscular mycorrhizal fungi; AMF+KH, arbuscular mycorrhizal fungi + potassium humate. Values indicate mean ± SE; different letters in the same row indicate significant difference (P < 0.05) between each treatment based on Tukey’s multiple range test

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3.3 Lipid Peroxidation and Hydrogen Peroxide

TBARS content did not significantly (P < 0.05) change with the AMF alone or AMF + KH treatments; however, KH alone decreased the TBARS level by 24.4% compared with the control plants (Fig. 3a). Similar to TBARS, there were no significant changes in H2O2 content with the AMF treatment. However, the KH and AMF + KH treatments significantly reduced H2O2 accumulation by 33.1 and 17.9%, respectively (Fig. 3b).

Fig. 3
figure 3

Elaeagnus angustifolia treatment results. Combined effects of arbuscular mycorrhizal fungi (AMF) inoculation and K-humate (KH) at 4 months on (a) lipid peroxidation (as indicated by TBARS content) and (b) hydrogen peroxide (H2O2). C, control; KH, potassium humate; AMF, arbuscular mycorrhizal fungi; AMF+KH, arbuscular mycorrhizal fungi + potassium humate. Values indicate mean ± SE; different letters in the same row indicate significant difference (P < 0.05) between each treatment based on Tukey’s multiple range test

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3.4 Antioxidant Enzyme Activities

In this study, activities of antioxidant enzymes, which play a key role in cell ROS homeostasis, were measured to reveal the reaction of E. angustifolia with fungal and humic substances (Fig. 4). The SOD activity increased with AMF (76.5%) and KH (2.4-fold) treatments, but the highest constitutive level of leaf SOD activity was determined at 3.1-fold in the AMF + KH soil-treated plants (Fig. 4a).

Fig. 4
figure 4

Elaeagnus angustifolia treatment results. Combined effects of arbuscular mycorrhizal fungi (AMF) inoculation and K-humate (KH) at 4 months on (a) superoxide dismutase peroxidase (SOD); (b) peroxidase (POX); (c) catalase (CAT); (d) ascorbate peroxidase (APX); (e) glutathione reductase (GR). C, control; KH, potassium humate; AMF, arbuscular mycorrhizal fungi; AMF+KH, arbuscular mycorrhizal fungi + potassium humate. Values indicate mean ± SE; different letters in the same row indicate significant difference (P < 0.05) between each treatment based on Tukey’s multiple range test

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The same trend of increasing with treatments was not observed with the POX activity (Fig. 4b). The KH treatment alone caused an activity increase of 28.3%, while AMF and AMF + KH decreased the POX activity in the E. angustifolia leaves by 34.8 and 65.2%, respectively, compared with the control plants.

Unlike SOD activity, CAT activities were reduced with the fungal and humate treatments (Fig. 4c). The highest decrease in CAT activity (70.8%) compared with the controls was determined with the AMF + KH treatment. Moreover, the AMF application alone caused a slight decrease (16.7%) in CAT activity, while the KH application alone decreased the activity by 41.7%.

The APX activity was sharply reduced in response not only to the AMF and KH applications but also to the simultaneous application of AMF and KH (Fig. 4d). The APX activity was noticeably decreased in the AMF-, KH-, and AMF + KH-treated E. angustifolia, by 89.5, 87.8, and 90.7%, respectively.

Unlike for APX activity, the AMF and KH applications resulted in increases in the activity of the GR enzymes (Fig. 4e). The KH alone displayed the highest GR activity (57.5-fold) in comparison with the control plants. Furthermore, AMF alone and AMF + KH also increased the GR activity by 6.5- and 16-fold compared with non-treated plants.

3.5 Total Phenolic Compound, Total Flavonoid Content, and Antioxidant Capacity Values

The TPC and TF contents of E. angustifolia treated by AMF and KH alone or their combination were determined as polyphenolic content, and the results are presented in Table 3. Moreover, the AC was evaluated by measuring the CUPRAC, DPPH, and FRAP (Table 3). The AMF + KH application resulted in significantly (P < 0.05) higher TPC content and AC values, while the TF content, interestingly, showed a decrease with all treatments. The KH alone and in combination with AMF exhibited a slightly lower TF content (by 28.1%) than with AMF alone (by 14.1%) when compared with the controls. Although single applications of AMF and KH induced increases in the TPC content and AC values, the highest TPC and AC results were obtained from the combination of AMF and KH. Under control conditions, the highest antioxidant capacity values were measured using DPPH assay. Similar to this result, the AMF + KH application increased the DPPH values by 24.6-fold compared with the control plants. On the other hand, 4- and 4.1-fold increases were observed in the CUPRAC and FRAP values with the combination of the fungus and K-humate. Significant (P < 0.05) TPC content level increases were detected (2.8-fold) with the AMF + KH-treated soil.

Table 3 Total phenolic compounds, total flavonoid contents, and antioxidant capacity values (CUPRAC, DPPH and FRAP) of Elaeagnus angustifolia affected by arbuscular mycorrhizal fungi inoculation and K-humate
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3.6 Statistical Analysis of Data

The correlation analysis evidenced significant relationships between the parameters analyzed in Russian olive plants subjected to the single and combined treatments of AMF and KH (Table 4). Significantly high positive correlations were observed within vegetative organ length (leaf, root, and shoot; range, r = 0.734 to 0.876; P < 0.01) and negative correlations between lengths and LFW (range, r = − 0.500 to − 0.676; P < 0.01). The LL, which was more stimulated than root length by the combined application of AMF + KH, significantly positively correlated with RL and SL (r = 0.734 and 0.826, respectively; P < 0.01), whereas negatively correlated with LFW and LDW (r = − 0.676 and − 0.466, respectively; P < 0.01). LFW displayed a positive correlation with LDW, CAT enzyme activity, and TF content (r = 0.757, 0.650, and 0.867, respectively; P < 0.01); however, LDW positively correlated only with TF (r = 0.759; P < 0.01). When TBARS showed a positive correlation with only H2O2 (r = 0.732; P < 0.05), H2O2 level of E. angustifolia leaves positively correlated with the CAT, POX, and TF contents and negatively correlated with TPC, CUPRAC, DPPH, and FRAP. The activity of SOD in leaves displayed negative correlation with APX, CAT, POX, and TF (r = − 0.784, − 0.826, − 0.830 and − 0.904, respectively; P < 0.01 and P < 0.05), whereas it positively correlated with GR, TPC, CUPRAC, DPPH, and FRAP (r = 0.835, 0.838, 0.819, 0.894 and 0.863, respectively; P < 0.01 and P < 0.05). APX activity did not show any correlations with other parameters, but positively correlated with TF (r = 0.768; P < 0.05). CAT and POX activities exhibited strong negative correlations with TPC, CUPRAC, DPPH, and FRAP (range for CAT, r = − 0.825 to − 0.878; P < 0.01 and range for POX, r = − 0.731 to − 0.778; P < 0.01). The activity of GR positively correlated with TF content (r = 0.611; P < 0.05). TPC, CUPRAC, DPPH, and FRAP were significantly high positively correlated with each other (range, r = 0.990 to 0.997; P < 0.01). However, TF content exhibited negative correlations with them.

Table 4 Correlations between variables determined using Pearson’s correlation coefficients (r) of Elaeagnus angustifolia as affected by arbuscular mycorrhizal fungi and K-humate
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4 Discussion

In addition to feeding the growing world population, plant studies aim to develop varieties more resistant to undesirable negative conditions as well as to reduce the inputs for sustainable agriculture. Therefore, in recent years, studies have been increasingly focused on the use of biostimulants (Calvo et al. 2014; Rouphael and Colla 2018). Although studies have generally concentrated on the effects of a single biostimulant on growth and development, studies on the effects of more than one biostimulant are limited. Because of this, the combined effects of AMF and KH as biostimulants on the woody plant Russian olive were emphasized in this study. In addition, the changes in the antioxidant capacity and enzyme activities with combined biostimulant applications were investigated under non-stress conditions. Moreover, AMF and KH applications were found to induce increases in the antioxidant defense system and to considerably increase antioxidant capacity.

Biotic and abiotic factors form the richness of the soil in terms of rhizosphere characteristics. These characteristics determine the physiological performance of plants. The AMF as a biotic factor and the KH as an abiotic factor were both applied to Russian olive, and the beneficial effects of both treatments, either singly or combined, on physiological and biochemical parameters were detected in this work. The effects of AMF and humic substances have been well documented separately for their growth promotion ability (Berruti et al. 2016; Canellas and Olivares 2014; Caser et al. 2019; Ertani et al. 2019; Nardi et al. 2016; Rouphael et al. 2015; Wang et al. 2019). Growth is a significant parameter for plants. In the current study, AMF and KH treatments exhibited a significant positive correlation (Table 4) with growth by enhancing vegetative organ length. Similarly, positive correlations between growth induction and biostimulant application and improvement in growth have been previously reported (Akinci et al. 2009; Wu et al. 2013). In contrast to the increased length, the FW of Russian olive was reduced by biostimulant treatment with both single and combined applications of AMF and KH. The reduction in leaf FW recorded in this study is in agreement with the findings of Asli and Neumann (2010), but not with those of Valdrighi et al. (1996), Wu et al. (2013), or Zhang et al. (2014). This decrease in FW with both biostimulant treatments of Russian olive may be related the excessive chemical activity of small growth regulatory biomolecules released from humic acid agglomerates after interactions with organic acids in the rhizosphere (Canellas et al. 2008; Muscolo and Sidari 2009). Although the roots of E. angustifolia were more directly exposed to AMF and KH than the leaves, these treatments caused reduction in leaf FW but did not reduce vegetative organ length.

Leaf RWC is one of the main indicators of plant water status and, like blood in humans, indicates the health status of the plant. Moreover, changes in the chlorophyll fluorescence of the photosynthetic parameters, expressed as Fv/Fm, indicate the effects of stress on the PSII mechanism and are also good indicators of plant health. In this study, neither the RWC nor the Fv/Fm ratio changed with the single or combined AMF and KH treatments. These results led us to think about the positive role of biostimulants in maintaining RWC and photosynthetic efficiency, and with these outcomes confirmed the findings of previous studies (Ozfidan-Konakci et al. 2018; Yang et al. 2015). On the other hand, leaf osmotic potential in both mycorrhizal- and KH-treated plants increased with the single and the combined applications; however, it was increased more with single applications of AMF and KH than with their combination. The similar alleviation with humic acid detected by Ozfidan-Konakci et al. (2018) could be related to maintaining the water uptake and content. Furthermore, in the same study, the water content in wheat leaves showed almost no effect when treated with HA alone, whereas osmotic potential and proline accumulation increased (Ozfidan-Konakci et al. 2018). As a result, as with RWC, the biostimulant treatments protected the water status of the leaves, in addition to possibly increasing osmolytes (low-molecular weight organic compounds). Proline is the best known of these osmolytes. Proline may be accumulated for the maintenance of the leaf water content, thus enabling the plant cells to withstand dehydration by maintaining turgor, buffering against ROS, and maintaining redox homeostasis (Szabados and Savouré 2010; Zegaoui et al. 2017). Besides, keeping leaf water content by AMF and KH-inoculated plants may also improve root water uptake and enhance stomatal control in plants.

When plants encounter undesirable environmental conditions, cellular homeostasis and cell membrane structures change (Couchoud et al. 2019; Gigon et al. 2004; Miller et al. 1971). As a result of this process, ROS such as H2O2, O2·−, 1O2, and OH. are produced. Meanwhile, the antioxidant defense system starts to protect cell membranes against lipid peroxidation, an indicator of oxidative damage (Ashraf and Foolad 2007). In our study, a correlation (r = 0.732; P < 0.05) between H2O2 accumulation and TBARS level was observed. In addition, H2O2 accumulation and lipid peroxidation were significantly lowered by KH treatment. Russian olive plants receiving combined mycorrhizal and KH treatment also had lower H2O2 content. However, their TBARS level reached that of the non-treated control plants. These results are consistent with the findings obtained by Yang et al. (2015) and Ozfidan-Konakci et al. (2018) in AMF-treated Robinia pseudoacacia and humic acid-treated wheat plants, respectively. This means that biostimulant applications of the AMF and KH treatments in this study might contribute to the alleviation of oxidative damage and may balance ROS production to play protective role in plant cells.

Recent studies have clearly demonstrated that ROS production occurs not only under stress. Mittler et al. (2004) reported that because of their dual role, ROS are important signal molecules even under non-stress conditions. Various antioxidant enzymes such as SOD, POX, CAT, APX, and GR are responsible for reducing excessive ROS production in plant cells. In this study, biostimulant applications induced intense changes in the antioxidant defense system enzyme activities. Both single applications and a combination of AMF and KH induced antioxidant enzyme activities; however, activity changes were more pronounced with the application of the combined biostimulants. One of these antioxidant enzymes is SOD, which is considered as the first defense against ROS production and catalyzes the dismutation reaction of O2·− into molecular oxygen (O2) and H2O2 (Hossain et al. 2009; Yang et al. 2015). The SOD activity increased in all biostimulant treatment groups, but more so in the combination of AMF and KH. This means that H2O2 production was higher than with the two other applications. Increases in SOD activity were also observed in Robinia pseudoacacia (Yang et al. 2015) and corn (Kazemi et al. 2019) treated with AMF and wheat treated with humic acid (Ozfidan-Konakci et al. 2018). However, in the current study, the H2O2 level detected was lower than in the control plants at that point. This result could be connected to the strong antioxidant defense in Russian olive leaves. Toxic levels of H2O2 were eliminated with the increase of the enzymatic activities of POX, CAT, APX, and GR or the non-enzymatic activities of ascorbate and glutathione (Mittler et al. 2004). In this study, H2O2 accumulation was eliminated by increased GR activity in both the mycorrhizal- and the K-humate-treated Russian olive plants. The GR converts GSSG to reduced glutathione (GSH) and scavenges H2O2 through the ascorbate-glutathione cycle (Gill et al. 2013; Santos et al. 2019). Moreover, an increase in GR activity contributes to a decrease in TBARS accumulation. It also helps in overcoming metabolic injuries from the radical scavenging capacity of the enzymes. These results may be attributed to the reduction in TBARS and H2O2 and could be connected with the protection of membrane permeability. The major scavengers of H2O2 are CAT and POX, although POX has a greater affinity to H2O2 than CAT (Jimenez et al. 1997; Yang et al. 2015). In this study, an increase in POX activity was exhibited with the KH treatment alone, correlating with the increased reduction of H2O2. Similar POX activity stimulation by humic substances was reported by Muscolo et al. (1993). This suggests that in Russian olive leaves, KH has a greater ability to protect cellular homeostasis than AMF. Thus, the combined effect of AMF and KH can provide clues for improving plant antioxidant defense systems. The system in the Russian olive leaves in this study was improved by SOD and GR activities, but not by POX, CAT, or APX activities. The reduced activities of CAT and APX in the Russian olive leaves might be attributed to the reduction of H2O2 content under the combined effects of AMF ad KH. According to Cordeiro et al. (2011), the accumulation of ROS is required for root growth. This suggests that the increase in root length of this woody species may be related to the increasing activity of CAT and the ROS content in the Russian olive roots. Therefore, to support this possible mechanism, more studies should be carried out to investigate roots and the combination of these biostimulants.

In the present study, a linear relationship (R2 = 0.908, P < 0.05) was observed between TPC content and antioxidant capacity (CUPRAC, DPPH, and FRAP). Russian olive exhibited the best antioxidant activity with the combination of AMF and KH. In particular, in addition to high SOD and GR activities, extreme accumulation of TPC content and CUPRAC, DPPH, and FRAP levels under both AMF and KH-treated plants may be more effective in reducing ROS levels. The studies involving antioxidant capacity values detected by CUPRAC, DPPH, and FRAP assays are limited. Despite this, similar significant increases in phenolics and antioxidant capacity values have been detected in AMF-treated lettuce (Avio et al. 2017), Libidibia ferrea (Dos Santos et al. 2017), and saffron (Caser et al. 2019). They have also been found in KH-treated kale (Abbey et al. 2018) and Plectranthus spp. ‘Nicoletta’ (Zhang et al. 2017), as well as in leonardite-humate-treated maize (Ertani et al. 2019). This huge enhancement in antioxidant capacity revealed in the studies might indicate that with combined treatment using these biostimulants, Russian olive could be a potential natural antioxidant supplement. Similarly, Sbrana et al. (2014) reported that AMF symbiosis could induce changes in plant secondary metabolism. These changes led to the enhanced biosynthesis of phytochemicals with health-promoting properties, such as polyphenols, carotenoids, flavonoids, and phytoestrogens. On the other hand, apart from the increase in TPC and antioxidant capacity values, the total flavonoid content decreased in the Russian olive leaves. Flavonoids are secondary metabolites derived from the phenylpropanoid pathway. They have been shown to accumulate in the plant host rooting media during the process of mycorrhization (Abdel-Lateif et al. 2012). Accumulation of flavonoid-stimulating arbuscular mycorrhiza formation has also been reported in melon (Akiyama et al. 2002), Medicago sativa (Larose et al. 2002), and Trifolium repens roots (Ponce et al. 2004). The reduction of total flavonoids in the Russian olive leaves may have been the result of their accumulation in the roots with AMF alone and with the combined AMF and KH treatment. Root flavonoid contents might also be considered and measured in further studies.

5 Conclusions

Our results indicated that a combination of arbuscular mycorrhizal fungus and potassium humate is involved in growth parameters, especially in the vegetative organ length, antioxidant capacity, and enzyme activities. In Russian olive, the mycorrhizae and humic substances when applied together greatly enhanced the defense system by increasing the antioxidant capacity and antioxidant enzymes. Both the single and the combined applications of arbuscular mycorrhizal fungus and potassium humate induced growth increases and maintained leaf water status and chlorophyll fluorescence. In addition, the application of both biostimulants combined decreased the hydrogen peroxide content in the leaf cells and strengthened cell membranes by alleviating accumulation of thiobarbituric acid reactive substances. Enzymatic defense was induced by increasing the activation of superoxide dismutase and glutathione reductase via the combined presence of mycorrhizae and potassium humate. Moreover, in Russian olive, under both applications, significantly marked changes in antioxidant capacity were determined by the cupric ion reducing antioxidant capacity, ferric reducing antioxidant power, and 2,2-diphenyl-1-picrylhydrazyl radical scavenging activities. Therefore, our findings potentially suggest that the application of mycorrhizae and/or potassium humate can stimulate plant growth with increasing antioxidant system, and moreover, a combined application was seen to be more effective.