**Biostimulants Application Alleviates Water Stress E**ff**ects on Yield and Chemical Composition of Greenhouse Green Bean (***Phaseolus vulgaris* **L.)**

**Spyridon A. Petropoulos 1,\*, Ângela Fernandes 2, Sofia Plexida 1, Antonios Chrysargyris 3, Nikos Tzortzakis 3, João C. M. Barreira 2, Lillian Barros <sup>2</sup> and Isabel C. F. R. Ferreira 2,\***


Received: 23 December 2019; Accepted: 25 January 2020; Published: 27 January 2020

**Abstract:** The increasing scarcity of water demands proper water management practices to ensure crop sustainability. In this study, the effect of drought stress and biostimulants application on the yield and chemical composition of green pods and seeds of common bean (*Phaseolus vulgaris* L.) was evaluated. For this purpose, four commercially available biostimulant products, namely Nomoren (G), EKOprop (EK), Veramin Ca (V), and Twin-Antistress (TW), were tested under two irrigation regimes: normal irrigation (W+) and water-holding (W-) conditions. The highest increase (20.8%) of pods total yield was observed in EKW+ treatment due to the formation of more pods of bigger size compared to control treatment (CW+). In addition, the highest yield under drought stress conditions was recorded for the GW- treatment (5691 ± 139 kg/ha). Regarding the effects of biostimulants on the protein and ash content of pods, the application of VW+ treatment (first harvest of pods; 201 ± 1 and 79 ± 1 g/kg dw for proteins and ash content, respectively) and GW+ (second harvest of pods; 207.1 ± 0.1 and 68.4 ± 0.5 g/kg dw for proteins and ash content, respectively) showed the best results. For seeds, the application of GW+ treatment resulted in the highest content for fat, protein, and ash content (52.7 ± 0.1, 337 ± 1, 56 ± 1 g/kg dw) and energetic value (5474 ± 3 kcal/kg dw). γ-tocopherol was the main detected tocopherol in pods and seeds, and it was significantly increased by the application of TWW- (first harvest of pods; 6410 ± 40 μg/kg dw), VW- (second harvest of pods; 3500 ± 20 μg/kg dw), and VW+ (seeds; 39.8 ± 0.1 g/kg dw) treatments. EKW- treatment resulted in the lowest oxalic acid content for both pod harvests (26.3 ± 0.1 g/kg dw and 22.7 ± 0.2 g/kg dw for the first and second harvest of pods, respectively) when compared with the rest of the treatments where biostimulants were applied, although in all the cases, the oxalic acid content was considerably low. Fructose and sucrose were the main sugars detected in pods and seeds, respectively, while the highest content was recorded for the TWW- (first harvest of pods) and GW- (second harvest of pods and seeds) treatments. The main detected fatty acids in pods and seeds were α-linolenic, linoleic, and palmitic acid, with a variable effect of the tested treatments being observed. In conclusion, the application of biostimulants could be considered as an eco-friendly and sustainable means to increase the pod yield and the quality of common bean green pods and seeds under normal irrigation conditions. Promising results were also recorded regarding the alleviation of negative effects of drought stress, especially for the application of arbuscular mycorrhizal fungi (AMF; G treatment), which increased the total yield of green pods. Moreover, the nutritional value and chemical composition of pods and seeds was positively affected by biostimulants application, although a product specific effect was recorded depending on the irrigation regime and harvesting time (pods and seeds).

**Keywords:** arbuscular mycorrhizal fungi; biofertilizers; common bean; *Glomus* spp.; organic acids; pod quality; seaweed extracts; seed quality; tocopherols; total sugars

#### **1. Introduction**

The increasing concerns for food security in a rapidly growing world population has rendered the necessary intensification of agricultural production for the achievement of higher crop yield and total production. Protected vegetables cultivation is the most intensified cropping system and requires high amounts of fertilizers and pesticides [1,2]. However, despite the fact that higher fertilizer rates result in increased total yield, this practice is not always favorable when the quality of the final product is also considered. On the contrary, it is very common for excessive fertilization to stimulate vegetative growth and increase susceptibility to pathogens, which may result in increased product losses, as well as high nutrient losses due to leaching [3].

In addition, the increasing scarcity of water availability for human activities and irrigation in particular is a worldwide phenomenon and demands appropriate water management practices to ensure crop sustainability and economic activities related to water, especially in semi-arid and arid regions [4]. The use of biostimulants can diminish effects of environmental abiotic stress factors such as water stress, improve soil water-holding capacity and root conformation, and increase root growth with beneficial effects on nutrient and water use efficiency and yield; hence, the past decade has witnessed tremendous growth in the use of biostimulants in the farming sector [5–7]. The use of biostimulants containing arbuscular mycorrhizal fungi (AMF), saprophytic fungi, or algae extracts is considered an environmental friendly technique for the alleviation of adverse impact of osmotic stress, by increasing wáter and the nutrient uptake of crops and tolerance to biotic and abiotic stress [8–10].

Plant biostimulants usually consist of amino acids and peptide mixtures [11]. They also contain a wide number of bioactive compounds that are able to improve various physiological processes that stimulate plant growth and increase nutrient use efficiency without adverse effects on crop yield and final product quality, while at the same time reduce chemical fertilizers inputs [5,12]. However, the effect of biostimulants may differ from species to species, while it greatly depends on environmental factors during and after application, as well as on the dose and time of application [13,14]. For example, the application of saprophytic fungi (*Trichoderma harzianum* ALL-42) was associated with increased shoot biomass production and the number of lateral shoots in *Phaseolus vulgaris* plants due to the beneficial effects of root colonization by fungi on plant root growth [15]. On the other hand, seaweed extracts (*Ascophyllum nodosum*) increased the plant growth and overall yield of leafy vegetables such as spinach and lettuce [16–18], while in bean plants, the application of extracts enhanced root growth and plant development, especially when water stress conditions were imposed [19]. The biostimulatory activity of symbiotic bacteria such as *Bacillus* sp. is mostly associated with adaptation mechanisms for improved water retention though alterations in plant cell wall composition and hormones production (e.g., indole-3-acetic acid (IAA)) [6]. Therefore, environmental friendly methods such as applying biostimulants for stimulating early growth in vegetable crops and ensuring high yields are innovative agricultural practices that have to be further investigated in order to improve our understanding of their functions and the involved mechanisms of action [20].

Common bean (*Phaseolus vulgaris* L.) is a drought-sensitive vegetable crop, and water stress may have a detrimental effect on crop yield [21] and the chemical composition of pods and seeds [22,23]. So far, there is limited literature regarding the use of biostimulants on common bean plants, whereas various studies have tested the effects of biostimulants on other legume species, especially under drought stress conditions. In particular, the application of *Pseudomonas aeruginosa* GGRJ21 strain on mung bean (*Vigna radiata* (L.) R. Wilczek) under greenhouse and field conditions up-regulated the expression of drought stress-responsive genes, which resulted in better plant growth and development under water stress conditions [24]. Foliar application of amino acids on faba beans (*Vicia faba* L.)

subjected to salt stress showed significant ameliorative effects that were mainly associated with the use of amino acids as carbon and nitrogen pools, which further increased photosynthetic apparatus efficiency [25]. Kumar et al. [26] reported the synergistic effects of *Pseudomonas putida* and *Bacillus amyloliquefaciens* on chickpea plants subjected to water stress through the evaluation of several traits, including the activation of plant defense and soil enzymes and plant growth parameters. Moreover, the inoculation of common bean plants with *Azospirillum brasilense* altered root morphology, which allowed plants to overcome water stress without increasing plant biomass compared to non-inoculated plants [27]. In another study, Klimek-Kopyra et al. [28] suggested that biostimulants application on seeds of seven winter pea cultivars (*Pisum sativum* L.) may increase frost tolerance through the increased germination percentage and growth rate of seedlings, although a varied response depending on biostimulant x cultivar combination was observed. In contrast, Galvão et al. [19] suggested that the application of *Bacillus amyloliquefaciens BV 03* and/or the combination of *B amyloliquefaciens BV 03* with *A. nodosum* extracts did not alleviate water deficit effects on common bean plants. According to Dourado-Neto et al. [29] the use of hormones with biostimulant activity (combination of kinetin, indole butyric acid, and gibberellic acid) on common bean plants through seed treatment, sowing, or foliar spraying may increase the number of grains per pod and grains yield. Moreover, licorice root extracts may have a beneficial role on mitigating the negative effect of salt stress on *P. vulgaris* plants' growth and yield, as well as on the total soluble carbohydrates, soluble sugars, and nutrients content [30]. The combined application of salicylic acid and *Moringa oleifera* leaves extracts has been also reported to mitigate salinity stress effects on common bean plants through the improvement of green pods and seeds yield and the physicochemical characteristics of pods and seeds [31]. Other examples of biostimulants use on common beans crop include the application of *Lolium perenne* foliage extracts as potent cell defense elicitors [32] and the positive effect of aqueous extracts of moringa leaves and garlic cloves on the yield and chemical composition of snap beans [33,34].

Most of the studies regarding the mitigating effects of biostimulants to abiotic stressors refer to high salinity or nutrient deficiency stress. The main goal of this study was to record the effects of natural biostimulants on the yield, nutritional value, and chemical composition under drought conditions. For this purpose, a drought-sensitive species, namely the common bean (*P. vulgaris*), was selected and grown in a protected environment under water stress conditions, and the use of commercially available biostimulants products was evaluated as an environmentally friendly and sustainable method for increasing the yield and quality of end-products through the improvement of the chemical composition of the final products (pods and/or seeds) without compromising yield.

#### **2. Materials and Methods**

#### *2.1. Plant Material and Growing Conditions*

The experiment was carried out during the growing period of summer–autumn 2017. Sowing took place on 11 August 2017 and seeds of bean (*Phaseolus vulgaris* L.) were sown directly in soil within the unheated plastic greenhouse at the experimental farm of the University of Thessaly, Greece. Seeds were sown in double rows with a spacing of 50 cm between the rows, and the plant density was 2 plants/m<sup>2</sup> (20,000 plants/ha), while each treatment consisted of 6 plants and was replicated three times (180 plants in total). The soil at 0–30 cm depth was clay (26% sand, 32% silt, and 42% clay); pH: 8.0 (1:1 soil/H2O); organic matter content: 3.1%; CaCO3: 10.8%; available P (Olsen method): 70.9 mg/kg; total N (Kjeldahl method): 1.8 g/kg; exchangeable K2O (ammonium acetate method): 195 mg/kg; electrical conductivity (ECe): 0.95 dS/m. The growth conditions throughout the experimental period are presented in Figure 1. Two factors were applied in a split-plot factorial design, namely water stress and biostimulants. Biostimulants treatments included: (1) Control (C: no biostimulants added), (2) Nomoren (G; Anthis S.A., Greece) (3) Twin-Antistress (TW; Microspore Hellas–Sacom Hellas, Greece), (4) Veramin Ca (V; Microspore Hellas–Sacom Hellas, Greece), and (5) EKOprop (EK; Anthesis S.A., Greece). Regarding the detailed composition of each product, Nomoren contains 20% arbuscular

mycorrhizal fungi (AMF) (*Glomus* spp.). Twin-Antistress contains natural microorganisms based on *Bacillus subtilis*, as well as yeast and *Ascophyllum nodosum* extracts, as well as N (organic): 1%, organic carbon: 10%, and organic matter (<50 kDa): 30%. Veramin Ca contains an amino acid complex of vegetable origin with *Aloe vera* extract, and CaO: 15.6%. EKOprop contains a mixture of arbuscular mycorrhizal fungi (*Glomus* spp: 1%), rhizospere symbiotic bacteria (*Bacillus* spp., *Streptomyces* spp., *Pseudomonas* spp.,: 1.6 × 109 CFU/g in total), and saprophytic fungi (*Trichoderma* spp.: 5 × 105 CFU/g) (Table 1).

**Figure 1.** Environmental conditions (mean, max, and min temperature and mean relative humidity (RH)) throughout the experimental period.

Water stress treatments were previously described by the authors and scheduled with the use of tensiometers (Irrometer-Moisture Indicator, Irrometer, Riverside, CA) including: (a) normally irrigated plants (W+) where irrigation was applied approximately twice a week and when tensiometer readings were between 10% and 15%, and (b) water-stressed plants (W-) where water holding was applied with irrigation being implemented approximately once a week and when tensiometer readings were between 40% and 50% [35,36]. Tensiometer readings are percent levels that correspond to soil moisture content ranging from field capacity (0%) to dry soil (100%). Irrigation was applied through a drip irrigation system with one dripper per plant and a water flow rate of 4.0 L/h for each dripper. The total amount of irrigation water was 350 m3/ha (17.5 L per plant) for normally irrigated plants and 210 m3/ha (10.5 L per plant) for water-stressed plants. Biostimulants were applied according to the directions for use of each product at 10, 20 and 30 days after sowing (DAS) as following: (G) was applied with irrigation water at 5 L/ha for each dose; (TW) was applied with irrigation water 5 L/ha for each dose; (V) was applied with foliar spraying at 500 g/100 L H2O for each dose; and (EK) was applied with irrigation water at 1 kg/ha for each dose. Water holding started after the second application of biostimulants (21 DAS). The harvest of green pods took place at marketable maturity at 60 DAS (first harvest) and 70 DAS (second harvest), while seeds were collected from fully mature green pods at 103 DAS. All harvests were carried out on the same plants. After harvest, the fresh and dry weight of pods, as well the fresh and dry weight of seeds, number of seeds per pod, and 100 seeds weight were recorded. The number of seeds per pod and the weight of 100 seeds was evaluated from 10 pods for each plot (30 pods per treatment). Batch samples of pods and seeds were put in deep-freezing conditions, lyophilized, ground with a mortar and pestle, and stored at freezing conditions (−80 ◦C) until further analyses.


**Table 1.** Detailed composition and application guides for the tested biostimulants.

#### *2.2. Chemical Analyses*

#### 2.2.1. Nutritional Value

Sample were analyzed in terms of nutritional compounds (moisture, fat, ash, proteins, and carbohydrates) according to the Association of Analytical Communities (AOAC) methods [37]. Moisture was determined by pods and seeds drying at 105 ± 5 ◦C until constant weight. Crude protein was evaluated by the macro-Kjeldahl method (N × 6.25) using an automatic distillation and titration unit (model Pro-Nitro-A, JP Selecta, Barcelona, Spain), ash content was determined by incineration at 550 ± 15 ◦C, and the crude fat was determined by extraction with petroleum ether using a Soxhlet apparatus (Behr Labor Technik, Dusseldorf, Germany). Total carbohydrates (g/kg dw) were determined by difference according to the equation: 1000 – (g moisture + g fat + g ash + g proteins), and energy (kcal/kg dw) was determined according to the equation: 4 × (g proteins + g carbohydrates) + 9 × (g fat).

#### 2.2.2. Minerals Composition

Mineral composition analysis was performed in forced-air dried (at 72 ◦C) and ground to powder pods and seeds, after dry ashing and extraction with 2 N HCl according to the method described by Chrysargyris et al. [38]. Atomic absorption spectrophotometry (PG Instruments AA500FG, Leicestershire, UK) was used for Ca, Mg, Mn, Zn, and Cu content determination, while flame photometry (Lasany Model 1832, Lasany International, Haryana, India) was used for and Na and K content determination. Nitrogen and phosphorus content were determined by Kjeldahl (Digest Automat K-439 and Distillation Kjelflex K-360, BÜCHI, Flawil, Switzerland) and spectrophotometry methods (Multiskan GO, Thermo Fisher Scientific, Waltham, Massachusetts, USA), respectively. The determination of minerals composition was performed only in pods of the second harvest and seeds. Pods of the first harvest were not evaluated due to insufficient amounts of samples for specific treatments, which did not allow a complete set of data. Results are expressed on a dry weight basis.

#### 2.2.3. Tocopherols

Tocopherols were determined in the lyophilized samples by HPLC fluorescence, following a procedure previously described using tocol (Matreya, Pleasant Gap, Pensylvania, USA) as internal standard [39]. Tocopherols standards (α-, β-, γ-, and δ-isoforms, Sigma-Aldirch, St. Louis, MO, USA) were used for compounds identification, and quantification was assessed by the internal standard method. Results were obtained using the Clarity 2.4 software (DataApex, Prague, Czech Republic) and expressed in μg/kg dw and mg/kg dw for pods and seeds, respectively.

#### 2.2.4. Organic Acids

Organic acids were determined in the lyophilized sample and determined by a high-performance liquid chromatography system equipped with a diode array detector (HPLC-DAD), following a procedure previously described [40]. Compounds were identified and quantified by comparison of the retention time, spectra, and peak area recorded at 245 nm and 215 nm (for ascorbic acid and remaining acids, respectively), with those obtained from commercial standards (oxalic, malic, fumaric, and ascorbic acids, Sigma-Aldrich, St. Louis, MO, USA). The results were recorded and processed using LabSolutions Multi LC-PDA software (Shimadzu Corporation, Kyoto, Japan) and were expressed in g/kg dw and mg/kg dw for pods and seeds, respectively.

#### 2.2.5. Free Sugars

Free sugars were determined by HPLC coupled to a refractive index (RI) detector (Knauer, Smartline system 1000, Berlin, Germany) using the internal standard (IS; melezitose). The lyophilized sample was extracted using a methodology previously described [40]. Compounds were identified by comparison with standards (Sigma-Aldrich, St. Louis, MO, USA), and quantification was performed by the IS method. Results were processed using the Clarity 2.4 software (DataApex, Prague, Czech Republic) and expressed in g per kg dw.

#### 2.2.6. Fatty Acids

Fatty acids profile was characterized after a transesterification procedure and according to the method previously described [41]. The analysis was carried out with gas-liquid chromatography with flame ionization detection (GC-FID; DANI1000, Contone, Switzerland). Fatty acids identification and quantification (Clarity DataApex 4.0 Software, Prague, Czech Republic) were performed by comparing the relative retention times of fatty acid methyl ester (FAME) peaks from samples with standards (reference standard mixture 47885-U, Sigma, St. Louis, MO, USA). Results were expressed in the percentage of each fatty acid.

#### *2.3. Statistical Analysis*

#### 2.3.1. Experimental Layout and Statistical Treatment

The experimental design was laid out in a split plot arrangement with each main plot consisting of water stress treatments (W+ or W-), while fully randomized sub-plots comprised the biostimulants treatments. Each subplot contained 6 plants and each main plot contained 30 plants. Pod yield components were evaluated in 18 plants for each treatment (*n* = 18), whereas for seed yield, 30 randomly selected pods from each treatment were measured. In order to constitute a representative and adequate sample of the tested treatments, batches of several samples of pods and seeds were taken at random from each plot in order to obtain three different samples. Then, these batches were powdered to obtain homogenous samples. For each methodology, three extractions were carried out, and the analyses were performed in triplicate. Statistical analysis was conducted with the aid of Statgraphics 5.1.plus (Statpoint Technologies, Inc., Warrenton, VA, USA). Data were evaluated by a two-way analysis of variance (two-way ANOVA), and significant interactions of the tested factors (water regime and biostimulant treatment) were observed. Therefore, all the means for each pod harvest and seeds were compared separately by using the Tukey's honestly significant difference (HSD) test (*p* = 0.05).

#### 2.3.2. Linear Discriminant Analysis (LDA)

Linear discriminant analysis (LDA) was applied to evaluate the overall effects of different biostimulants, independently of water level, in each phenological stage (first and second harvest of pods and seeds). The stepwise technique, considering the Wilk's ʎ test with the usual probabilities of F (3.84 to enter and 2.71 to be removed) for variable selection, was employed. With this procedure, it was aimed to estimate the association between the single categorical dependent variables (biostimulant treatments: C, G, TW, V, EK) and the quantitative independent variables (analyzed parameters: proximate composition, organic acids, tocopherols, sugars, fatty acids). In all cases, a leaving-one-out cross-validation procedure was carried out to assess the model performance.

#### **3. Results and Discussion**

#### *3.1. Yield and Growth Parameters*

The yield and growth characteristics of pods and seeds are presented in Table 2. Yield was positively affected by the application of EK treatment in normally irrigated plants (EKW+) where higher yields compared to the control and the rest of biostimulant treatments were observed (5284 ± 120 kg/ha, 3701 ± 88 kg/ha, and 8985 ± 196 kg/ha, for the first harvest, second harvest, and total yield, respectively). This increase in pod yield was attributed to the higher number of pods harvested from both harvests in plants treated with the specific biostimulants, while the mean pod weight was also the highest in EKW+ treatment only for aggregated results. On the contrary, the application of V treatment in water-stressed plants (VW-) resulted in the lowest yields for the first and second harvest and consequently in the total yield of green pods (2213 ± 90 kg/ha, 1749 ± 59 kg/ha, and 3962 ± 147 kg/ha for the first harvest, second harvest, and total yield, respectively). Additionally, the number of pods per plant and consequently total yield was higher in normally irrigated plants comparing to water-stressed ones, while GW- and TWW- treatments were the most effective at alleviating the negative effects of stress conditions. Similar results have been reported by Aimo et al. [42], who suggested that the *Crocus sativus* yield was increased after AMF application due to the higher number of flowers. This was also the case in our study, where the products containing AMF (G) or a mixture of AMF, saprophytic fungi, and rizosphere bacteria (EK) resulted in higher yields in water-stressed and normally irrigated plants, respectively. According to German et al., the inoculation of common bean plants with *Azospirillum brasilense* increased tap root length as well as the proportion of long and thin roots at the early growth stages, which are critical for plant adaptation to water stress conditions [27]. Moreover, Weber et al. reported an increase of fruit setting and total yield in strawberry plants as the result of *Ascophyllum nodosum* extracts application [43]. In the present study, the application of products containing *A. nodosum* extracts had a positive effect on total yield under water stress conditions (TWW-) when compared to non-biostimulant treated plants (CW-), although the product containing AMFs (GW-) was shown to be more effective. According to Arthur et al. [44], biostimulants such as seaweed extracts may contain plant hormones (cytokins and auxins) that induce flower formation. Seaweed extracts (*Ascophyllum nodosum*) have been also reported to have a positive effect on the plant growth of lettuce, carrot, and strawberry through increased hormone activity and K uptake [18,45,46]. Moreover, the increased yield for plants treated with biostimulants is associated with improved plant tolerance to abiotic stress conditions, which according to Battacharyya et al. [47] could be attributed to various protective mechanisms such as the regulation of related genes, the accumulation of osmolytes, the improvement in water-use efficiency, and other effects on the plant rhizosphere. Moreover, Ahmad et al. [10] reported that the inoculation of Indian mustard plants with *Trichoderma harzianum* alleviated osmotic stress effects through the activation of plant antioxidant mechanisms.


**Table 2.** Yield and growth characteristics of bean plants in relation to water stress and biostimulants application (mean

¥ column followed by different Latin letters are significantly different according to Tukey's honestly significant difference (HSD) test (*<sup>p</sup>* = 0.05).

Regarding the weight of 100 seeds, the highest value was observed for the EK treatment under water stress conditions (EKW-), while normally irrigated plants with no added biostimulants (CW+) presented the lowest values (122 ± 2 and 101 ± 2 for EKW- and CW+ treatments, respectively) (Table 2). Moreover, the weight of 100 seeds and number of seeds per pod were higher and lower, respectively, for water-stressed plants regardless of biostimulants treatment when compared to the control treatment of normally irrigated plants, indicating that water stress may affect the fertilization process and consequently the number of seeds per pod. The beneficial use of plant-growth promoting rhizobacteria under water stress conditions has been also reported by Sarma and Saikia [24], who suggested that the inoculation of mung bean plants with *Pseudomonas aeruginosa* strains alleviated water stress effects through the scavenging of oxidative enzymes. Moreover, Korir et al. [48] reported a synergistic effect of plant growth promoting bacteria with common bean rhizobia that enhanced plant growth and development, while Farouk and Abdul Qados [49] suggested that folic acid application increased the seed yield and chemical composition of pea plants (*Pisum sativum*).

#### *3.2. Nutritional Value*

The nutritional value of the pods and seeds is presented in Table 3. The application of biostimulants did not have a beneficial effect on the moisture content of pods in the first harvest when compared with the control treatment for either normally irrigated (CW+) or water-stressed plants (CW-). Similar trends were observed under prolonged water stress (second harvest), while for normally irrigated plants, the VW+ treatment resulted in the highest moisture content values. For both harvests, the lowest values were recorded for pods harvested from water-stressed plants treated with the V treatment (VW-). These findings could be probably attributed to a functional allocation equilibrium where under biostimulant treatments, plants allocated resources and biomass in fruit; thus, a reduction in moisture content (or similarly an increased dry matter content) was observed [50]. Moreover, plants under stress tend to accumulate minerals and metabolites as a means to maintain high water potential [5]. However, considering that these trends were observed both in water-stressed and normally irrigated plants highlights the need for further investigation. On the other hand, the moisture content of seeds was beneficially affected by the various biostimulants in plants grown under water stress conditions, especially for G treatment (GW-), where the highest values were observed. Regarding the rest of the nutritional value parameters, a varied effect of biostimulants and irrigation treatments was observed in terms of fat, protein, ash, and carbohydrates content and the energetic value of pods and seeds. In particular and for the first harvest of pods, protein, ash content, and energetic value were beneficially affected under the normal irrigation regime, while carbohydrates content was the highest for water-stressed plants with no added biostimulants. Similarly, a beneficial effect of GW+ treatment on protein and ash content was observed for the pods of the second harvest, whereas fat and carbohydrates content were the highest for water-stressed plants that received no biostimulants (CW-) or the G treatment (GW-). The energetic value was the highest for normally irrigated plants that received no biostimulants. Seeds' nutritional value was beneficially affected by the application of G treatment under normal irrigation conditions (GW+) when proteins, fat, and ash content were considered, whereas EK and G treatments increased energetic value under the same irrigation treatments (5468 ± 6 kcal/kg dw and 5474 ± 3 kcal/kg dw, respectively). The highest carbohydrates content values were recorded for normally irrigated plants where no biostimulant or the V treatment was applied (CW+ and VW+, respectively), as well as for water-stressed plants that received the EK treatment (EKW-). Significant differences were also observed between normally irrigated and water-stressed plants in a biostimulant treatment-specific manner, although no direct comparisons between the corresponding treatments were performed due to the presence of significant interactions among the tested factors. Nevertheless, it is worth highlighting the beneficial effects of biostimulants on the protein content of green pods under water stress conditions compared to the corresponding control treatment (CW-). The effect of biostimulants on plant metabolism and the quality of end-products has been previously reported by Colla et al. [51], while Przygocka-Cyna

and Grzebisz [52] have associated the use of biostimulants with the improvement in plant nutrient uptake and therefore with the better nutritional value of the end products. Moreover, according to Elsheikh et al., the inoculation of faba bean (*Vicia faba*) plants with arbuscular mycorrhiza increased the protein content in the seeds, regardless of irrigation conditions, suggesting the improved nutritional status of plants as the main reason for this increase [53]. Similarly, Farouk and Abdul Qados [49] suggested that folic acid and hydrogen peroxide application may improve the nutritional value of pea seeds through the increase of protein and carbohydrates content, while Elsheikh and Mohamedzein reported that the inoculation of groundnut with *Glomus* sp. and *Bradyrhizobium* sp. increased the protein content of seeds. Regarding the biostimulatory activity of seaweed extracts, Kocira et al. [54] and Castellanos-Barriga et al. [55] reported contrasting effects of *Ecklonia maxima* and *Ulva lactuca* extracts on the nutritional value of common bean (*P. vulgaris*) and mung bean (*Vigna radiata*) seeds, respectively. These differences in the literature reports highlight the variable biostimulatory effects of seaweed extracts, which contain a wide range of compounds associated with improved plant nutrient uptake, phytohormone-like activities, tolerance to abiotic stressors, and the modulatory effects of plant metabolism and physiology [47].

**Table 3.** Nutritional (g/kg dw) and energetic value (kcal/kg dw) of the studied pods and seeds of beans in relation to irrigation regime (mean ± SD).


<sup>¥</sup> W+: indicates normal irrigation regime; W-: indicates water-holding irrigation regime; C: Control; V: Veramin Ca; EK: EKOprop; G: Nomoren; TW: Twin-Antistress. Means in the same column and the same harvest (first and second harvest of pods and seeds) followed by different Latin letters are significantly different according to Tukey's honestly significant difference (HSD) test (*p* = 0.05).

#### *3.3. Mineral Composition*

The mineral composition of pods and seeds is presented in Table 4. The combination of biostimulants application and irrigation regime had a varied effect on mineral content of pods and seeds with no specific trends being observed. In particular, the application of V treatment under water stress conditions (VW-) increased the nitrogen content of pods without being significantly different from normally irrigated control plants (CW+). Similarly, the highest values of nitrogen content in seeds were recorded for GW- and TWW-. Positive effects were observed for K content in the pods of normally irrigated plants that received G treatment (GW+), whereas no significant differences were observed in the K content of seeds for the tested treatments. EKW+ and GW+ resulted in the highest values for P content in pods and seeds, respectively, whereas contrasting effects of the irrigation regime × TW treatment combination were observed on Ca content where TWW- and TWW+ increased its content in pods and seed, respectively. The Na content of pods was the highest when EK treatment was applied regardless of the irrigation regime, while similar results were observed in seeds for the control and V treatment. The Mg content of pods and seeds increased when no biostimulants or the V treatment were applied on water-stressed plants, respectively. Moreover, the Cu content of pods was beneficially affected by the EK treatment, regardless of the irrigation regime, whereas for seeds, the highest values were recorded for the TWW+ treatment. Regarding the Zn content, the highest values in pods and seeds were observed for the EKW+ and VW- treatments, respectively. Finally, the Mn content of pods increased when V and G treatments were applied, regardless of the irrigation regime, whereas for seeds, the application of biostimulants had a negative effect on Mn content when compared to control treatments (CW- and CW+). The impact of biostimulants on the nutrient content of agricultural products could be attributed to the fact that they usually contain various minerals in their composition [5]. Mineral uptake from plants may help in maintaining high stomatal conductance and leaf water potential; therefore, biostimulants may improve the nutritional status of plants and induce tolerance to abiotic stress factors such as drought stress [56]. In addition, according to Chrysargyris et al. [18], the application of *Ascophyllum nodosum* seaweed extracts alleviated the negative effects of K deficiency on lettuce plants, while the beneficial effect of AMFs as biostimulants has been associated with higher P uptake from plants [57]. This was the case in our study under normal irrigation conditions where the P content of pods was the highest for the EK treatment. The results from the study of Colla et al. [58] confirm the beneficial effect of biostimulant application on the Ca content of tomato fruit for the seaweed extracts treatment, which was also observed in our study for TW treatment, regardless of the irrigation regime. Moreover, it has been reported in several studies that the inoculation of plants with mixtures of bacteria has better results in nutrient mobilization and uptake compared to inoculation with a single bacterium [26], which was also the case in our study where EK treatment increased the P, Na, Cu, and Zn content in the pods of normally irrigated plants.



¥ samecolumn and the same plant part (pods and seeds) followed by different Latin letters are significantly different according to Tukey's honestly significant difference (HSD) test (*<sup>p</sup>* = 0.05).

#### *3.4. Tocopherols*

The main detected tocopherols in pods were γ-tocopherol, followed by α-tocopherol, while seeds contained mainly γ-tocopherol and less amounts of δ- and α-tocopherol (Table 5). The application of TW treatment resulted in a significant increase of γ-tocopherol (104% and 18.3% for the TWW+ and TWW- treatments, respectively) and total tocopherols (82.3% and 19.4% for the TWW+ and TWWtreatments, respectively) in the pods of the first harvest compared to the control treatments (CW+ and CW-). In contrast, the application of G treatment had a negative effect on the γ-tocopherol content in pods of the first harvest under water stress conditions (reduced by 45.7%), while EK treatment resulted in the lowest content of α-tocopherol for the same irrigation regime (1800 ± 40 μg/kg dw). In the second harvest, the highest values for γ- and total tocopherols were recorded for the VW- treatment (3500 ± 20 μg/kg dw and 5250 ± 10 μg/kg dw, respectively), while pods from the control treatment (CW-) contained the highest amounts of α-tocopherol (1810 ± 20 μg/kg dw). In addition, drought stress increased the individual and total tocopherols content for all the biostimulant treatments, except for G treatment, where normally irrigated plants had a higher content of tocopherols compared to water-stressed ones. The observed increase of tocopherols in pods under prolonged water stress conditions could be attributed to the induction of self-defense mechanisms by biostimulants application and the production of antioxidant compounds such as tocopherols [59]. However, the variable effects of the tested biostimulants indicate a diverse plants x biostimulant interaction, as well as the induction of different mechanisms in each combination depending on the biostimulant composition and the severity of stress [60]. Therefore, although in the first harvest TWW- treatment induced tocopherols biosynthesis as a non-enzymatic antioxidant mechanism, under prolonged stress conditions, VW- (second harvest) and VW+ (seeds) treatments were beneficial to tocopherols content. Regarding seeds, the presence of α- and γ-tocopherol has been previously reported in common bean seeds by Kan et al. [61]. In our study, γ-, δ-, and the total tocopherols content was the highest under normal irrigation conditions and for those plants that did not receive biostimulants or the V treatment was applied. In contrast, EK treatment had a negative effect on tocopherols content under water stress conditions. According to the literature, Ca and amino acids supplementation (as in the case of V treatment in our study) may induce the biosynthesis of non-enzymatic antioxidants such as tocopherols and increase tolerance against drought stress [62–65].


**Table 5.** Composition in tocopherols of the studied pods (μg/kg dw) and seeds (mg/kg dw) of beans in relation to the irrigation regime (mean ± SD).


**Table 5.** *Cont.*

¥ W+: indicates normal irrigation regime; W-: indicates water-holding irrigation regime; C: Control; V: Veramin Ca; EK: EKOprop; G: Nomoren; TW: Twin-Antistress. Means in the same column and the same harvest (1st and 2nd pod harvests and seeds) followed by different Latin letters are significantly different according to Tukey's honestly significant difference (HSD) test (*p* = 0.05).

#### *3.5. Organic Acids*

The main detected organic acids in pods were malic and oxalic acid, while ascorbic acid was detected in less amounts in specific treatments of the first harvest (Table 6). On the other hand, malic and oxalic acid were the main organic acids detected in seeds, followed by ascorbic acid and traces of fumaric acid. The application of the VW+ treatment resulted in the highest content of oxalic and malic acid and total organic acids in pods of the first harvest (26.3 ± 0.1 g/kg dw, 23.1 ± 0.1 g/kg/dw, and 49.4 ± 0.1 g/kg/dw, respectively). Similarly, in the second harvest, the highest content of oxalic and malic acid was recorded for the GW+ and VW- treatments (22.7 ± 0.2 g/kg/dw and 24.6 ± 0.2 g/kg/dw, respectively), while total organic acids content was most abundant in the GW+ treatment (46.9 ± 0.1 g/kg/dw). Regarding seeds, VW- treatment resulted in the highest content of oxalic and malic acid, and total organic acids (752 ± 1 mg/kg/dw, 1440 ± 40 mg/kg/dw, and 2780 ± 30mg/kg/dw, respectively), whereas the highest amounts of ascorbic acid were detected in GW+ treatment (715 ± 4 mg/kg dw). Considering the antinutritional properties of oxalic acid, it is worth mentioning that EKW- treatment resulted in the lowest content for both pod harvests when compared with the rest of the treatments where biostimulants were applied, although in all the cases, the oxalic acid content was considerably low. On the other hand, in the case of seeds, the application of EKW+ and TWW+ treatments significantly reduced the oxalic acid content compared to the control and the rest of the biostimulant treatments. Although there are reports in the literature that suggest that organic acids increase under stress conditions, according to Zushi and Matsuzoe [66], this increase could be attributed only to a concentration effect due to the increase in dry matter content under stress conditions. According to other studies, the composition of biostimulants may significantly affect the organic acids composition, especially those biostimulant products that contain microorganisms such as Twin-Antistress and EKOprop in our study [36,67]. However, although the total organic acids

content of pods harvested from water-stressed plants was in general higher in biostimulant-treated plants compared to the control treatment (CW-), the application of the EKW- treatment resulted in a significant reduction of organic acids content. This finding is reflected to the reduced total pod yield for this treatment (see Table 2), suggesting a non-effective alleviating mechanism against water stress related to biostimulant product composition. Moreover, the effect of V treatment on the oxalic acid content of seeds under water stress conditions (VW-) could be attributed to Ca addition, which is associated with calcium oxalate formation for the removal of excessive calcium or oxalic acid [67].

#### *3.6. Free Sugars*

The sugars composition of pods and seeds is presented in Table 6. The main detected sugar in the pods of both harvests was fructose, followed by glucose and sucrose, whereas in seeds, only sucrose was detected. Similarly with our study, Kan et al. [61] detected sucrose as the main sugar in common bean seeds, while they also detected the presence of glucose; this difference could be attributed to the different harvesting stages (fully dried seeds comparing to fully developed green seeds in our study), which may affect hydrolysis and the transformation of sugars after harvest [68]. In the first harvest, the highest content of individual and total sugars were recorded in TWW- (fructose: 198 ± 1 g/kg dw), CW- (glucose: 135 ± 5 g/kg dw; total sugars: 333 ± 7 g/kg dw), and VW- (sucrose: 7.6 ± 1 g/kg dw) treatments. Under prolonged water stress (second harvest), the application of G treatment (GW-) resulted in the highest content of fructose, glucose, and total sugars (232 ± 7 g/kg dw, 140 ± 7 g/kg dw, and 380 ± 10 g/kg dw, respectively), while the sucrose content was the highest for the TWW- treatment (15.3 ± 0.8 g/kg/dw). Similarly, the highest content of sucrose in seeds was recorded for the GWtreatment (22.9 ± 0.7 g/kg dw). The low levels of sucrose in pods could be attributed to the inhibitory activity of hexose sugars (fructose and glucose) to sucrose synthase activity [68]. Moreover, for most of the tested biostimulants and control treatments, the total and individual sugars content was higher in water-stressed plants than normally irrigated plants, especially for G treatment—that resulted in the highest total sugars content, which could be associated with osmoprotective effects against water stress [69]. Considering the involvement of soluble sugars in plant defense mechanisms as well as in the regulation of stress and growth-related genes, the findings of our study suggest an efficient defense mechanism against water stress for the AMF-containing biostimulant product (G treatment), as already justified by the increased pods yield under water stress conditions for the same treatment (see Table 2). According to the literature, inoculation with AMF is associated with increased soluble sugars content in *Ipomea batatas* and *Vigna subterranea* under drought stress, since sugars may serve as organic carbon pools to be used for photosynthates and biomass production [70]. Apart from the osmoregulatory role of sugars in plant defense mechanisms against stress, sucrose content is also related with secondary metabolites biosynthesis, which may also contribute to the overall non-enzymatic tolerance of plants under stress [71].




**Table 6.** *Cont.* W+:column and the same harvest (1st and 2nd pod harvests and seeds) followed by different Latin letters are significantly different according to Tukey's honestly significant difference (HSD)test (*<sup>p</sup>* = 0.05). Tr: traces.

¥

#### *3.7. Fatty Acids*

The main fatty acids composition is presented in Table 7. Seventeen individual fatty acids were detected in pods and seeds regardless of the irrigation treatment and harvest (data not shown). Pods were abundant in α-linolenic (C18:3n3), linoleic (C18:2n6c), and palmitic acid (C16:0) followed by stearic (C18:0), oleic (C18:1n9c), behenic (C22:0), and lignoceric acid (C24:0), which were detected in lower amounts. Similarly, in seeds, the most abundant fatty acids were α-linolenic, linoleic, and palmitic acid, followed by stearic and oleic acid. In the first harvest of pods, GW- and EKW- treatments had a beneficial effect on palmitic and linoleic acid content in water-stressed plants (25.6 ± 0.3% and 35.02 ± 0.01%, respectively), whereas in normally irrigated plants, TWW+ treatment resulted in the highest content of α-linolenic acid (42.76 ± 0.06%). In the second harvest, fatty acids composition showed a varied response, with control and TW treatment resulting in the highest content of linoleic and palmitic acid for normally irrigated plants (43.3 ± 0.1% and 28.90 ± 0.06, respectively), whereas α-linolenic acid content was the highest for the GW- treatment (34.33 ± 0.09%). For seeds, the highest amounts of α-linolenic, linoleic and palmitic acid were recorded in the treatments of GW-, CW+, and VW+ (59.08 ± 0.02%, 29.54 ± 0.02%, and 11.86 ± 0.03%, respectively). Polyunsaturated fatty acids (PUFA) were the most abundant fatty acids class, followed by saturated (SFA) and monounsaturated fatty acids (MUFA) in both seeds and pods due to the high amounts of α-linolenic and linoleic acids. Overall, the ratios of PUFA/SFA and n-6/n-3 fatty acids were higher than 0.45 and lower than 4.0 for all the tested treatments, respectively, which according to Petropoulos et al. [72] is indicative for the good nutritional value of a food product. Moreover, the increase of PUFAs under water stress conditions comparing to the control treatment (CW-) for all the biostimulant treatments except for GW- (first harvest) and EKW- (second harvest) treatments indicates the stimulation of plant antioxidant mechanisms which effectively quenched the reactive oxygen species (ROS) that appear after stress initiation and induce lipid peroxidation and decrease fatty acids content [10,73]. Fatty acids may also serve as organic carbon pools to be used for photosynthates and biomass production [70]. Therefore, considering that inoculation with AMFs and bacteria induces synergistic effects between plants and symbionts that may improve plant nutrient and water uptake, this could be the reason for the increased content of fatty acids.


*Agronomy* **2020** , *10*, 181

honestly significant difference (HSD) test (*<sup>p</sup>* = 0.05).

#### *3.8. LDA Analysis*

#### 3.8.1. First Harvest

In the first harvest, the linear discriminant analysis (LDA) selected PUFA, C17:0, ascorbic acid, glucose, α-tocopherol, C16:1, C14:0, C16:0, carbohydrates, and C22:1 as variables with discriminant ability, which is equivalent to say that these were the parameters showing the most profound changes in result of using different biostimulants (Figure 2). Function 1 separated primarily samples treated with G, which was placed in the farthest position in the negative side of the axis; in turn, the biostimulants with the most similar effect according to this function (which was the most important, as it included 85.1% of the observed variance) were V and TW. In turn, Function 2 separated mainly C samples, which was mostly due to their higher contents in carbohydrates and glucose (in this case, specifically in Wsamples). The most noticeable effect of Function 3 was the individualization of markers corresponding to EK samples, which was mostly due to the levels of PUFA, C16:0, and C14:0.

**Figure 2.** Canonical discriminant functions coefficients defined from the evaluated parameters plotted to show the effect of biostimulants treatments on *Phaseolus vulgaris* green pods of the first harvest under different irrigation regimes (normal irrigation and water stress).

#### 3.8.2. Second Harvest

Concerning the second harvest, the LDA selected C22:0, fructose, organic acids, C21:0, proteins, C16:1, C24:0, C20:1, C18:0, C18:1n9c, MUFA, and sugars as the variables with the highest differences as a result of using different biostimulants (highest discriminant ability) (Figure 3). Function 1 was especially effective in separating samples treated with TW or EK, which was mostly due to their higher C18:1n9c contents (regardless of the irrigation treatment). On the other hand, Function 2 separated samples treated with G, which was placed in the farthest position in comparison to control samples. Considering all the functions together, it was possible to conclude that the biostimulant treatment V was the one that induced the least differences in comparison to the control.

**Figure 3.** Canonical discriminant functions coefficients defined from the evaluated parameters plotted to show the effect of biostimulants treatments on *Phaseolus vulgaris* green pods of the second harvest under different irrigation regimes (normal irrigation and water stress).

#### 3.8.3. Seeds

The effects of the tested biostimulants on seeds were also more pronounced for fatty acids, as indicated by the variables classified as being discriminant: C20:1, C20:0, sucrose, C22:1, lipids, C17:0, C15:0, organic acids, PUFA, C16:0, C18:2n6c, and C16:1 (Figure 4). According to Function 1, all biostimulants had similar effects (markers are almost vertically aligned), while untreated samples (C) were completely individualized (negative side of the axis); among the selected variables, the one showing the highest correlation with this function was C20:1, which showed higher percentages in C samples, independently of water level. Function 2, in turn, was mostly correlated to C16:1 and sucrose, contributing mainly to separate samples treated with EK, while Function 3 was more highly correlated with lipids content, contributing to separate samples treated with G.

In all the former LDAs, the classification performance was 100% accurate both for originally grouped cases as well as for cross-validated ones.

**Figure 4.** Canonical discriminant functions coefficients defined from the evaluated parameters plotted to show the effect of biostimulants treatments on *Phaseolus vulgaris* seeds under different irrigation regimes (normal irrigation and water stress).

#### **4. Conclusions**

The results of the present study showed a varied effect of biostimulants and water treatments on pod yield and the quality of common bean green pods and seeds, while significant differences were also observed between normally irrigated and water-stressed plants in a biostimulants treatment-specific manner. Promising results were also recorded regarding the alleviation of negative effects of drought stress where the application of arbuscular mycorrhizal fungi (AMF; G treatment) increased the crop yield of green beans. Moreover, the nutritional value and chemical composition of pods and seeds was positively affected by biostimulants application, although a product specific effect was recorded depending on the irrigation regime and harvesting time (pods and/or seeds). In conclusion, the application of biostimulants could be considered as an eco-friendly and sustainable tool to increase the pod yield and quality of common bean green pods and seeds under normal irrigation and/or drought stress conditions. Considering that the tested biostimulants contain beneficial microorganisms such as AMF, symbiotic rizosphere bacteria, and saprophytic fungi, its application not only could benefit crops but it could also improve soil properties and preserve soil quality. However, future research is needed to investigate in depth the mechanisms of action of biostimulant product, the application dose efficiency, as well as the most effective application regime and the possible effect of genotype x biostimulants interactions.

**Author Contributions:** S.A.P. conceived and designed the research, administered and supervised the project, carried out the cultivation, wrote the original draft, and reviewed and edited the final manuscript; A.F. performed chemical analyses, data curation, and methodology; S.P. carried out the cultivation and prepared the original draft; A.C. performed chemical analyses, data curation, and methodology; N.T. performed chemical analyses, data curation, prepared the original draft, and edited the final manuscript; J.C.M.B. performed the LDA analysis of the data and the interpretation of the statistical analysis results; L.B. performed chemical analyses, data curation, and

methodology, wrote the original draft, and reviewed and edited the final manuscript; I.C.F.R.F. obtained funding, administered and supervised the project, and reviewed and edited the final manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2013), A. Fernandes and L. Barros contract.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Changes in Biochemistry and Yield in Response to Biostimulants Applied in Bean (***Phaseolus vulgaris* **L.)**

**Anna Kocira 1,\*, Joanna Lamorska 1, Rafał Kornas 1, Natalia Nowosad 1, Marzena Tomaszewska 1, Danuta Leszczy ´nska 2, Katarzyna Kozłowicz <sup>3</sup> and Sylwester Tabor <sup>4</sup>**


Received: 22 December 2019; Accepted: 28 January 2020; Published: 31 January 2020

**Abstract:** Biostimulants are preparations that favorably impact the growth, development, and yield of plants. The research objective was to examine the effect of the frequency of use of Kelpak, Terra Sorb Complex and Fylloton biostimulants on improving the yield and nutritional properties of beans. Been seeds (variety Oczko) were sown in the first week of May in 2015, 2016, and 2017. During the growing season, Fylloton (1%), Terra Sorb Complex (0.5%), and Kelpak (1%) biostimulants were applied by single (BBCH 12-13) and double spraying of plants (BBCH 12-13, BBCH 61). All variants of treatment with biostimulants were compared with the control. Single application of Kelpak had a positive effect on increasing the number of pods. The double application of Kelpak increased the number and yield of seeds and protein contents. Double application of Fylloton increased the number of seeds, and application of Terra Sorb Complex increased the protein content in the beans. Application of all biostimulants increased the flavonoid content. Biostimulants containing seaweed (Kelpak–*Ecklonia maxima* extract) or amino-acid extracts (Fylloton–*Ascophyllum nodosum* extract and amino acids or Terra Sorb Complex–amino acids) increased the seed yield, while improving its quality by increasing the content of protein, polyphenols, and flavonoids. It was found that the double application of Kelpak biostimulant stimulated the yield and quality of beans to a greater extent.

**Keywords:** bean; biostimulants; amino acids; seaweed extract; yield; protein; phenols; flavonoids

#### **1. Introduction**

Agricultural production seeks technological solutions to improve the quality of yields. Therefore, biostimulants are increasingly popular as preparations that favorably impact the growth, development, and yield of plants [1,2], and they are safe for humans and environmentally friendly at the same time. Du Jardin [3] defines biostimulants as "any substance or microorganism applied to plants with the aim of enhancing nutrition efficiency, abiotic stress tolerance, and/or crop quality traits, regardless of its nutrient content". In addition, in order to develop legal provisions regarding the registration of biostimulants based on their specificity of operation, the European Biostimulants Industry Council (EBIC) was created. Currently, however, their registration is based on legal provisions on fertilizers and pesticides, and, for some of them, there is a marketing gap in many European Union (EU) member states [3–6].

Depending on the origin, there are natural or synthetic biostimulants. The former are obtained from biological material, and the latter are structurally similar and functionally identical to biological material [7]. The group of natural biostimulants includes preparations based on free amino acids, humic compounds, seaweed or fruit extracts, chitin and its derivative, chitosan, or microbial inoculants (free-living bacteria, fungi, and arbuscular mycorrhizal fungi) [8–10]. Of this group, biostimulants containing seaweed extract and protein hydrolysates are the most important category of substances that stimulate plant growth and development [11,12].

Biostimulants affect the metabolic processes occurring in the plant, stimulating the synthesis or activity of phytohormones, facilitating the growth of the root system, and improving the uptake, translocation, and utilization of nutrients, which determines the quality of the obtained yield [3,8–12]. Moreover, biostimulants increase plant resistance to abiotic stress factors such as drought, frost, salinity, and environmental contamination with heavy metals, which is probably caused by changes in the enzymatic activity of antioxidant compounds and their increased synthesis [8,13].

In bean cultivation, the most commonly used are extracts of brown algae, e.g., of the species *Ascophyllum nodosum*, *Laminaria* spp., *Ecklonia maxima*, *Sargassum* spp., and *Fucus* spp. [14–17]. The positive effect of seaweed extracts on plant growth, development, and yield is attributed to the presence of phytohormones and low-molecular-weight compounds [18,19]. Some authors suggest that the polysaccharides and polyphenols present in the extract are also important, since they are allelochemicals which increase plant resistance to stress conditions [20–22]. Generally, organic and mineral compounds occur in seaweed extracts, the content of which depends on the algae species, their harvest date, and the applied extraction process [8]. The most important bioactive ingredients include proteins, enzymes, and amino acids (glycoproteins, metalloproteins, exogenous amino acids such as aspartic acid, glutamic acid, alanine), phytohormones (auxins, cytokinins, gibberellins, abscisic acid, polyamines, betaine, ethylene, brassinosteroids, brasinolide, castasterone), polyphenols (florotanins, ecol, floroglucin), phytalexins, vitamins (C, B2, B12, D3, E, K, niacin, panthotenic and folic acid), oligosaccharides, polysaccharides (agar, hyaluronic acid, alginic acid and its salts, carrageenans, fucans, mannitol, sorbitol, laminarin), macro- and microelements (Mg, Cu, Fe, Br, Zn, I, Mn), and essential unsaturated fatty acids (arachidonic, eicosapentenoic, γ-linolenic). Although vitamin A is not present in seaweed extracts, the presence of its precursors, i.e., carotene and possibly fucoxanthin was detected [14,23–31].

The positive effect of seaweed extracts on plants is visible in the stimulation of phytohormone synthesis, the uptake and translocation of nutrients, and soil conditioning, which is done by improving water–air conditions and the activity of beneficial soil microorganisms [32,33]. It was demonstrated that, even in low concentrations, seaweed extracts induce a number of physiological processes in the plant, contributing to their better growth, flowering, yield size, and quality, and improving the nutritional and storage quality of crop plants. In addition, the use of seaweed extracts increases the tolerance of plants to unfavorable growing conditions, for example, salinity, drought, or extreme temperatures [12,34,35]. The use of seaweed extract has a positive effect on plant growth and the size and quality of the obtained yield of tomato, eggplant, pepper, lettuce, beans, soybean, and wheat [13,25,36–46]. Undoubtedly, the beneficial effects of biostimulants on plants, under both optimal and stressful growing conditions, can be associated with stimulation of enzymatic activity related to carbon and nitrogen metabolism, the Krebs cycle, and glycolysis. Treatment of plants with these preparations may induce activity similar to that of phytohormones (auxin, gibberellin), which in effect improves their nutrition by modifying the structure of the root system [8,9,47].

The biostimulant Kelpak (Kelp Products Ltd.) is based on an extract from *Ecklonia maxima* (Osbeck) Papenfuss and contains auxins (11 mg·dm<sup>−</sup>3), cytokinins (0.031 mg·dm<sup>−</sup>3), alginates (1.5 g·L<sup>−</sup>1), amino acids (total 441.3 mg·100 g<sup>−</sup>1), mannitol (2261 mg·L<sup>−</sup>1), neutral sugars (1.08 g·L<sup>−</sup>1), and small amounts of macroelements (N 0.09%, P 90.7 mg·kg<sup>−</sup>1, K 7163.3 mg·kg<sup>−</sup>1, Ca 190.4 mg·kg<sup>−</sup>1, Mg 337.2 mg·kg<sup>−</sup>1, Na 1623.7 mg·kg−1) and microelements (mean composition: Mn 17.3 mg·kg−1, Fe 40.7 mg·kg−1, Cu 13.5 mg·kg−1, Zn 17.0 mg·kg−1, B 33.0 mg·kg−1) [14,48]. The very high auxin-to-cytokinin ratio is

responsible for stimulating the growth and development of the root system, which in turn contributes to better uptake and translocation of macro- and microelements, and it is associated with a significant increase in crops [48].

Biostimulants based on protein products and protein hydrolysates consist of a mixture of peptides, animal or vegetal amino acids, and single amino acids. Amino acids are a building material for proteins, but they are also precursors of phytohormones. They are involved in the synthesis of, e.g., vitamins, enzymes, terpenes, amines, purines, pyrimidines, and alkaloids [49,50]. They also play an important role in the process of pollination and fruit formation [51]. The application of exogenous amino acids, which are active in metabolic signaling (glutamate, histidine, proline, glycine, betaine), induces plant defense mechanisms by increasing their resistance to abiotic stress factors [8,52]. Due to the presence of specific peptides and precursors of phytohormone biosynthesis (tryptophan, which is the main precursor of IAA (Indole-3-acetic acid) biosynthesis and bioactive peptides), protein hydrolysates affect the hormonal balance of plants, which is related to stimulating plant growth [53]. The use of these biopreparations positively impacts the quality of agricultural produce, increasing the content of carotenoids, flavonoids, polyphenols, and ascorbic acid [54–57], and reducing the amount of undesirable compounds, e.g., nitrates [56].

Terra Sorb Complex (Bioiberica, S.A.U.) is a biostimulant containing 20% free vegetal amino acids and 5.5% total N (including 5% organic N), 0.8% MgO, 1.5% B, 1% Fe, 0.1% Mn, 0.001% Mo, 0.1% Zn, and 25% organic matter.

The beneficial effect of biostimulants with seaweed extracts or amino acids on plant growth and development, and on the quantity and quality of the yield, regardless of its developmental stage, was confirmed in numerous studies [13,34,35,39–41,57–62]. An interesting solution is to combine these two components in a single preparation, as in the case of the Fylloton biostimulant (Biolchim Poland), which contains the extract of *Ascophyllum nodosum* (L.) Le Jolis, as well as vegetal amino acids. The composition of this preparation includes *Ascophyllum nodosum* extract, amino acid complexes of vegetal origin 37.5%, organic nitrogen 6%, organic carbon of biological origin 11%, and organic substance 35%.

Bean is an economically important legume that is sensitive to low temperatures in the early stages of its development and flowering. The use of biostimulants that positively affect the metabolic processes occurring in the plant, especially in the time of climate change, which causes stress factors for this sensitive plant, can be one of the elements contributing to the improvement in the quantity and quality of bean yield. Plant response to the biostimulant often depends on the variety, as demonstrated in earlier studies [15,39,59,60]. There are also no reports regarding the reaction of two-colored coat seed of bean to treatment with biostimulants. In view of the above and based on the importance being given to the improving crop yields, the research objective was to investigate the effect of the use of Kelpak, Terra Sorb Complex, and Fylloton biostimulants on improving the yield and nutritional properties of common bean (*Phaseolus vulgaris* L.) variety Oczko.

#### **2. Materials and Methods**

#### *2.1. Plant Materials and Growth Conditions*

The research material came from field studies carried out in the year 2015–2017 in Perespa (50◦66' north (N); 23◦63' east (E)), Poland, on common beans (*Phaseolus vulgaris* L.), variety Oczko. The experiment was established in a random block system, in four replications, on an area of 10 m2. The experiment was established on an alkaline (pH in 1 M KCl-7.4) soil of the brown rendzina subtype. Soil fertility level was as follows: phosphorus medium (12.6–14.2 mg P2O5 in 100 g of soil), potassium medium (15.3–17.1 mg K2O in 100 g of soil), and magnesium medium (6.2–6.8 mg Mg in 100 g of soil). In each year of research, the forecrop for common beans was winter wheat. Tillage for bean was carried out in accordance with good agricultural practice [63]. Pre-winter plowing was performed in the first week of November. In the spring, soil treatment combined with mineral fertilization was performed. Mineral fertilizers were in the following doses: 30 kg N·ha<sup>−</sup>1, 60 kg P2O5·ha<sup>−</sup>1, and 120 kg K2O·ha<sup>−</sup>1. Mineral fertilization was used at a constant level throughout all experimental combinations. Beans of common bean, variety Oczko (with a red and white bean coat), were sown with a mechanical precision bean drill in the first week of May (2 May in 2015, 2016, and 2017) at a depth of 3–4 cm, in rows 45 cm apart, using 30 plants per 1 m2. Biostimulants Terra Sorb Complex, Kelpak, and Fylloton were used during the growing season, according to the experiment design (Table 1), and the obtained results were compared with the control, in which pure water was used for double spraying the plants. In individual years of research, biostimulants were used in time frames dependent on the development phase of plants, as shown in Table 1. Plants single sprayed with the biostimulant in BBCH 12-13, in the second period (plant stage of BBCH 61), were sprayed with pure water.


**Table 1.** Overview of biostimulant application in bean variety Oczko cultivation.

Abbreviations: single spraying BBCH 12-13—single spraying at the 2–3-leaf stage; double spraying BBCH 12-13, BBCH 61—double spraying first at the 2–3-leaf stage and second at the beginning of bean blooming.

The plants were sprayed using a GARLAND FUM 12B backpack sprayer. The Lechler LU 120–03 atomizer was used, at a working pressure of 0.30 MPa, using 300 L of the working liquid per 1 ha. All variants of treatment with biostimulants were compared with the control, where plants were treated with the same volume of water (no biostimulant was applied). No pesticides were used in the cultivation, as pathogens, pests and weeds did not exceed the damage threshold. Plants were weeded manually. The average temperature and rainfall during the bean growing season are shown in Table 2. The weather station (W200P, Vector Instruments Ltd., Rhyl, UK) was located in the experimental field, in which the experiment was carried out, at 210 m above sea level.

After harvesting plants in the third week of August (22 August 2015; 27 August 2016; 24 August 2017), 20 plants were randomly selected from each plot, and the number of pods, number of seeds, seed yield, and weight of one thousand seeds was determined. The beans obtained from each plot were dried, ground in a laboratory mill, and sieved with a 0.310-mm sieve. The flours were stored at −20 ◦C and used for further chemical analysis.


**Table 2.** Conditions during the growing seasons in bean variety Oczko cultivation in 2015–2017.

Abbreviation: T—temperature.

#### *2.2. Determination of Polyphenols*

A ground sample of bean seeds of 0.25 g was weighed, to which 4 cm<sup>3</sup> of extraction solution (acetone:water:hydrochloric acid 70:29:1) was added. The solutions were shaken for 1 h. Then, 100 μL of distilled water and 0.4 mL of Folina–Ciocalteu reagent were added to 100 μL of extract, and, after 10 min, 2 mL of 10% Na2O3 solution was added. After 30 min, the absorbance of methanol was measured at <sup>λ</sup> = 725 nm. The polyphenol content was calculated in mg·100 g−1, from the gallic acid calibration curve (1 mg·mL<sup>−</sup>1).

#### *2.3. Determination of Flavonoids*

A ground sample of bean seeds of 0.25 g was weighed, to which 4 cm<sup>3</sup> of extraction solution (acetic acid:methanol 1:19) was added. The solutions were shaken for 1 h. Then, 0.1 mL of a 2% AlCl3·6H2O methanolic solution was added to 1 mL of the extract, together with 1.4 mL of CH3COOH methanolic solution (1:19). The sample was then incubated at 20 ◦C for 30 min. Absorbance was measured at <sup>λ</sup> <sup>=</sup> 425 nm against methanol. The flavonoid content was calculated in mg·100 g<sup>−</sup>1, from the calibration curve for quercetin (0.2 mg·mL<sup>−</sup>1).

#### *2.4. Determination of Proteins*

The protein content of bean extracts was determined using the Bradford reagent, according to the method of Redmile-Gordon et al. [57] with modifications. The Bradford reagent (150 μL) was applied on a microplate and 50 μL of assay or standard protein (BSA, bovine serum albumin) was added. The samples were shaken at room temperature for 15 min. Absorbance at 595 nm was measured using an Epoch Microplate Spectrophotometer (BioTek-USA). The resulting protein was expressed in mg·g−<sup>1</sup> of dry weight (DW).

#### *2.5. Statistical Analysis*

The statistical analysis was performed using the Statistica 10PL program by StatSoft®. The normal distribution of variables was tested using the Shapiro–Wilk test. The one-way (for 2015, 2016, and 2017) and the two-way (for average 2015–2017) analysis of variance was used. The significance of the mean was determined using the Tukey test, at a significance level of *p* < 0.05.

#### **3. Results**

Based on the two-way ANOVA analysis, the effect of the number of applications and biostimulant treatment on seed yield, number of seeds, and phenol content in bean seeds was found (Table 3). The effect of biostimulant treatment on the number of pods, the weight of one thousand seeds, and the content of proteins and flavonoids in bean seeds were demonstrated. The interaction of biostimulant treatment with its number of applications regarding the impact on the number of seeds, as well as the content of protein and phenols in bean seeds, was found.



Number of applications (1 or 2); treatment (Fylloton; Terra Sorb Complex, Kelpak, control).

Treating plants with the biostimulants had a positive effect on increasing the number of pods (Table 4). In 2015, no significant effect of the biostimulants on the studied trait was identified; however, a tendency to increase the number of pods was observed after foliar application of biostimulants, especially Kelpak. On the other hand, in 2016, after treating the plants with biostimulants based on seaweed extract, i.e., both after a single application of Kelpak (*Ecklonia maxima* extract) and after a single or double application of Fylloton (*Ascophyllum nodosum* extract, amino acids), a significant increase in this trait was identified (increases by 36%, 28%, and 33%, respectively, as compared to the control). Treatment of plants with Kelpak in 2017 significantly increased the number of pods, regardless of the number of applications (increase by 18% for a single application, and by 19% for a double application, as compared to the control). A synthesis of the three years of research (2015–2017) confirmed that a single spraying of plants with the Kelpak biostimulant in the BBCH 12-13 phase significantly increased the number of pods (by 26%), as compared to the control.


**Table 4.** Effect of Fylloton, Terra Sorb Complex, and Kelpak biostimulants treatment on number of pods and seeds of bean variety Oczko.

Abbreviations: F\_1, single spraying of Fylloton; F\_2, double spraying of Fylloton; TS\_1, single spraying of Terra Sorb Complex; TS\_2, double spraying of Terra Sorb Complex; K\_1, single spraying of Kelpak; K\_2, double spraying of Kelpak; C, control; n.s., not significant. Means in the columns, concerning the selected traits, followed by different small letters are significantly different at *p* < 0.05.

Analysis of variance showed that, regardless of the number of applications, the treatment of plants with Fylloton significantly increased the number of seeds in 2015, as did the double spraying with Kelpak (increases by 29%, 34%, and 32% respectively, as compared to the control) (Table 4). In the second year of research, the best results were obtained after double spraying with biostimulant based on the extract of *Ecklonia maxima*, with a significant increase of 37%, as compared to the control. In 2017, it was found that spraying plants with biostimulants containing seaweed extracts, i.e., Fylloton and Kelpak, significantly increased the number of seeds, by 30% and 27%, respectively, as compared to the control. The average of three years of research demonstrated that an increase of this trait, by 31% and 32%, respectively, as compared to the control, was obtained after a double foliar application of Fylloton and Kelpak.

Double application of Kelpak, in 2015 and 2016, had the most beneficial effect on seed yield, increasing this trait by 22% and 38%, respectively, as compared to the control (Figure 1). In 2017, the best effects in increasing the bean crop were obtained after double treatment of plants with Fylloton and Kelpak biostimulants, when a 25% increase of this trait was obtained, as compared to the control. A synthesis of the three years of research demonstrated that double spraying plants with Kelpak biostimulant was most beneficial for increasing the bean yield (increase by 28% as compared to the control).

**Figure 1.** Effect of Fylloton, Terra Sorb Complex, and Kelpak biostimulant treatment on seed yield of bean variety Oczko. Abbreviations: F\_1, single spraying of Fylloton; F\_2, double spraying of Fylloton; TS\_1, single spraying of Terra Sorb Complex; TS\_2, double spraying of Terra Sorb Complex; K\_1, single spraying of Kelpak; K\_2, double spraying of Kelpak; C, control. Means over the study years followed by different small letters are significantly different at *p* < 0.05.

Analysis of variance showed that the use of biostimulants in bean cultivation resulted in a reduction of the weight of one thousand seeds (Figure 2). In the first year of research, a significant increase in the weight of one thousand seeds was obtained in the control, by 11%–15%, as compared to the combination with Fylloton or Terra Sorb Complex. However, in 2016 there were no significant differences in the weight of one thousand seeds between the combinations that included the biostimulants and the control. Plants in the control plot in 2017 were characterized by a higher weight of one thousand seeds by 8%, as compared to the double application of the Terra Sorb Complex. In turn, the average of three years of research confirmed that the highest weight of one thousand seeds (an increase of 9%, as compared to the double use of Fylloton) was obtained in the control.

Foliar application of biostimulants increased the protein content in the seeds (Table 5). In 2015 and 2017, the best effects were obtained after applying Terra Sorb Complex as a single or double spraying of plants, which increased this trait by 12% and 13% (in 2015), and by 9% and 10% (in 2017), respectively, as compared to the control. Double treatment of plants with the Terra Sorb Complex biostimulant in 2016 had the most beneficial effect on increasing the protein content by 13%, as compared to the control. A synthesis of the three years of research showed that, regardless of the number of applications, the use of an amino acid-based biostimulant significantly increased the protein content, as did the double application of the biostimulant containing the of the *Ecklonia maxima* extract (11%, 12%, and 10% increases, respectively, as compared to the control).

**Figure 2.** Effect of Fylloton, Terra Sorb Complex, and Kelpak biostimulant treatment on the weight of one thousand seeds of bean variety Oczko. Abbreviations: F\_1, single spraying of Fylloton; F\_2, double spraying of Fylloton; TS\_1, single spraying of Terra Sorb Complex; TS\_2, double spraying of Terra Sorb Complex; K\_1, single spraying of Kelpak; K\_2, double spraying of Kelpak; C, control. Means over the study years followed by different small letters are significantly different at *p* < 0.05.


**Table 5.** Effect of Fylloton, Terra Sorb Complex, and Kelpak biostimulant treatment on protein, phenol, and flavonoid content of bean seeds.

Abbreviation: F\_1, single spraying of Fylloton; F\_2, double spraying of Fylloton; TS\_1, single spraying of Terra Sorb Complex; TS\_2, double spraying of Terra Sorb Complex; K\_1, single spraying of Kelpak; K\_2, double spraying of Kelpak; C, control; n.s., not significant; DM, dry matter. Means in the columns, concerning the selected traits, followed by different small letters are significantly different at *p* < 0.05.

Double application of biostimulants containing seaweed extracts most favorably impacted the content of phenols in the seeds (Table 5). In 2015, an increase in the phenolic compound content in beans was noted after a double application of Fylloton (an increase of this trait by 82%, as compared to the control). However, after a single application of Kelpak, this trait was found to be reduced, as compared to the control. In turn, in 2016 and 2017, the best effects in increasing the phenol content were observed after a double application of Kelpak (increases by 42% and 41%, respectively, as compared to the control). No significant differences were found for this trait in the average for the years 2015–2017, but only a tendency to increase after a double application of biostimulants containing seaweed.

The application of preparations containing seaweed had the most beneficial effect on increasing the flavonoid content in the seeds (Table 5). In the first year of research, this characteristic was increased by 87%, as compared to the control, after a double application of Kelpak. In 2016, a single application of Fylloton had the most beneficial effect on the increase of the flavonoid content (by 98%, as compared to the control). On the other hand, in 2017, a significant increase in the flavonoid content was noted both with a single application of Fylloton and double of Kelpak (increases by 90% and 82%, respectively, as compared to the control). The average of three years of research showed that the application of all biostimulants significantly increased the flavonoid content, by 64%–88%, as compared to the control.

#### **4. Discussion**

The results of our research show a positive effect of natural biostimulants on bean yield, as well as on its quality. A more beneficial effect in modifying the yield components (number of pods and seeds, seed yield) was obtained after using biostimulants based on seaweed extract, especially upon double application of the *Ecklonia maxima* (Kelpak) extract. Only in the case of the weight of one thousand seeds was a reduction of this trait observed as a result of using biostimulants. The use of other biostimulants, i.e., Fylloton, which contains an *Ascophyllum nodosum* extract and amino acids, as well as Terra Sorb Complex containing amino acids, also had a positive effect on the yield components. In turn, the conditions favorable for setting pods and seeds, and increasing the weight of seeds were the most beneficial in 2016, when the double use of Kelpak resulted in the largest increase in these characteristics compared to control. This year, there were also favorable temperature and humidity conditions conducive to flowering, and setting pods and seeds. The significant increase in protein content was most positively influenced by weather conditions in 2015 and 2016, when the highest value of this feature was obtained after double application of Terra Sorb Complex. The application of biostimulants based on marine algae had a positive effect on the flavonoid content in all years of research. In 2015, however, a significant increase in polyphenols was found after double application of Fylloton.

Numerous studies conducted on arable crops confirmed the beneficial effect of seaweed extracts on increasing yield components [11,12,47,64–66]. Previous research on other bean and soybean varieties confirmed the stimulating effect of the *Ecklonia maxima* or *Ascophyllum nodosum* extracts on the number of pods and seeds, and the weight of beans [39–41,67]. The lack of effect of Kelpak on the one-thousand-seed weight of bean was also confirmed by previous studies on common bean [66]. Use of extracts from *Kappaphycus alvarezii* and *A. nodosum* increased the number of pods, seeds, and yield in soybean [68,69], even under stress conditions (reduced NPK fertilization) [70]. Bean plants reacted favorably to the foliar application of *Caulerpa racemosa* and *A. nodosum* extract by increasing the number of pods and seeds, one thousand weight of bean, and seed yield in common beans [71,72], mung beans [73], and broad beans [74].

The foliar application of amino acids positively affects the yield of many plants, even growing under stress [11,75,76]. In previous studies, the studied bean responded positively to foliar application of the Terra Sorb Complex; however, the yielding effect depended on the variety, concentration, and number of applications of the biostimulant, as well as climatic conditions prevailing in a given study year [59]. The Aura variety (with white seeds) responded more favorably to a single application of a 0.5% concentration, and the Toska variety (with red seeds) responded more favorably to a single application of a 0.3% concentration of this biostimulant. The plants increased the number of pods and seeds, as well as the seed yield; however, no effect of this biostimulant was found for the weight of one

thousand seeds. Foliar application of biostimulants containing amino acids increased the number of pods and seeds, weight of one thousand seeds, and yield in seeds of beans [71,77], peas [78], and broad beans [79,80].

The positive effect of seaweed extracts on plant growth and development and, as a result, on the increase in yield is undoubtedly associated with the presence of phytohormones, especially cytokinins [81]. Together with auxins, cytokinins regulate many physiological processes, including those affecting plant growth and development [82–84]. Aremu et al. [85] and Masondo et al. [86] also observed a positive effect of Kelpak on increasing the content of cytokinins. In addition, Kulkarni et al. [87] found an increase in the content of *cis*-zeatin, dihydrozeatin, and isopentenyladenine after using the *Ecklonia maxima* extract (Kelpak). The many active substances and compounds included in Kelpak suggest that it is not only cytokinin that is responsible for the growth and development of plants, but also probably the cross-reactions of these compounds with other bioactive molecules included in biostimulants that are based on seaweed extracts [70].

Thanks to the content of endogenous auxins, seaweed extracts have a positive effect on the growth and development of the root system [88]. This improves the uptake of water and nutrients and, in effect, stimulates the growth and development of plants, contributing to the improvement of yield quantity and quality [81]. The application of biostimulants based on seaweed extracts also has a positive effect on plant growth and development due to the content of gibberellins (GA1, GA3, GA4, GA5, GA6, GA7, GA13) [14], which affect seed germination, stem elongation, leaf expansion, and flower and seed development [89–91], as well as of gibberellin-like substances, e.g., terpenoids and tocopherol [92,93]. In the seaweed extract (Kelpak), the presence of brassinosteroids, brassinolide and castasterone [10], was identified. As phytohormones, brassinosteroids promote cell division and elongation, stimulate stem and root growth, and initiate flowering and flower development, as well as fruit development and increases in seed yield. Under stress conditions, they protect plants against abiotic and biotic stress [94,95].

The positive effect of amino acid-based preparations on plant growth and development probably results from the fact that, at the molecular level, they stimulate the plant's defense response to biotic and abiotic stress factors [96]. The amino acids contained in them are easily absorbed by plants. They participate in the synthesis of a number of organic compounds and affect the uptake of macro- and microelements [97]. Garcia et al. [98] showed that foliar application of amino acids and peptides together with nutrients increases the content of potassium, calcium, magnesium, iron, copper, and zinc in leaves, affecting their nutritional condition and promoting improved growth and development of plants. Applied on a leaf, preparations of this type exhibit phytohormone-like effects, comparable to that of auxin and gibberellin [54]. They also contribute to increasing the content of phytohormones (gibberellins, cytokinins, auxins) [76]. In addition, the use of protein hydrolysates in plant cultivation has a beneficial effect on the uptake of water and nutrients, resulting in increasing crop yielding thanks to the increased microbial and enzymatic activity of the soil, improved mobility and solubility of microelements (iron, zinc, manganese, copper), modified structure of the root system (its length, compaction, and number of lateral roots), or the increased synthesis of nitrate and glutamine reductase, as well as the activity of iron reductase [11,54,99–102]. Numerous reports confirmed that protein hydrolysates, such as auxins and gibberellins, have hormone-like effects, stimulating root and shoot growth. This, in turn, has a positive effect on crop productivity [11,53,54,100,102–106].

In our research, the use of biostimulants based on amino acids and seaweed extracts had a positive effect on the nutritional value of beans by increasing their protein content. Application of seaweed extract and amino acids had a positive effect on increasing the protein content in bean, pea, and faba bean seeds [59,71,74,78,80]. This was confirmed by Rouphael [62]; in their research, the increase in protein content in spinach plants was obtained after using biostimulants containing an extract of *Ecklonia maxima* and *Ascophyllum nodosum* and legume-derived protein hydrolysate. The protein content of legumes leaves was also determined by seaweed extracts. Numerous authors found an increase in this trait after the application of *Ulva rigida, Fucus spiralis, Hypnea musciformis,* and

*Colpomenia sinuosa* extracts in bean leaves [16,107,108]. However, the use of biostimulants is not always beneficial for the protein content in beans [15,109]. Schubert and Mengel [110] demonstrated that amino-acid uptake is an important mechanism for recovering carbon and nitrogen that was lost in the rhizosphere. Ertani et al. [100] found that the stimulation of nitrogen assimilation is responsible for accelerating the growth and metabolism of nitrogen in plants treated with protein hydrolysates. This is due to an increase in the activity of two key enzymes, nitrate reductase and glutamine synthetase, thereby contributing to increasing protein firmness.

After the application of biostimulants containing seaweed extracts, researchers observed an increase in the phenolic content. Ertani [56] showed that the treatment of plants with biostimulants stimulates numerous metabolic pathways in plants. The pathways are also associated with the synthesis of secondary metabolites, including phenolic compounds, which play an important role in protecting plants against stress factors. In turn, a frequent indicator of plant resistance to biotic factors is the content of phenolic compounds, which are precursors to more complex phenolic structures, such as flavonoids and lignins [60,111]. The presence of bioactive compounds in biostimulants, including phytohormones, amino acids, protein, and phenols, is responsible for the physiological response of plants treated with these preparations [56,112,113]. Eckol (phenolic compound) found in seaweed extracts affects the phenylpropanoid pathway in the biochemical synthesis of phenolic acids [114]. Aremu et al. [85,115] showed that the timing of eckol plants increases the content of phenolic compounds, such as *p*-hydroxybenzoic and ferulic acids. In turn, the Kelpak application increases the content of caffeic acid, ferulic acid [85,116], protocatechuic acid, *p*-hydroxybenzoic acid, gentisic acid, *p*-coumaric acid, and *trans*-cinnamic acid in *Eucomis autumnalis* [85]; however, the content of phenolic compounds depended on the biostimulant concentration. In turn, Rouphael et al. [62] found an increased content of phenolic compounds after the treatment of plants with biostimulants containing alginians, fucoidans, and laminarins that affect endogenic hormonal homeostasis [117]. Treatment of plants with eckol also had a positive effect on the flavonoid content, increasing the amount of kempferol in plants several times. However, foliar application of Kelpak increased the content of kaempferol in tubers and entire plants, as well as taxifolin in leaves of *Eucomis autumnalis* [85,115]. Increasing the content of bioactive compounds in plants treated with biostimulants is associated with a mechanism that includes the stimulation of the chalcone isomerase enzyme, involved in the biosynthesis of flavanone precursors [117].

Paul et al. [118] found that tomato plants treated with protein hydrolysates were characterized by a higher content of, e.g., low-molecular-weight phenolic compounds, phytohormones (polyamines), hydroxy-carotenoids, poly-hydroxy fatty acids, and membrane lipids (glycoand phospholipids). They suggest that the metabolic changes caused by treating plants with protein hydrolysates can be correlated with a relatively small number of processes that converge toward the ROS-related (reactive oxygen species-related) plant signaling network. Increasing the content of secondary metabolites, such as phenols and carotenoids, which play a key role in protecting plants against oxidative stress [62,102,119], suggests fine-tuning of ROS signaling in plants after the application of protein hydrolysates [118]. In addition, the use of animal protein hydrolysates had a positive effect on the content of protein, phenols, and flavonoids in bananas [120], and vegetal protein hydrolysates stimulated an increase in the content of phenolic compounds and anthocyanins in grape [55].

So far, there are few reports confirming the beneficial effect of a preparation consisting of a combination of seaweed extract and amino acids. In our research, combining the Fylloton biostimulant, which induces the extract's effect, with *Ascophyllum nodosum* and amino acids increased the yield components, particularly the number of seeds after its double application. Moreover, previous studies, conducted on three soybean varieties, confirmed that this preparation has a positive effect on the number of pods and seeds, as well as seed yield [40]. The application of Fylloton in the cultivation of winter oilseed rape positively influenced the increase in the number of pods, the yield of seeds, and the weight of one thousand seeds, especially after applying the biostimulant together

with the Perfektmikro micronutrient fertilizer containing EDTA-chelated (ethylenediaminetetraacetic acid-chelated) manganese, copper, iron, and zinc, as well as molybdenum, boron, and nitrogen [121].

#### **5. Conclusions**

All studied biostimulants had a positive effect on quantity and quality of bean yield. Double application of Kelpak biostimulant (*Ecklonia maxima* extract) stimulated morphological features and seed yield, as well as the content of polyphenols and flavonoids in seeds to a greater extent. In turn, the biostimulant containing amino acids (Terra Sorb Complex) significantly increased the protein content in beans. In contrast, Fylloton containing *Ascophyllum nodosum* extract and amino acids had also a more favorable effect on the number of seeds. In 2015 and 2017, biostimulants containing seaweed extracts had the most beneficial effect on bean yield and its quality. On the other hand, in 2016, treatment of plants with Kelpak had a more beneficial effect on the studied features.

**Author Contributions:** All authors read and agreed to the published version of the manuscript. A.K. and J.L. conceptualized and designed the research; R.K. and M.T. performed the field experiments; J.L, N.N., and K.K. analyzed the plant material; S.T. carried out statistical analysis of the data; A.K., R.K., and D.L. wrote the paper; M.T. and S.T. revised the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **Appraisal of Combined Applications of** *Trichoderma virens* **and a Biopolymer-Based Biostimulant on Lettuce Agronomical, Physiological, and Qualitative Properties under Variable N Regimes**

**Youssef Rouphael 1,\*, Petronia Carillo 2, Giuseppe Colla 3,\*, Nunzio Fiorentino 1, Leo Sabatino 4, Christophe El-Nakhel 1, Maria Giordano 1, Antonio Pannico 1, Valerio Cirillo 1, Edris Shabani 1,5, Eugenio Cozzolino 6, Nadia Lombardi 1, Mauro Napolitano <sup>1</sup> and Sheridan L. Woo 7,8,9**


Received: 7 January 2020; Accepted: 28 January 2020; Published: 1 February 2020

**Abstract:** The current research elucidated the agronomical, physiological, qualitative characteristics and mineral composition of lettuce (*Lactuca sativa* L. var. *longifolia*) after treatments with a beneficial fungus *Trichoderma virens*(TG41) alone or in combination with a vegetal biopolymer-based biostimulant (VBP; 'Quik-link'). The experiment consisted of lettuce plants grown in three N conditions: sub-optimal (0N kg ha−1), optimal (70N kg ha−1), and supra-optimal (140N kg ha−1) N levels. Lettuce grown under 0N fertilization showed a significant increase in fresh yield when inoculated with TG41 alone (45%) and a greater increase with TG41 + VBP biostimulant (67%). At 48 days after transplanting, both the TG41 alone or TG41+VBP biostimulant induced higher values of CO2 assimilation in comparison to the control. The mineral concentrations in leaf tissues were greater by 10% for K and 12% for Mg with the TG41+VBP treatments compared to the untreated lettuce. The lettuce plants receiving either TG41 alone or TG41+VBP biostimulants had a significantly lower nitrate content than any of the untreated controls. In non-fertilized conditions, plants treated with TG41+VBP biostimulants produced lettuce of higher premium quality as indicated by the higher antioxidant activity, total ascorbic acid (+61%–91%), total phenols (+14%) and lower nitrate content when compared to the untreated lettuce.

**Keywords:** microbial biostimulant; non-microbial biostimulant; Lactuca sativa L. var. *longifolia*; mineral profile; physiological mechanism; photosynthesis; nitrate; functional quality

#### **1. Introduction**

Rapid growth in the world population will determine an increase in global food demand that is expected to double by 2050 [1]. The intensification in agricultural production appears to be the only useful strategy to meet the rapidly growing food demand in the future, although this imposes stress to the agroecosystem [1], presents serious problems to the ecosystem and health [2–4], since it requires high-input resource cropping systems (such as greenhouse horticulture), that are not ecologically sustainable [5]. In actual fact, greenhouse farming systems use the highest amount of synthetic nitrogen (N) fertilizers per unit area of cultivated produce than any other cropping system [6–9].

Nitrogen-containing compounds are typically applied as chemical fertilizers in agriculture [10,11]. Nitrogen overuse and/or the imbalance between N and other nutrients, such as phosphorus, increases N losses while reducing nitrogen use efficiency (NUE) by the plant, which affects yield and, consequently, profit margins for farmers [12]. Moreover, the accumulation of excess nitrate in edible plant parts can be reduced to the nitrite form, which can cause diseases, such as methemoglobinemia, to which children are particularly at risk [13,14]. However, to date, the efforts to reduce N fertilizer use while at the same time attempting to increase NUE have been proven ineffective. This can be attributed to the inability of crop plants to adapt to low N availability conditions, which limit the activation of the physiological processes necessary for increasing crop production [7,15].

Recently, promising strategies that could aid a shift from N-intensive agriculture to a more eco-friendly approach that reduces the use of N fertilizers while simultaneously increasing NUE and yields, proposes the integrated use of non-chemical plant biostimulants (PBs) in cropping systems [16–20]. PBs are products able to enhance plant growth and development that include several substances with bioactive properties (seaweed and plant extracts, humic and fulvic acids, protein hydrolysates, and silicon), as well as some plant growth-promoting microorganisms (mycorrhizal fungi and plant growth-promoting rhizobacteria) [21–24]. Other plant beneficial microbes include fungi, such as *Trichoderma,* that have multiple plant beneficial capabilities, such as pathogen/pest control, increased nutrient uptake, stimulation of photosynthesis, and carbohydrate metabolism processes, that positively influence crop productivity and quality [25–29]. Several *Trichoderma* spp. are registered as microbial biological control agents in Plant Protection Products commercialized for the control of a broad-spectrum plant diseases [27,30]. Biocontrol mechanisms include direct antagonism with the production of secondary metabolites (i.e., hydrolytic enzymes, antibiotics), competition, and induced plant resistance [26–28,31–33]. Furthermore, many species, among *T. harzianum*, *T. virens, T. asperellum,* and *T. atroviride*, also act as plant biostimulants, able to enhance nutrient uptake and plant growth, or conferring plant tolerance to abiotic stress [25,34–38]. The direct and indirect benefits to the plants depend upon *Trichoderma*-plant molecular crosstalk, and exchange of diverse chemicals and small peptides that stimulate various plant responses [39,40]. These include the fungi metabolites, proven to have auxin and ethylene-like activity, that induce a reorganization of gene expression patterns in shoots and roots with significant changes in the plant metabolic machinery and a consequent improvement in plant resilience and yield [26,40–42]. These released compounds specifically modify plant root architecture, increasing root length, density and branching, and nutrient uptake (P, Fe, Mn, and Zn), in addition to acting as mediators in the plant microbiome for communication, warning signals, and pest management [25–27,43,44]. Recently, experiments conducted by Fiorentino and co-workers [25] on lettuce and rocket, grown under three different N fertilization rates and inoculated with two *Trichoderma* strains, demonstrated that, in particular, one strain of *T. virens* G41 (ex-*Gliocladium virens* GV41) was able to enhance NUE in lettuce, also favoring the uptake of native N present in the soil. Specifically, the benefits of inoculating plants with this *Trichoderma* strain were more evident when cultivation was performed under sub-optimal N conditions [25].

Another prominent category of PBs that has demonstrated beneficial effects on root stimulation, similar to those exerted by *Trichoderma*, is that of vegetal biopolymer-based products (VBP) that contain lateral root promoting peptides (LRPP) and lignosulphonates. In particular, the lignosulfonates obtained from sulfite pulping processes during cellulose extraction from wood are used in a variety

of industries, but they have also been used as fertilizers in crops [45]. They have proven auxin and gibberellin-like activities, probably due to the biological action of phenol metabolites able to interact with plant phytohormones and enzymes affecting carbon–nitrogen metabolism [45]. Lucini et al. [46] indicated that when the vegetal-based biopolymer was applied as a drench to melon, it altered the plant hormone profile by inducing an increase in ABA intermediates, brassinosteroids, and cytokinins in a dose-dependent manner. This mechanism stimulated root growth and consequently resulted in a 'nutrient acquisition response' improving resources use efficiency (RUE), thus enhancing plant biomass production and resistance to transplant stress. In addition, the authors reported that brassinosteroids may play a key role both in root system architecture changes as well as in shoot interference with hormone signaling and secondary metabolites, such as phenolic acids and carotenoids, plus the modulation of photosynthesis.

Romaine lettuce requires varying levels of N during the 65–75-day production cycle that depends upon the plant growth and development stages, plus N availability in the rhizosphere. N availability affects the morphological and physiological plant attributes [47] that influence the marketability of the leafy produce (i.e., leaf size) and consumer perception (i.e., visual green color). From this perspective, depending on the farming conditions, growing season, and genotypes, the combined application of *Trichoderma* and vegetal-biopolymer biostimulants could be particularly useful to enhance lettuce production due to their abilities to increase NUE, favoring nutrient uptake and utilization efficiency. Furthermore, the appropriate incorporation of N in the plant is important since the nitrate content in vegetable products must be within the limits established by the market according to EU regulation no. 1258/2011, whereby the levels should not exceed 3000–5000 mg kg−<sup>1</sup> fw.

In a recent opinion, Rouphael and Colla [23], indicated that the scientific community and private companies should focus on exploiting the potential synergistic biostimulatory action of microbes with non-microbial PBs combinations to design and develop second-generation plant products (biostimulant 2.0) with specific targeted biostimulant actions. A few experimental investigations have demonstrated the beneficial effects on crop performance of combining microbial inoculants (i.e., *Rhizophagus intraradices* or plant growth promoting bacteria or *R. irregulare* and *T. atroviride*) with humic acids [48,49] or protein hydrolysates [50]. Previous indications by Fiorentino et al. [25] suggested that the nutrient content of leafy horticulture crops could vary according to cultivation in diverse fertilizer conditions and in the presence/absence of a fungal inoculant. However, to date, nothing is known about the effects of *Trichoderma* alone or in combination with a vegetal biopolymer based-biostimulant on the agronomical, physiological and qualitative responses of an important leafy vegetables, such as Romaine lettuce (*Lactuca sativa* L. var. *longifolia*). This study will investigate the effect of a beneficial microbe (*T. virens* TG41) when used alone or in combination with a VBP biostimulant ('Quik-link'), under supra-optimal, optimal, and suboptimal N regimes, on Romaine lettuce production and marketability characteristics. This study will increase understanding of the processes involving these two different types of plant biostimulants and the effects on plant N acquisition response, for which the comprehension is pivotal to increasing NUE, as well as attempting to decrease N environmental inputs and reduce risks to consumer health.

#### **2. Materials and Methods**

#### *2.1. Experimental Setup, Design, and Crop Management*

An experiment was performed on lettuce (*Lactuca sativa* L. var. *longifolia* cv. 'Romana Bionda Lentissima a Montare'—Esasem, Casaleone, Verona) from November 4, 2015 to January 19, 2016, in a protected greenhouse structure (unheated) at the Department of Agricultural Sciences, University of Naples Federico II located at Portici, Italy. The soil was classified as a sandy loam texture (73% sand, 19% silt, 8% clay), with a pH of 7.0, electrical conductivity of 0.5 dS m−1, an organic matter of 1.25% (w/w) and a total N of 1.1 g kg<sup>−</sup>1. The NO3-N, NH4 <sup>+</sup>-N, available P, and exchangeable K were 95, 7, 35, and 950 mg kg<sup>−</sup>1, respectively.

A split-plot design with three replicates (randomized blocks) was adopted with fertilization (3 levels) as the main factor and biostimulant applications (3 levels) as the sub-factor. The three N fertilization levels were suboptimal (0 kg ha<sup>−</sup>1; 0N), optimal (70 kg N ha−1; 70N) and supra-optimal (140 kg N ha<sup>−</sup>1; 140N), while the three biostimulant applications were non-inoculated control, inoculated *Trichoderma virens* G41 (TG41), and *T. virens* + vegetal biopolymer-based biostimulant (TG41 + VBP). The cultivated area of each experimental plot (27 experimental plots in total) was 3.5 m2. Lettuce were transplanted on November 4th (at the 3 true-leaf stage) in double rows with a plant density of 14 plants per square meter. A biodegradable black mulch film (15 μm thick MaterBi®, Novamont, Novara, Italy) was used and maintained throughout the entire greenhouse experiment.

N total amount was applied as ammonium nitrate (NH4NO3 34%) into two identical doses, at 6 and 27 days after transplanting (DAT) by fertigation using a drip irrigation system with in-line emitters (flow rate: 3.3 L h−1; distances: 35 cm). Foliar pests, such as cutworms, were controlled with two applications of Decis Evo (active ingredient 25 g L−<sup>1</sup> of deltamethrin—Bayer Crop Science, Milano, Italy) at the rate of 0.4 L ha−1, whereas a copper-based fungicide (Cupravit 35 WG containing 350 g kg−<sup>1</sup> of copper as copper oxychloride—Bayer Crop Science, Milano) was sprayed twice at the rate of 2.5 kg ha−<sup>1</sup> to control downy mildew caused by *Bremia lactucae* Regel.

#### *2.2. Fungal and Vegetal Biostimulants*

A spore suspension of *T. virens* strain G41 (final concentration 1 <sup>×</sup> 107spores mL−1; TG41) was used to inoculate the lettuce seedlings at time of transplant by using a root dip method (with submergence for 10 min); then a repeated inoculation was conducted at 18 DAT by watering 25 mL of the inoculum plant<sup>−</sup>1. The vegetal biopolymer-based (VBP) biostimulant ('Quik-link®', Italpollina, Rivoli Veronese, Italy) was used in the current experiment. The product has a density of 1.21 kg L<sup>−</sup>1, a pH (1:5) of 4.7, an electrical conductivity; EC (1:5) of 20 mS cm<sup>−</sup>1, 25 g kg−<sup>1</sup> of organic N as peptides and free amino acids, 160 g kg−<sup>1</sup> of organic C, lignosulphonates, and micronutrients, such as iron, manganese, zinc, copper, and molybdenum, in the following concentrations 10.0, 7.0, 3.0, 1.0, and 0.2 g kg<sup>−</sup>1, respectively [46]. Peptides and free amino acids were obtained through enzymatic hydrolysis of a vegetal source of proteins, as reported by Carillo et al. [7]. The peptides in the product have a high biological activity being signaling molecules (e.g., lateral root promoting peptides—LRPP). The commercial product was applied at the base of each plant (100 mL, containing 6 L ha−<sup>1</sup> of 'Quik-link') at transplant, plus 17 (stage BBCH41-head beginning to form) and 45 DAT (stage BBCH45%–50% of the expected head size).

#### *2.3. Fungal Colony Forming Units in Soil Rhizosphere and Trichoderma-VBP Compatibility*

Soil samples were collected from the plant rhizosphere at the time of harvest. The number of fungal colonies forming units was determined, as indicated in Fiorentino et al. [25]. Briefly, a 1% (w/v) soil suspension was prepared in water, in serial dilutions, then 100 μL aliquots of each sample were spread on the surface of 90 mm culture plates containing Rose Bengal-Chloramphenicol agar (HiMedia Pvt. Ltd., Mumbai–India) supplemented with 0.1% (v/v) Igepal (Sigma–Aldrich, Milano, Italy), and incubated for 3–7 days at 25 ◦C. The emerging fungal colonies were counted daily.

In vitro tests were performed with varying doses of the VBP biostimulant and the *Trichoderma* inoculum, including the doses used for the field treatments to determine if the 'Quik-link' product inhibited the germination and growth of the fungus.

#### *2.4. Fresh and Dry Yield, SPAD index and CIE (lab) Measurements*

At harvest (76 DAT), the lettuce fresh yield was assessed in sampling areas of 2 m<sup>2</sup> from the center of the 27 experimental plots. The shoot dry biomass was determined (after oven drying at 80 ◦C for 72 h). The dried leaf tissues were conserved for mineral analysis. At 45 and 75 DAT, the soil plant analysis development (SPAD) index (i.e., non-destructive measurement of chlorophyll content) was measured on undamaged and expanded lettuce leaves using a portable SPAD-502 chlorophyll meter (Konica-Minolta, Tokyo, Japan). Twelve measurements were conducted on four randomly picked lettuce plants per experimental plot, then averaged to a single SPAD value for each replicate [51]. Subsequently, on the same date, measurements were performed using a Minolta CR-300 Chroma Meter (Minolta Camera Co. Ltd., Osaka, Japan) to evaluate the *Commission Internationale de L'Eclairage* (CIE) color space parameters for L\* (lightness) and chroma coordinates: a\* (−a\* greenness) and b\* (+b\* yellowness). In each experimental plot, 10 healthy leaves were measured and averaged to represent a single color value [52].

#### *2.5. Net CO2 Assimilation Rate and Stomatal Resistance Measurements*

At 33, 40, and 48 DAT, measurements of leaf gas exchange were carried out within 2 h across solar noon on the youngest fully expanded lettuce leaves, using nine replicates for each treatment. Measurements of net CO2 assimilation rate (Aco2; μmol CO2 m−<sup>2</sup> s−1) and stomatal resistance (rs; m<sup>2</sup> s−<sup>1</sup> mol<sup>−</sup>1) were recorded using a portable gas exchange analyzer (LCA-4; ADC BioScientific Ltd., UK). Photosynthetically active radiation, relative humidity, and carbon dioxide concentration (PAR, <sup>850</sup> <sup>±</sup> 100, 1000 <sup>±</sup> 100, and 600 <sup>±</sup> <sup>100</sup> <sup>μ</sup>mol m−<sup>2</sup> <sup>s</sup><sup>−</sup>1, RH 60 <sup>±</sup> 5, 55 <sup>±</sup> 5, and 60 <sup>±</sup> 5%, and 400 <sup>±</sup> 5, 410 <sup>±</sup> 5, and 400 ± 5 ppm, at 33, 40, and 48 DAT, respectively) were set at ambient value, and the airflow rate was 400 mL s<sup>−</sup>1.

#### *2.6. Mineral Composition Analysis*

Plant material was dried and pulverized using a cutting–grinder head (IKA, MF10.1, Staufen, Germany), then the powder was extracted in Milli-Q water (Merck Millipore, Darmstadt, Germany) for 10 min at 80 ◦C in a thermostatic bath (ShakeTemp SW22, Julabo, Seelbach, Germany) and centrifuged at 6000 rpm for 10 min as indicated in Rouphael et al. [50]. A Dionex ICS-3000 system (Sunnyvale, CA, USA) equipped with suppressed conductivity detection was used to determine the ion content of the samples. The ion separation of the samples was carried out with two different ion-exchange columns: An IonPac CS12A column (250 × 4 mm) was used for the cation separation eluted with 20 mM methanesulfonic acid (flow rate 1 mL min<sup>−</sup>1), and an IonPac AS11-HC column (250 <sup>×</sup> 4 mm) was used for the anion separation eluted with a potassium hydroxide gradient (flow rate 1.5 mL min<sup>−</sup>1). Nitrogen (total N) concentration in leaf tissue was determined according to the Kjeldahl method [53].

#### *2.7. Antioxidant Capacity, Total Phenols, and Total Ascorbic Acid Analysis*

Lipophilic and hydrophilic antioxidant capacity and total phenols were determined on freeze-dried tissue samples, whereas the total ascorbic acid was assessed on fresh material and measured using a spectrophotometer (Hach DR 2000, Hach Co., Loveland, CO, USA) according to the protocols of Re et al. [54], Fogliano et al. [55], Singleton et al. [56], and Kampfenkel et al. [57], respectively. Solution absorbances were assessed at 505, 734, 525, and 765 nm for the lipophilic and hydrophilic antioxidant fractions, total polyphenols, and total ascorbic acid, respectively.

#### *2.8. Data Elaboration, Statistical Analysis, Principal Component Analysis, and Heat Map*

The statistical analyses were all carried out using the software IBM SPSS Statistics 21. All data were subjected to two-way analysis of variance, and mean values were separated according to Duncan test with *p* < 0.05. Principal component analysis (PCA) was performed on the whole morphological and physiological data set, and the eigen values, total variance of the first three principal components (PCs) as well as the loading scores and plots were determined [58–60]. A heat map summarizing the agronomical, physiological, and qualitative responses of lettuce to plant biostimulant applications and N fertilization levels was also generated using the https://biit.cs.ut.ee/clustvis/ online program package with Euclidean distance as the similarity measure and hierarchical clustering with complete linkage [6].

#### **3. Results**

#### *3.1. Fungal Concentration in the Soil*

The total number of fungal colonies (including *Trichoderma*) recovered from soil rhizosphere in the nine treatments ranged between 2.0 <sup>×</sup> 105 and 6.5 <sup>×</sup> 105 colony forming units (CFU) g−<sup>1</sup> of soil and was significantly (*p* < 0.05) influenced by the interaction of the two tested factors: N fertilization level (N) and VBP biostimulant application. In particular, results indicated that the highest fungal CFU was observed in soils from lettuce plants inoculated with TG41 under suboptimal 0N conditions (6.5 <sup>×</sup> 105 CFU g−<sup>1</sup> of soil), in comparison to any of non-inoculated plants under suboptimal, optimal, or supra-optimal N conditions (average 2.5 <sup>×</sup> <sup>10</sup><sup>5</sup> CFU g−<sup>1</sup> of soil), whereas the treatments with TG41 (at 70N and 140N) or TG41 +VBP biostimulant (at 0N and 70N) exhibited intermediate values (average 3.9 <sup>×</sup> 105 CFU g−<sup>1</sup> of soil) (data not shown). Moreover, the in vitro tests performed with the beneficial microbe (TG41) and non-microbial VBP biostimulant at the dose applied in the greenhouse experiment did not demonstrate any inhibition of the germination and growth of the fungi concentration (69.2 CFU in the absence and 68.5 CFU in the presence of the 'Quik-link-product), suggesting compatibility between the two biostimulants.

#### *3.2. Growth Responses, SPAD Index and Leaf Colorimetry*

A significant (*p* < 0.01) interaction between N fertilization level and biostimulant application was observed on fresh yield and dry biomass. For instance, the use of the TG41-based biostimulant alone or in combination with the VBP biostimulant positively affected both fresh and dry yield of lettuce plants under both sub-optimal (0 kg ha<sup>−</sup>1) and optimal (70 kg ha−1) N conditions, but the beneficial effect was not apparent in the over N fertilization condition (140 kg ha<sup>−</sup>1) (Figure 1). Lettuce grown in the absence of N fertilization demonstrated a highly significant increase in fresh yield of 67% when inoculated with the combined TG41+VBP biostimulants. Instead, a more moderate increase of 45% was observed over the untreated 0N condition with the inoculation of *T. virens* G41 alone. Moreover, under optimal N fertilization (70 kg ha−1), only lettuce plants inoculated with TG41 alone exhibited significantly higher fresh yields. Treatments with TG41 alone or TG41+VBP increased marketable dry yield by 16% when compared to the untreated control, but no significant differences were noted between the two different biostimulant inoculations (Figure 1). No effects on lettuce yield were observed with either of the biostimulants at the supra-optimal 140N fertilization.

The SPAD index in *Lactuca sativa* L. var. *longifolia*, as an indication of chlorophyll content, was significantly affected by N fertilization levels (at 75 DAT) and by biostimulant applications (at 45 and 75 DAT), with effects in the N × T interaction (Table 1). At 75 DAT, the highest SPAD index values were recorded with TG41 + VBP biostimulant combination (Table 1). The visual appearance, particularly the greenness of leaf color, is a primary parameter used by the consumer in product preference and selection choice [61]. In general, neither the N fertilization level nor biostimulant application had a significant effect on the leaf greenness (−a\* values) in lettuce (Table 1). Overall, the N application levels resulted in a greater lightness in the color of the lettuce leaves, with the lowest L\* values recorded in the 140 kg N ha−<sup>1</sup> treatment, which also corresponded to a decrease in the chroma coordinate (b\*; Table 1).

**Figure 1.** Fresh yield (**A**) and dry biomass (**B**) of Romaine lettuce grown in greenhouse in relation to N fertilization level (0N = 0 kg ha<sup>−</sup>1, 70N = 70 kg ha<sup>−</sup>1, 140N = 140 kg ha<sup>−</sup>1) and biostimulant application (Untreated = Control, TG41 = *T. virens* G41, and TG41+VBP = vegetal biopolymer-based biostimulant). Mean values with the same letter were not different, according to Duncan's test (*p* < 0.05).



0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; NS, not significant. Mean values with the same letter in each column were not different according to Duncan's test (*<sup>p</sup>* < 0.05). DAT: days transplanting.

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#### *3.3. Leaf Gas Exchange: Net CO2 Assimilation Rate and Stomatal Resistance*

The physiological parameters, in particular, the net CO2 assimilation rate (ACO2) and stomatal resistance (rs) in the Romaine lettuce plants throughout the cultivation cycle in the greenhouse, were evaluated as a function of N fertilization level and biostimulant application as displayed in Table 2. The ACO2 was significantly affected by the biostimulant treatments for all measured data, and to a lesser degree, by the N fertilization level (only at 48 DAT). Irrespective of the N fertilization level (N × B interaction= ns) at 33 and 40 DAT, both the TG41 alone or in combination with the VBP-based biostimulant induced higher values of ACO2 in comparison to the control treatment that was not significantly different between the two biostimulant treatments. At 48 DAT, the ACO2 increased in the following order with the applications: TG41+VBP > TG41 > control (Table 2). On the other hand, augmenting the N fertilization level resulted in a linear increase in ACO2 from 0 to 140 kg ha−<sup>1</sup> but only at 48 DAT (Table 2).

Contrary to ACO2, the rs was not affected neither by N fertilization level nor by biostimulant application at 33 and 48 DAT, while at 40 DAT, the rs was only influenced by the two biostimulant applications (Table 2). Particularly, on this date, the rs was significantly lower on average by 26% when lettuce plants were inoculated with *Trichoderma* alone or in combination with the commercial product 'Quik-link' (Table 2).

#### *3.4. Mineral Composition in Leaf Tissue*

The results regarding the mineral profile in Romaine lettuce leaves are presented in Table 3. For all the macronutrients and sodium analyzed, no significant differences were observed in the N fertilization level and biostimulant application interaction. In particular, neither N fertilization rate nor biostimulant treatment had a significant effect on Ca and Na concentrations in lettuce leaves (average 7.0 and 1.4 g kg−<sup>1</sup> dry weight, respectively; Table 3). The concentrations of N and P in leaf tissues were significantly affected by N fertilization rate. Concentrations of N and P increased as the N fertilization level increased, with the highest values recorded at 140 kg ha−<sup>1</sup> for N and at 70 and 140 kg ha−<sup>1</sup> for P (Table 3).

The effects of TG41 and TG41+VBP biostimulant, when averaged over all N fertilization rates, affected the K and Mg concentrations in leaf tissues which were higher by 10% and 12%, respectively, than in untreated lettuce plants, but with no significant difference noted between the two biostimulant treatments (Table 3).


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#### *Agronomy* **2020** , *10*, 196

\* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; NS, not significant. Mean values with the same letter were not different according to the Duncan's test (*<sup>p</sup>* < 0.05). DAT: days after transplanting.

#### *3.5. Nitrate, Antioxidant Capacity, and Bioactive Content*

The registered nitrate content among the 9 experimental conditions (890–1496 mg kg−<sup>1</sup> fresh weight) was within the limits imposed by the European Regulation No. 1258/2011 for the commercialization of fresh lettuce (3000–5000 mg kg−<sup>1</sup> on a fresh weight basis). In our study, nitrate content was affected by both N fertilization level and biostimulant application, without significant effects in the N×B interaction (Table 4). As expected, our results demonstrated that increasing N fertilization from 0 to 140 kg ha−<sup>1</sup> elicited a significant increase in nitrate content compared to non-fertilized plants, whereas lettuce plants cultivated under optimal N fertilization (70 kg ha−1) exhibited intermediate values (Table 4). Interestingly, the nitrate content was significantly lowered in lettuce plants receiving treatments of either TG41 alone and the combined TG41+VBP biostimulants (not significant between them) compared to the untreated control (Table 4).

The hydrophilic and lipophilic antioxidant fractions of greenhouse lettuce ranged from 1.44 to 1.61 mmol ascorbic acid eq. 100 g−<sup>1</sup> dw and from 2.69 to 4.62 mmol trolox 100 g−<sup>1</sup> dw, respectively. Neither N fertilization level nor the biostimulant application had a significant effect on the hydrophilic antioxidant activity. Moreover, significant effects were noted on lipophilic antioxidant activity (LAA) with both N and biostimulant treatments, but not the N×B interaction. Irrespective of N fertilization treatments, the application of TG41+VBP demonstrated a significant increase in LAA (+13%) compared to the treatment of TG41 alone and the non-inoculated control (Table 4). Moreover, antioxidant molecules, in particular, total phenols and total ascorbic acid, were significantly influenced by either tested factors of N fertilization and biostimulant application. When averaged over the nitrogen treatments, the lettuce plants cultivated under supra-optimal conditions (i.e., 140 kg ha−1) were characterized by low-quality bioactive compounds in terms of both total phenols and total ascorbic acid (Table 4). Interestingly, the biostimulants-treated plants with TG41 alone and particularly in the combination of TG41+VBP, produced a major amplification of total phenols (+14%) and total ascorbic acid (+61%–91%) in comparison to untreated lettuce plants (Table 4).



#### *3.6. Heat Map Analysis of all Measured Plant Parameters*

An aggregated data heat-map analysis of the measured agronomic and physiological parameters was conducted to produce a visual comparison of the effects determined by the tested treatment factors on the Romaine lettuce plants. In Figure 2, the analysis revealed two dendrograms: on the top (Dendrogram 1), a classification that corresponded principally to the biostimulant applications, and on the left (Dendrogram 2), the parameters that influenced this distribution. Dendrogram 1 revealed two main groups: on the left, the cluster corresponded to controls for each of the three N levels that were all untreated with the biostimulant conditions; then on the right of the heat map, two clusters that contained the other six treatments, consisting of a mix of the N levels receiving the biostimulant applications (Figure 2).

In particular, in the left cluster of Dendrogram 1, the 140N Control was well separated from the other two controls (0N and 70N) due to the higher rs at 40 and 48 DAT; nitrate, Na, P, and dry biomass (in the first/highest cluster of Dendrogram 2), as well as the lower values for the parameters in the second cluster, mainly for the parameters of L\*, total phenols, Mg, and K content. On the right side of Dendrogram 1, two clusters were identified, the first on the left included treatments 70N TG41+VBP biostimulant, separated from the 140N level with the biostimulants TG41 or TG41+VBP, that showed in particular lower Na, rs at 40 and 48 DAT, hydrophilic (HAA), b\* and total phenols parameters, but higher LAA, a\* value, P and N content, SPAD index and ACO2 at 33 and 48 DAT. The grouping on the right included 70N TG41, 0N TG41, and 0N TG41+VBP treatments. Within this cluster, the 0N treatments with the biostimulants were clearly separated from the 70 N TG41 by higher HAA, leaf number (LN), and lower LAA in this latter treatment. Instead, the two 0N levels receiving the biostimulants were distinguished by the parameter groupings found in Dendrogram 2, whereby 0N TG41 could be attributed to the lower values for the parameters found in the third cluster (mainly due to a\*, SPAD Index, N), as well as the lower rs 40 DAT and nitrate, but higher b\*; whereas 0N TG41+VBP biostimulant could principally be identified by the all the higher parameters found in the second cluster—specifically total ascorbic acid (TAA). Interestingly, the first cluster in Dendrogram 2 clearly demonstrated the differential effects of the biostimulant treatments (untreated ones had high parameters for all N levels), while the second cluster clearly revealed the consequence of supra-optimal N levels (all parameter values were low), and the outcome of the combined biostimulants in the low N level condition (all parameter values were high), comparatively to the *Trichoderma* alone (i.e., TG41) at 0N.

**Figure 2.** Cluster heat map analysis summarizing greenhouse lettuce plant responses to a factorial experiment with three N fertilization levels (0N = 0 kg ha<sup>−</sup>1, 70N = 70 kg ha<sup>−</sup>1, 140N = 140 kg ha<sup>−</sup>1) and biostimulant application (Untreated=Control, TG41=*T. virens* G41, and TG41+VBP=vegetal biopolymer-based biostimulant). Control plants were not treated with TG41 and/or VBP. The figure was generated using the https://biit.cs.ut.ee/clustvis/ online program package with Euclidean distance as the similarity measure and hierarchical clustering with complete linkage. ACO2: net CO2 assimilation rate; rs: stomatal resistance; HAA: hydrophilic antioxidant activity; LAA: lipophilic antioxidant activity; TAA: total ascorbic acid; SPAD: soil plant analysis development; DAT: days after transplanting.

#### *3.7. Principal Component Analysis of all Measured Plant Parameters*

Principal component analysis was carried out on the whole experimental data set, and the loading plot and scores are reported in Figure 3. The analysis indicated that the variables in the first three principal components (PCs) were highly correlated, with eigen values greater than 1, thus explaining for 80.4% of the total variance, with PC1, PC2, and PC3 accounting for 36.1%, 32.0%, and 12.4%, respectively. The variable distribution along PC1 was clearly attributed to the biostimulant treatments, while N fertilization levels contributed to that on PC2 (Figure 3). TG41 and TG41+VBP biostimulant treated plants were distributed in the positive quadrants of PC1 except for 0N TG41, while all control treatments (untreated lettuce plants) were distributed in the negative side of PC1. In particular, 0N TG41+VBP biostimulant and 70N TG41 were in the upper right quadrant, while

70N TG41+VBP biostimulant, 140N TG41+VBP biostimulant, and 140N TG41 were in the lower right quadrant. Moreover, in PC2, 0N TG41 was positioned in the positive side of the upper left quadrant, with 0N and 70N untreated control treatments, while the 140N control was in the lower left negative quadrant (Figure 3). PC1 was positively correlated to ACO2 at 33, 40, and 48 DAT, K, Mg, and Ca content, yield (fresh weight), dry biomass, and SPAD index. PC1 was also negatively correlated with rs at 33, 40, 48 DAT, and also with nitrate content. PC2 was positively correlated with L\* and b\* colorimetric parameters, total phenols, and TAA, while it was negatively correlated to P content and a\* colorimetric parameter. In addition, the treatments with 70N TG41+VBP biostimulant and 140N TG41 produced lettuce with a higher yield, leaf number, SPAD index, and ACO2 at 48 DAT. Interestingly, the non-fertilized 0N lettuce plants treated with TG41+VBP produced lettuce with higher premium quality (higher total phenols and TAA and lower nitrate content) (Figure 3). Finally, the upper and lower left quadrant depicted the three non-treated control treatments with the lowest quality characteristics (high Na and nitrate content; Figure 3).

**Figure 3.** Principal component loading plot and scores of principal component analysis of all morpho-physiological and qualitative parameters analyzed in Romaine lettuce plants submitted to a factorial experiment with three N fertilization levels (0N = 0 kg ha<sup>−</sup>1, 70N = 70 kg ha<sup>−</sup>1, 140N = 140 kg ha<sup>−</sup>1) and biostimulant application (Untreated = Control, TG41 = *T. virens* G41, and TG41+VBP = vegetal biopolymer-based biostimulant). ACO2: net CO2 assimilation rate; rs: stomatal resistance; HAA: hydrophilic antioxidant activity; LAA: lipophilic antioxidant activity; TAA: total ascorbic acid; SPAD: soil plant analysis development; DAT: days after transplanting.

#### **4. Discussion**

Our findings indicated that the suboptimal fertilizer condition (0 kg N ha−1) sharply reduced yield, dry biomass, and ACO2, particularly at 48 DAT, whereas rs and sodium content increased. In fact, at 0 and 70 kg ha−1, the lower leaf N availability may affect photosynthetic performance and rate due to N remobilization from photosynthetic enzymes and pigments [62]. The decreased SPAD index, which is significantly correlated to chlorophyll concentration as indicated by absorbance measurements [63], corresponded to the decrease in photosynthetic capacity, and an increase in the sensitivity to photo-inhibition [64]. However, the application of TG41 alone, but especially in combination with the VBP, to lettuce grown in sub-optimal N induced significant changes in morphology and physiology, as noted with increased yield and dry biomass. Therefore, under low-input conditions (0 kg N ha−1), the combination of the microbial inoculant with the biopolymer-based biostimulant exhibited an important synergistic effect, thus confirming the beneficial effects on crop productivity as

previously reported by several authors [48–50]. Both PB treatments enhanced photosynthetic activity, SPAD index, and leaf nutritional status, as reflected by higher K and Mg and lower Na concentrations, that indicate a more efficient accumulation and translocation of assimilates to photosynthetic sinks that improve crop performance, but are not associated to the external N fertilization level applied [50]. Under optimal N conditions (i.e., recommended rate of 70 kg ha<sup>−</sup>1), the treatment with TG41 alone had the best effect on fresh yield, combined with high Mg and antioxidant contents, as well as low nitrate and Na. In this N regime, the addition of the VBP-based biostimulant to the fungal inoculant did not improve the morpho-physiological parameters, nor the mineral profile in the leaves. As mentioned above, growth under suboptimal N conditions increased leaf cell susceptibility to light-induced oxidative damages, a condition that plants are not capable of overcoming. However, the application of the combined microbial and VBP PBs induced a strong production of TAA, phenols, and probably glutathione, a metabolite that works cooperatively with ascorbic acid to generate antioxidant effects that safely detoxify accumulated reactive oxygen species (ROS), thus protecting the plant and increasing the photosynthetic rate [6,7].

The application of 140 kg N ha−<sup>1</sup> to lettuce was an excess condition that determined a plateau in yield and dry biomass but not in the N content, although there was an increase in nitrate and Na, as well as rs at 40 DAT. This demonstrated that plants supplied with high levels of N were not able to assimilate and reduce all the nitrate supplied, risking negative consequences by the accumulation of these compounds in the vacuoles. This was also reported by Di Mola et al. [65] in rocket plants and by Wang et al. [66] in leafy vegetables, whereby optimal and particularly supra-optimal N treatments were not always characterized by the best quality traits in the produce, but on the contrary, resulted in damage to the commercial, nutritional, and functional quality traits. These effects were similar to those noted in our lettuce plants under supra-optimal N conditions, i.e., low macronutrients and total ascorbic acid, high nitrate and sodium content. The application of both PBs under supra-optimal N level (e.g., 140 kg ha−1) significantly enhanced the N content and SPAD index while reducing nitrate content without affecting the CO2 assimilation rate and the accumulation of beneficial nutrients. The strong increase in the SPAD index at 140 kg ha−<sup>1</sup> in plants inoculated with TG41 or TG41+VBP biostimulants was also observed at 70 kg ha<sup>−</sup>1, suggesting that the biostimulants were able to increase the number and efficiency of photosynthesis systems and light-harvesting complexes (LHC), that allowed plants to "fine-tune" photosynthesis in the fluctuating spectral quality and light intensity conditions, thus avoiding ROS formation and photo-oxidation. This also allowed a higher use efficiency of nitrate, as confirmed by the lower concentration of this ion in leaf tissues when compared to the untreated control because of a more efficient reduction and assimilation processes [6,7].

Our results correspond to previous findings on the plant growth-promoting effect of fungi inoculants containing *Trichoderma* [25,26,29,30,33,38,67]. The presumed mechanisms behind the beneficial morpho-physiological effects on lettuce plants by TG41 could be due to the release of signaling molecules with auxin and ethylene-like activity [28], in particular, bioactive volatile compounds [43], which increased nutrient bioavailability to the plant, that improved their uptake, translocation, and accumulation within the plant [35]. In addition, it has also been demonstrated that *Trichoderma* in the rhizosphere stimulates root growth and reshapes its architecture, morphological changes which are pivotal for improving nutrient uptake, in particular, nitrate, Ca, Mg, and K [29,30,35,41]. The synergistic action of TG41 with the VBP biostimulant is of particular interest because it resulted in the production of premium quality lettuce traits, as is clearly exhibited by the PCA. The vegetal-biopolymer biostimulant action was probably due to the presence of phenol metabolites with auxin and gibberellin-like activities, that interacted with phytohormones and enzymes stimulating the activity of carbon–nitrogen metabolism and plant development [45,46]. Another putative mechanism behind the stimulation of plant growth and yield in response to VBP drench application could involve the increased presence of bioactive molecules, such as signaling peptides (LRPP) and lignosulphonates, which are typical compounds present in VBP [46]. A previous study reported that lignosulphonate treatments can improve N uptake and assimilation in plants through the stimulation of glutamate synthase and

glutamine synthetase, as well as by triggering photosynthetic activity through the stimulation of both rubisco enzyme activity, thus improving plant performance [45]. The improved NUE in lettuce treated with PBs enhanced not only the chlorophyll content (as represented by the increased SPAD index) but also the synthesis of antioxidant metabolites that were capable of re-activating photosynthetic activity that under sub-optimal N conditions without PBs, would be severely compromised. Finally, the synergistic beneficial effect on root system architecture, as previously shown by Colla et al. [38,68], determined a '*nutrient acquisition response*' improving resource use efficiency (RUE) that enhanced plant biomass production and the quality of the produce.

#### **5. Conclusions**

Our study on the leafy vegetable crop Romaine lettuce confirmed that inoculations with *Trichoderma* TG41 under optimal N conditions (70 kg ha−1) were able to improve the leaf nutritional status as indicated with the higher potassium and magnesium content and lower sodium content, plus providing the best yield performance of all tested conditions in terms of plant fresh and dry weight. Interestingly, the combined biostimulant applications of *Trichoderma* with the vegetal biopolymer-based product, in suboptimal fertilizer conditions of low N availability (0N kg ha−1), was more effective than the treatment of the microbial inoculant alone not only in improving yield but also in producing a premium quality marketable lettuce with higher lipophilic antioxidant activity and total ascorbic acid content. Together these biostimulants positively influenced plant morpho-physiological processes that improved the assimilation of nitrate and macronutrients and stimulated root system architecture reshaping, thus permitting increased bioabsorption or '*nutrient acquisition response*'. Moreover, the assimilatory pathways were stimulated, for which nitrate was used to synthesize chlorophyll (increased SPAD index) and the antioxidant metabolites, which, in turn, re-activated the CO2 assimilation activity normally decreased under sub-optimal N conditions. Therefore, the combination of microbial and non-microbial plant biostimulants represents a promising, efficient, and sustainable strategy for improving yield and quality of horticultural crops, such as lettuce, as well as improving cultivation in N compromised fields or low fertilizer input scenarios.

**Author Contributions:** Conceptualization, Y.R., G.C., and S.L.W.; methodology, Y.R., G.C., N.F., and S.L.W.; software, L.S., C.E.-N., A.P., M.G., and V.C.; validation, L.S., C.E.-N., A.P., M.G., E.S., N.L., M.N., and V.C.; formal analysis, A.P. and V.C.; data curation, C.E.-N., A.P., E.C., M.G., E.S., N.L., M.N., and V.C.; writing—original draft preparation, P.C. and Y.R.; writing—review and editing, Y.R., P.C., G.C., N.F., and S.L.W.; visualization, Y.R., P.C., G.C., and S.L.W.; supervision, Y.R., P.C., G.C., and S.L.W.; project administration, Y.R. and S.L.W.; funding acquisition, G.C. and S.L.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the following projects: MIURPON [grant number Linfa 03PE\_00026\_1; grant number Marea 03PE\_00106]; POR FESR CAMPANIA 2014/2020- O.S. 1.1 [grant number Bioagro 559]; European Union Horizon 2020 Research and Innovation Program, ECOSTACK [grant agreement no. 773554]; ProBio IZSM (Portici), n. D01 6309, 14/12/2016.

**Acknowledgments:** The authors would like to thank Antonio Pignataro and Mauro Senatore for their technical assistance in the greenhouse.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **A Composite Bioinoculant Based on the Combined Application of Beneficial Bacteria and Fungi**

**Henrietta Allaga 1, Bettina Bóka 1, Péter Poór 2, Viktor Dávid Nagy 1, Attila Sz ˝ucs 1, Istvánné Stankovics 3, Miklós Takó 1, László Manczinger 1, Csaba Vágvölgyi 1, László Kredics 1,\* and Péter Körmöczi <sup>4</sup>**


Received: 20 December 2019; Accepted: 31 January 2020; Published: 3 February 2020

**Abstract:** A composite soil bioinoculant containing beneficial bacteria and fungi was developed for biocontrol of plant pathogens, phosphorous mobilization, stem degradation, humification, and nitrogen fixation. A *Trichoderma asperellum* isolate with outstanding in vitro antagonistic abilities toward a series of plant pathogenic fungi was included as a potential biocontrol component. The selected strain was also shown to promote growth and increase photosynthetic activity of tomato plants. For phosphorous mobilization and stem degradation, a *Trichoderma atrobrunneum* strain was selected, which produced cellulose-degrading enzymes even in the absence of stem residues, while this ability increased 10–15-fold in the presence of ground maize stem. The strain was also shown to produce large amounts of enzymes liberating organically bound phosphorous, as well as cellulase and xylanase activities in solid-state fermentation on various plant residues. A *Streptomyces albus* strain with excellent peroxidase-producing abilities was selected as a potential humus-producing component, while an *Azotobacter vinelandii* strain with the potential to provide excess nitrogen for crops was included for nitrogen fixation. The assembled soil bioinoculant had positive effect on the uptake of certain important macro- and microelements (potassium, sodium, and manganese) from the soil by field-grown tomato plants. The applied screening strategy proved to be applicable for the assembly of a composite soil bioinoculant with notable application potentials.

**Keywords:** biocontrol; plant growth promotion; soil inoculant; *Trichoderma*; *Azotobacter*; *Streptomyces*

#### **1. Introduction**

Chemical pesticides and fertilizers are applied world-wide in agricultural production. Pesticides are used for the prevention and control of plant pests and diseases in order to reduce or eliminate yield losses and maintain product quality. However, there are serious concerns regarding the risks resulting from occupational exposure to them, as well as from environmental pollution leading to the presence of their residues in the food-chain and drinking water [1]. Chemical fertilizers are used to supply plants with necessary elements (primarily phosphorous and nitrogen), thereby improving crop productivity; however, their application is resulting in pollution with phosphates and nitrates. The agricultural run-off of phosphates deriving from fertilizers contributes to the eutrophication of fresh water bodies

and also presents a serious threat to the biodiversity in terrestrial ecosystems [2], while the increased run-off of nitrogen fertilizers results in nitrate pollution of surface and groundwater [3]. Therefore, the need for alternative, environment-friendly, microbial soil treatment strategies with favorable effects on crop plants is emerging all over the world. Microbial abilities of biocontrol, plant growth promotion, stem degradation, phosphorous solubilization, humification, and nitrogen-fixation can be exploited for the development of microbial soil inoculants to be applied in sustainable agricultural production.

One of the main challenges in the agricultural use of beneficial microorganisms as plant growth promoters and/or biocontrol agents (BCAs) is their frequently inconsistent field performance [4,5], which may be due to a series of biotic and abiotic factors. Among the abiotic factors, physicochemical properties of the rhizosphere such as pH, temperature, water activity, and the chemical composition of the soil are varying in space and time, which substantially influences the performance of biocontrol and plant growth promoting microorganisms. Particular agents may exert different activities under different soil environmental conditions. Inconsistent field performance has long been identified as the major impediment to the wide-scale commercialization of beneficial microorganisms for agricultural applications [6]. A possible strategy to counteract inconsistencies due to varying environmental conditions is the development of consortial soil inoculants consisting of multiple beneficial organisms. The combination of efficient plant growth promoting microorganisms and BCAs may result in an increased consistency of field performance during different periods of the growing season, thereby enabling a more predictable increase in crop yields [7].

The aim of this study was to assemble a consortial soil bioinoculant based on the combined application of beneficial bacteria and fungi with the potential of increasing pathogen control, plant growth and crop yield, stem residue degradation, phosphorous mobilization, humification, and nitrogen fixation in treated agricultural soils.

#### **2. Materials and Methods**

#### *2.1. Examined Strains*

The microbial strains involved in this study derived from the Szeged Microbiology Collection, Szeged, Hungary (SZMC). The *Trichoderma* strains included in the study (*Trichoderma asperellum* SZMC 20866, and SZMC 20786; *Trichoderma harzianum* species complex (THSC) members SZMC 20761, SZMC 20762, and SZMC 20869; *Trichoderma atroviride* SZMC 20780 and SZMC 20781; *Trichoderma virens* SZMC 20779; *Trichoderma gamsii* SZMC 20783; and *Trichoderma hamatum* SZMC 20784) were isolated from Hungarian agricultural soil samples and initially identified by sequence analysis of the internal transcribed spacer (ITS) region [8]. However, as ITS sequence analysis is not able to discriminate between species belonging to THSC, the species level identity of strains SZMC 20761, SZMC 20762, and SZMC 20869 was determined during this study by the sequence analysis of a fragment of the *tef1* alpha gene [9] as *Trichoderma guizhouense, T. guizhouense* and *Trichoderma atrobrunneum* (GenBank accession numbers MN750371, MN750372, MN750373), respectively. Fungal isolates were maintained on yeast extract-glucose medium (5 g L−<sup>1</sup> glucose, 5 g L−<sup>1</sup> KH2PO4,1gL<sup>−</sup><sup>1</sup> yeast extract, 20 g L−<sup>1</sup> agar).

*Streptomyces* sp. isolates (SZMC 0282, SZMC 0232, 00001, 00002, 00004, 00005, 00006, 00007, 00008, 00009, 00010, 00012, 00013, 00014, 00015, 00017, 00019, 00020, 00021, 00022, 00023, 00024, 00025, 00026, 00027, 00028, 00029, 00030, 00031, 00032, 00033, 00034, 00035, 00036, 00037, 00038, 00039, 00040, 00041, 00042, 00043, 00044, 00045, 00046, and 00047) and *Azotobacter vinelandii* SZMC 22195 were derived from soil samples. *Streptomyces microflavus* DSM 40561 was derived from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) strain collection and was included as control in the peroxidase-producing assays. Bacterial strains were maintained on glucose-yeast extract-malt extract *Streptomyces* medium (GYM-STR: 4 g L−<sup>1</sup> glucose, 4 g L−<sup>1</sup> yeast extract, 10 g L−<sup>1</sup> malt extract, 2 g L−<sup>1</sup> CaCO3, and 20 g L<sup>−</sup><sup>1</sup> agar).

#### *2.2. Determination of Biocontrol Index (BCI) Values*

Dual confrontation tests were performed in vitro in Petri dishes (90 mm in diameter) according to the method described by Szekeres et al. [10]. During the experiments, 11 different strains of plant pathogenic fungi were confronted with the *Trichoderma* strains, including 3 *Armillaria* species (*Armillaria mellea* SZMC 23638, *Armillaria ostoyae* SZMC 23080, and *Armillaria gallica* SZMC23076), 4 *Fusarium solani* species complex (FSSC) isolates (SZMC 11057F, SZMC 6241J, SZMC 11067F, and SZMC 11070F), as well as *Phoma cucurbitacearum* (SZMC 16088), *Alternaria alternata* (SZMC 16085), *Botrytis cinerea* (SZMC 6244J), and *Rhizoctonia solani* (SZMC 6252J). The tests were carried out in three replicates on malt extract agar (MEA) medium (10 g L−<sup>1</sup> glucose, 2.5 g L−<sup>1</sup> yeast extract, 20 mL L−<sup>1</sup> 20% malt extract, 20 g L−<sup>1</sup> agar). After the incubation period, digital photos were taken with a Nikon Coolpix P7700 camera (Nikon, Tokyo, Japan) from each Petri plate, and the area visibly covered by the *Trichoderma* strain as well as the area covered by *Trichoderma* and the pathogen together were calculated for each plate with the aid of the Image J software (http://imagej.nih.gov/ij). BCI values were calculated with Excel 2010 (Microsoft, Redmond, WA, USA) according to the formula: BCI = (area of *Trichoderma* colony/total area occupied by the colonies of both *Trichoderma* and the plant pathogenic fungus) × 100 [10].

#### *2.3. Liquid and Solid-State Fermentations*

For the investigation of cellulase and phosphatase activities, culturing was performed in 100 mL flasks containing 20 mL liquid minimal (10 g L−<sup>1</sup> glucose, 5 g L−<sup>1</sup> (NH4)2SO4,5gL<sup>−</sup><sup>1</sup> KH2PO4, 0.1 g L<sup>−</sup><sup>1</sup> MgSO4 <sup>×</sup> 7H2O) or maize stem medium (2 g L<sup>−</sup><sup>1</sup> dried maize stem ground with a coffee grinder (Bosch, Gerlingen, Germany), 1 g L−<sup>1</sup> NaNO3). The minimal medium was inoculated with *Trichoderma* conidia to a concentration of 2 <sup>×</sup> <sup>10</sup><sup>5</sup> mL<sup>−</sup>1, while the maize stem medium was inoculated with the total amount of 5-days-old fungal mycelium pre-grown in 20 mL liquid minimal medium, filtered with the aid of filter paper and vacuum pump and washed with sterile distilled water. After 5 days of incubation at 25 ◦C in an IKA KS 4000 IC Control shaker (ProfiLab24, Berlin, Germany) at 180 rpm, samples were filtered, and the culture filtrates were used for extracellular enzyme activity measurements.

Extracellular enzyme activities of *T. atrobrunneum* SZMC 20869 from THSC and the industrially important *Trichoderma reesei* strain QM9414 (SZMC 22616) were also compared in solid-state fermentation (SSF) experiments using maize, wheat, sunflower, and canola stem residues as substrates. One gram amounts of ground plant residues were placed into 50 mL Erlenmeyers flask and moisturized with 5 mL distilled water. After sterilization, the substrates were inoculated with 2 <sup>×</sup> 105 *Trichoderma* conidia. On the 8th day of fermentation, extractions were performed by adding 20 mL distilled water to the cultures and incubating for 3 h at 4 ◦C. The fluid phases were filtered through sterile gauze sheets into 15 mL centrifuge tubes and centrifuged at 4600 rpm for 10 min, 2 times by transferring the fluid phase to a new centrifuge tube. The 8× dilution of the fluid samples in distilled water were used for extracellular enzyme activity measurements.

#### *2.4. Enzyme Activity Measurements*

Cellobiohydrolase, β-glucosidase, β-xylosidase, and acidic phosphatase enzyme activities were measured with the chromogenic substrates p-nitrophenyl-β-d-cellobioside, p-nitrophenyl-β-d-glucopyranoside, p-nitrophenyl-β-d-xylopyranoside, and p-nitrophenyl-phosphate (Sigma-Aldrich, Budapest, Hungary), respectively. Enzyme reactions were carried out in three replicates in the wells of 96-well microtiter plates (Sarstedt, Nümbrecht, Germany) by mixing 100 μL culture filtrate or SSF extract with 100 μL substrate solution (1 mg mL−<sup>1</sup> in distilled water). After 1 h of incubation at room temperature, enzyme reactions were stopped with 10% (w/v) Na2CO3 and the optical densities measured at 405 nm with a Spectrostar Nano microplate reader (BMG Labtech, Ortenberg, Germany).

Peroxidase assays of bacteria were carried out in liquid *Streptomyces* induction medium (STR-IND) 6gL−<sup>1</sup> yeast extract, 8 g L−<sup>1</sup> xylan, 0.1 g L−<sup>1</sup> (NH4)2SO4, 0.3 g L−<sup>1</sup> NaCl, 0.1 g L−<sup>1</sup> MgSO4, 0.02 g

L−<sup>1</sup> CaCO3, 0.6 mL L−<sup>1</sup> TE (0.1 g L−<sup>1</sup> FeSO4, 0.002 g L−<sup>1</sup> MnSO4 <sup>×</sup> 7H2O, 0.09 g L−<sup>1</sup> ZnSO4 <sup>×</sup> 7H2O) inoculated with the examined *Streptomyces* strains. After 7 days of incubation (28 ◦C, 150 rpm), the samples were centrifuged at 7000 rpm for 10 min. The reaction mixture contained 0.2 mL phosphate buffer (100 mM, pH 7.2), 0.2 mL 2,4-dichlorophenoxy acetic acid (25 mM), 0.2 mL 4-aminoantipyrine (4AAP, 16 mM), 0.2 mL ferment broth, and 0.2 mL hydrogen peroxide (50 mM). The reaction mixtures were put into a 53 ◦C thermostat for 1 min, which was followed by the measurement of the optical density at 510 nm with a Spectrostar Nano microplate reader (BMG Labtech, Ortenberg, Germany). All measurements were carried out in three replicates.

In order to investigate the peroxidase enzyme production, dye decolorization assays were also performed. For this purpose, we used STR-IND medium supplemented with 20 g L−<sup>1</sup> agar and Remazol Brilliant Blue (RBB), Methyl Orange (MO), or Neutral Red (NR) dyes at a concentration of 1gL<sup>−</sup>1. The plates were inoculated with *Streptomyces* isolates with the aid of inoculation loop onto the middle of the Petri plates. Color changes were observed around the colonies after 1 week of incubation.

#### *2.5. Growth Assay in Nitrogen-Free Medium*

The growth kinetics of *A. vinelandii* strain SZMC 22195 were tested in nitrogen source-free liquid medium (5 g L−<sup>1</sup> glucose,5gL−<sup>1</sup> mannitol, 0.1 g L−<sup>1</sup> CaCl2 <sup>×</sup> 2H2O, 0.1 g L<sup>−</sup><sup>1</sup> MgSO4 <sup>×</sup> 7H2O, 0.005 g L−<sup>1</sup> Na2MoO4 <sup>×</sup> 2H2O, 0.9 g L−<sup>1</sup> K2HPO4, 0.1 g L−<sup>1</sup> KH2PO4, 0.01 g L−<sup>1</sup> FeSO4 <sup>×</sup> 7H2O, pH 7.3). During an incubation period of 1 week (30 ◦C, 120 rpm), the optical densities of the liquid cultures were measured on days 1, 2, 4, and 7 at 620 nm with a Spectrostar Nano microplate reader.

#### *2.6. Plant Material for Growth Chamber Experiments*

Seeds of tomato (*Solanum lycopersicum* Mill. L. cvar. Ailsa craig) were germinated at 26 ◦C for 3 days in the dark, and the seedlings were subsequently transferred to 6 × 6 cm pots filled with vermiculite (Terracult GmbH, Siegburg, Germany) for 6 weeks. Plants were irrigated every third day with nutrient solution containing 2 mM Ca(NO3)2, 1 mM MgSO4, 0.5 mM KCl, 0.5 mM KH2PO4, and 0.5 mM Na2HPO4, pH 6.0. The concentrations of micronutrients were 0.001 mM MnSO4, 0.005 mM ZnSO4, 0.0001 mM (NH4)6Mo7O24, 0.01 mM H3BO4, and 0.02 mM Fe(III)-EDTA. The plants were grown in a controlled environment under 300 μmol m−<sup>2</sup> s−<sup>1</sup> light intensity (emitted F36W/GRO lamps, Feilo Sylvania, Erlangen, Germany), with 12/12-h light/dark period, day/night temperatures of 24/22 ◦C, and relative humidity of 55–60%. Plants were treated with 20 <sup>μ</sup>L of *Trichoderma* suspension (1 <sup>×</sup> 10<sup>6</sup> conidia mL<sup>−</sup>1) after the 3-days-long germination. Samples for measurements were prepared in each replicate from the second, fully expanded young leaves of tomato plants. After harvest, the plant height and root length as well as the biomass production were recorded in 5 replicates.

#### *2.7. Measurement of Stomatal Conductance, CO2 Assimilation, and Total Soluble Sugar Content*

Stomatal conductance and CO2 assimilation were measured in 3 replicate samples by a portable photosynthesis system (LI-6400, LI-COR Inc., Lincoln, NE, USA), as described by Poór et al. [11]. Data were recorded after 15 min light adaptation on 300 μmol m−<sup>2</sup> s−<sup>1</sup> light intensity and under constant conditions (25 ◦C, 65 <sup>±</sup> 10% relative humidity, and controlled CO2 supply of 400 <sup>μ</sup>mol mol<sup>−</sup>1).

Total sugar contents were determined according to Dubois et al. [12]. One gram of leaf samples was homogenized in 10 mL distilled water and incubated in a 90 ◦C water bath for 45 min. Samples were centrifuged (12,000× *g* for 15 min, at 4 ◦C), and 40 μL of the supernatant was mixed with 400 μL of 1.8% phenol and 2 mL of concentrated sulfuric acid. The absorbance was measured from 5 replicate samples by a spectrophotometer at 490 nm.

#### *2.8. Chlorophyll a Fluorescence Measurements*

Chlorophyll a fluorescence was detected in 3 replicate samples with the portable photosynthesis system (LI-6400, LI-COR Inc., Lincoln, NE, USA) described above [11]. Leaves were dark-adapted for 15 min before the measurement of the minimal fluorescence (F0) using weak measuring light. The maximal fluorescence (Fm) was measured by applying a pulse (800 ms) of saturating light (12,000 μmol m−<sup>2</sup> s<sup>−</sup>1). The leaves were then illuminated continuously with actinic light (300 μmol m−<sup>2</sup> s<sup>−</sup>1). After 20 min, the light-adapted steady-state fluorescence (Fs) was recorded and the maximum fluorescence level (Fm') in the light-adapted state was determined with saturating pulses. The actinic light was next turned off and the minimum fluorescence level in the light-adapted state (F0') was determined by illuminating the leaf with 3-s far-red light (5 μmol m−<sup>2</sup> s<sup>−</sup>1). The following chlorophyll fluorescence parameters were calculated: the maximal quantum yield of PSII photochemistry, Fv/Fm = (Fm − F0/Fm); the actual quantum yield of PSII electron transport in the light adapted-state, ΦPSII = (Fm' − Fs)/Fm' [13]; the photochemical quenching coefficient, qP = (Fm' − Fs)/(Fm' − F0') [14]; and the non-photochemical quenching NPQ = Fm/Fm' − 1 [15].

#### *2.9. Pigment Analysis*

For pigment analysis, a two-step extraction was applied. Fifty milligrams of leaf samples were homogenized in ice-cold 100% (v/v) acetone (1 mL) and extracted for 24 h. Samples were centrifuged (12,000× *g* for 15 min at 4 ◦C). The pellet was extracted again with 80% (v/v) acetone (1 mL) for 24 h. After spinning down (12,000× *g*, 15 min, 4 ◦C), the supernatants were collected. The pigment composition was measured in 5 replicate samples as according to Lichtenthaler and Wellburn [16].

#### *2.10. Field Experiment*

A field study was performed in tomato culture (*Solanum lycopersicum* cvar. ACE-55) on sandy loam soil according to the yarn number of Arany (KA), which is a humus-rich soil with good nutrient availability and water management. The GPS coordinates of the examined area are 46◦05 01.05 N, 19◦26 28.83 E. Three-week-old tomato seedlings were planted on 11 May 2019. A total of 220 seedlings were planted in the field with 40 cm row distance and 40 cm plant distance, with 20 seedlings in a row. At the beginning of the experiment, a bioinoculant preparation consisting of a mixture of two *Trichoderma* strains and two bacteria was prepared. The concentration of the bioinoculant was adjusted to 106 conidia mL−<sup>1</sup> for both selected *Trichoderma* components, and 108 cells mL−<sup>1</sup> for both selected bacteria. The application of the soil inoculant was performed after 100× dilution at a concentration of 100 mL L<sup>−</sup>1, while the control area was not treated. Three control and eight treated rows were examined. No organic manure or chemical fertilizer was applied to the area during the soil preparation. Changes in the contents of soil macro- and microelements were measured three times during cultivation (I: 22 June, II: 6 July, and III: 3 August 2019) from rhizosphere samples taken from a depth of 15 cm. The total numbers of the control and treated plants were 60 and 160, respectively. During the experiment, the same plant protection measures were applied both in the control and treated area: Cuproxat FW (5 mL L−1), Topaz (0.5 mL L−1), Mospilan (200 mg L−1), Wuxal (2 mL L−1), Humusz (1 mL L−1), as well as calcium (5 mL L<sup>−</sup>1) and magnesium (5 mL L−1) in the form of foliar fertilizer were used 3 times during the cultivation (20 May, 8 June, and 13 July 2019). Control and treated plants were examined separately.

#### *2.11. Soil Examination Methods*

Soil sampling and analysis were carried out according to the test methods prescribed by the Hungarian Standard [17,18]. The soil tests were carried out by the Fels˝o-Bácskai Agrolabor Ltd., Bácsalmás, Hungary. Carbonate content was determined with a Scheibler's calcimetre (Bovimex, Székesfehérvár, Hungary). The total salt content was measured by the electric conductivity using a HI98311 conductivity meter (HANNA Instruments, Szeged, Hungary). These methods were based on the Hungarian Standard (MSZ) MSZ-08-0206-2:1978. The soil texture was determined by the yarn number of Arany (MSZ-08-0205:1978). The macro- and microelement content and the humus content were measured by a Lambda 25 UV/VIS Spectrophotometer (PerkinElmer, Waltham, MA, USA) according to the Hungarian standards MSZ 20135:1999 and MSZ-21470-52:1983, respectively. N-content was measured by the Kjeldahl method, K2O with flame emission spectrophotometry (FES), while Mg and Ca content with flame atomic absorption spectroscopy (FAAS) after acidic (H2SO4-HClO4) digestion [19]. Samples were prepared with microwave digestion for microelement analysis (Cu, Mn, Zn, and Fe content) measured with FAAS [17,19,20].

#### *2.12. Statistical Analysis*

Data presented as average values resulted from at least 3 independent experiments. Statistical analyses were carried out for the measurement data with Sigma plot v11.0 software (Systat Software Inc., Erkrath, Germany) using Student's t test, and the differences were considered significant if *p* < 0.05 (\*), *p* < 0.01 (\*\*), or *p* < 0.001 (\*\*\*). The statistical analyses of the soil results and crop yield data were performed with the GraphPad Prism v8.3.0 software (GraphPad Software Inc., San Diego, CA, USA) applying two-way ANOVA, and the results were considered significant if *p* < 0.05 (\*), *p* = 0.02 (\*\*), *p* = 0.001 (\*\*\*), and *p* < 0.0001 (\*\*\*\*).

#### **3. Results**

#### *3.1. Selection of the Components for the Soil Inoculant*

The potential biocontrol component of the soil inoculant was selected based on the results of dual confrontation tests between *Trichoderma* strains and plant pathogenic fungi. As shown in Table 1 and Figure 1, the two examined *T. asperellum* strains were the most effective against many of the tested pathogens. The BCI values of *T. asperellum* SZMC 20786 were the highest against FSSC SZMC 11067F and SZMC 11070F and *Alternaria alternata* 16085, while against *Rizoctonia solani* and *Armillaria gallica* they even reached 100, which means that the *Trichoderma* could completely overgrow and inhibit these plant pathogens. Except for *T. atrobrunneum* SZMC 20869 and *T. virens* SZMC 20779, all other *Trichoderma* strains could completely inhibit the *R. solani* strain SZMC 6252J. Based on the results, strain *T. asperellum* SZMC 20786 was selected as the potential biocontrol component of the soil inoculant.

Eight *Trichoderma* strains were included in the screening for cellulose-degrading and phosphatase-producing abilities (Figure 2). High levels of β-glucosidase and cellobiohydrolase enzyme activities could be measured in the case of three *Trichoderma* strains, which included two isolates of THSC (*T. guizhouense* SZMC 20761, *T. atrobrunneum* SZMC 20869) and one isolate of *T. hamatum* (SZMC 20784). The cellulolytic activities of SZMC 20869 were inducible with maize stem powder (Figure 2A,B), while the other two strains possessed high enzyme activity values only in liquid minimal medium. Only low phosphatase enzyme activities could be detected for the examined *Trichoderma* strains, except for the above mentioned *T. atrobrunneum* SZMC 20869 strain (Figure 2C), for which increased enzyme activity levels could be observed in liquid medium supplemented with maize stem powder. Based on the results, strain *T. atrobrunneum* SZMC 20869 from THSC was selected as the potential stem-degrading and phosphate-mobilizing component of the soil inoculant.

In the dye decolorization assays performed with *Streptomyces* isolates, color changes could be observed only when RBB was applied. Only the isolates *S. albus* SZMC 0232 and SZMC 0282 and *S. microflavus* DSM 40561 gave positive reactions with the RBB dye. The results of peroxidase assays are shown in Figure 3, indicating that only the isolates *S. albus* SZMC 0232 and SZMC 0282 showed an increased peroxidase production. The activities of these two strains were about twice as high as those of the control strain *S. microflavus* DSM 40561. Based on the results, strain *S. albus* SZMC 0282 was selected as the potential humus-producing component of the soil inoculant.



*Agronomy* **2020** , *10*, 220

**Figure 1.** In vitro antagonism of *Trichoderma* strains against different plant pathogenic fungi examined in dual confrontation tests.

**Figure 2.** Extracellular enzyme activities of *Trichoderma* strains in liquid minimal and maize stem medium (mean ± SE, *n* = 3). (**A**): β-glucosidase, (**B**): cellobiohydrolase, (**C**): phosphatase.

**Figure 3.** Peroxidase activities of *Streptomyces* isolates (mean ± SE, *n* = 3).

As the growth kinetics of strain *A. vinelandii* SZMC 22195 revealed that it reached the concentration of 108 cells ml−<sup>1</sup> after 4 days of incubation in nitrogen-free liquid medium and the cell concentration increased further until day 7 (Figure 4), this strain was selected as the nitrogen-fixing component of the soil inoculant.

**Figure 4.** Growth kinetics of *Azotobacter vinelandii* SZMC 22195 in nitrogen-free liquid medium (mean ± SE, *n* = 3).

#### *3.2. Influence of T. asperellum Strain SZMC 20786 on the Shoot and Root Growth and Photosynthetic Activity of Tomato Plants*

In the case of the *T. asperellum* strain SZMC 20786 selected for the composite bioinoculant, direct plant growth promotion and effects on photosynthetic activity were examined on tomato plants. Significant increases in the fresh weight of the roots and shoots could be recorded in comparison to the control plants (Figure 5). The results deriving from the measurements of stomatal conductance and CO2 assimilation are shown in Figure 6. Treatment with strain SZMC 20786 resulted in a non-significant

increase in stomatal conductance (Figure 6A), coupled with a significant increase in both the CO2 assimilation (Figure 6B) and the total sugar content (Figure 6C), indicating an increased photosynthetic activity in the plants treated with *T. asperellum*.

**Figure 5.** Effect of *T. asperellum* SZMC 20786 treatment on the growth parameters of tomato plants. (**A**) Root and shoot growth (mean ± SE, *n* = 10), (**B**) root and shoot biomass (mean ± SE, *n* = 10). Treated samples marked with asterisks are significantly different from the untreated control at *p* ≤ 0.01 (\*\*), or *p* ≤ 0.001 (\*\*\*).

**Figure 6.** Changes in stomatal conductance (**A**), CO2 assimilation (**B**), and total sugar content (**C**) in the leaves of tomato plants 6 weeks after treatment with *T. asperellum* SZMC 20786 (mean ± SE, *n* = 5). Treated samples marked with asterisks are significantly different from the untreated control at *p* ≤ 0.05 (\*), ns: not significant.

This is also supported by the changes of the chlorophyll a fluorescence induction parameters: the maximal quantum yield of PSII photochemistry (Fv/Fm), the actual quantum yield of PSII electron transport in the light-adapted state (ΦPSII), the photochemical quenching coefficient (qP), and the non-photochemical quenching (NPQ) were increased in plants treated with *T. asperellum* strain SZMC 20786; however, a significant change could be measured only for qP (Figure 7). The *T. asperellum* treatment also resulted in non-significant increases in the levels of chlorophyll a + b and carotenoids (Figure 8).

**Figure 7.** Changes of chlorophyll *a* fluorescence induction parameters ((**A**): Fv/Fm; (**B**): ΦPSII; (**C**): qP; (**D**): NPQ) in the leaves of tomato plants 6 weeks after treatment with *T. asperellum* SZMC 20786 (mean ± SE, *n* = 5). Treated samples marked with asterisks are significantly different from the untreated control at *p* ≤ 0.05 (\*), ns: not significant.

**Figure 8.** Changes in the content of chlorophyll a + b (**A**) and carotenoids (**B**) in the leaves of tomato plants 6 weeks after treatment with *T. asperellum* SZMC 20786 (mean ± SE, *n* = 5). ns: not significant.

#### *3.3. Solid State Fermentation of Plant Stem Residues with T. atrobrunneum in Comparison with T. reesei*

The extracellular enzyme activities of *T. atrobrunneum* SZMC 20869 (THSC) selected for the composite bioinoculant were compared with those of the industrially important, hypercellulolytic *T. reesei* strain QM9414 (SZMC 22616) in SSF experiments performed on the residues of 4 different crop plants (wheat, maize, sunflower, and canola) as substrates (Figure 9). Strain SZMC 20869 was able to produce β-glucosidase, cellobiohydrolase, β-xylosidase, and phosphatase activities on all four examined stem residues. Although the industrial strain of *T. reesei* produced larger amounts of β-glucosidase, cellobiohydrolase, and β-xylosidase—the three examined plants' cell-wall-degrading enzymes (PCWDEs)—on maize stem and canola stem residues, and was also a better producer of the two cellulolytic enzyme activities on sunflower stem residues than *T. atrobrunneum* SZMC 20869; the selected *T. atrobrunneum* strain was more efficient in production of all examined PCWDEs on wheat straw as the substrate. Regarding phosphatase activities, strain SZMC 20869 had been proved to be better than *T. reesei* on sunflower stem residues and equal to it on maize stem residues.

**Figure 9.** Extracellular enzyme activities of *T. atrobrunneum* SZMC 20869 and *Trichoderma reesei* SZMC 22616 after 8 days of solid-state fermentation on stem residues of wheat, maize, sunflower, and canola plants as substrates. (**A**) β-glucosidase, (**B**) cellobiohydrolase, (**C**) β-xylosidase, and (**D**) phosphatase (mean ± SE, *n* = 3). -: *T. atrobrunneum*; : *T. reesei.* Columns of *T. reesei* marked with asterisks are significantly different at *p* ≤ 0.01 (\*\*), or *p* ≤ 0.001 (\*\*\*).

#### *3.4. Field Experiment with the Combination of the Selected Bioinoculant Strains in Tomato Culture*

Soil initial chemical and physical characteristics of the experimental area were as follows: pH: 7.6 (KCl), KA: 35, carbonate: 10.6 m/m%, humus content: 1.84 m/m%, P2O5: 2423 mg/kg, K2O: 669 mg/kg, NOX-N: 11 mg/kg; SO4-S: 39 mg/kg, Cu: 4.4 mg/kg, Zn: 7.6 mg/kg, Mn: 13 mg/kg, Mg: 233 mg/kg, and Na: 99 mg/kg.

Changes in the soil macroelement, microelement, and humus content were monitored three times during the cultivation (Table 2). The initial potassium, sodium, and manganese contents of the total area determined from soil samples taken on 11th May were 669 mg kg<sup>−</sup>1, 99 mg kg−1, and 13 mg kg−1, respectively. According to the statistical analysis, the K2O, Na, and Mn content of the soil changed significantly in the treated area compared to the control area. This could be the result of a more efficient uptake of K2O, Na, and Mn in the case of the treated plants; however, it is also possible that the elements P, Na, and Mn could have been sequestered in the microbial biomass. The average tomato fruit crop yields per row were 22,470 g and 22,810 g for the control and treated rows, respectively; however, the increase in the crop size in the case of the treated rows did not prove to be significant. The average total green mass of control and treated rows were 11,613 g and 11,032 g respectively, with no significant difference.


**Table 2.** Changes in the macro- and microelement content of the soil during the cultivation period of tomato.

Soils samplings were performed on I: 22 June, II: 6 July, and III: 3 August 2019. Data marked with asterisks are significantly different from the untreated control at *p* < 0.05 (\*), *p* = 0.001 (\*\*\*), *p* < 0.001 (\*\*\*\*).

#### **4. Discussion**

The soil inoculant developed during this study contains two *Trichoderma* strains (*T. asperellum* and *T. atrobrunneum*) and two bacteria (*A. vinelandii* and *S. albus*) with potentially synergistic beneficial effects.

Members of the genus *Trichoderma* are geographically widespread filamentous ascomycetes from Hypocreales [21], which have long been known as agriculturally important, beneficial fungi with antagonistic abilities toward plant pathogenic fungi. *Trichoderma* antagonism is based on a series of different mechanisms including the competition for space and nutrients, antibiosis, mycoparasitism [22], plant growth promotion [23], enhancement of plant resistance to diseases [24,25], and relieving abiotic stress in plants [26]. These properties make many representatives of the genus *Trichoderma* (e.g., the THSC, *T. asperellum*, *T. atroviride*, or *T. virens*) to potential ingredients of soil inoculant and biocontrol preparations. However, when the practical application of a *Trichoderma* strain is planned, an exact, sequence-based, species-level identification is important to prevent the spread of species known as the causal agents of the green mold disease in mushroom cultivation [27–29] or of opportunistic infections in immunocompromised humans [30].

Strain *T. asperellum* SZMC 20786 was selected as a component of the composite soil bioinoculant due to its good in vitro antagonistic performance against different plant pathogenic fungi and its abilities to promote the growth of tomato plants and increase their photosynthetic activities. According to the literature, one of the mostly studied strains of "*T. asperellum*" for plant growth promotion is strain T203 [31], which, however, was recently reidentified as *T. asperelloides* [32]; thus, the number of studies about the plant growth promoting activities of *T. asperellum* sensu stricto is restricted. Qi and Zhao [33] demonstrated the plant growth promoting activities of *T. asperellum* srain Q1 on cucumber plants, and the positive effects were detected even when the plants were subjected to salt stress. In our study, *T. asperellum* strain SZMC 20786 showed positive effects on the CO2 assimilation, total sugar content, and the photochemical quenching coefficient of tomato leaves. Similar results were obtained by Doni et al. [34], who found plant growth promotion as well as increased stomatal conductance and CO2 assimilation in rice plants treated with *Trichoderma* sp. isolates. Other studies reported about the positive effects on the photosynthetic pigments exerted by *T. harzianum* strains on tomato [35] and wheat plants [36], as well as by *T. hamatum* on mungbean [37].

Another beneficial trait of many *Trichoderma* strains is their efficient ability to produce PCWDEs including cellulases and xylanases, which can be exploited both in the biotechnological industry and in the agriculture for the degradation of cellulose and xylan-containing materials, e.g., stem residues [38,39]. In accordance with these, another *Trichoderma* component, a *T. atrobrunneum* isolate possessing good cellulase, xylanase, and phosphatase enzyme production capabilities has been included in the assembled soil inoculant.

The species *A. vinelandii* involves Gram-negative, aerobic, free-living soil-inhabiting gamma-proteobacteria from the *Pseudomonadaceae* family. This species is capable of direct nitrogen fixation from the atmosphere by three distinct nitrogenase systems under fully aerobic conditions, thereby providing plant roots with bioavailable nitrogen source [40]. This aerobic bacterium possesses various protection mechanisms for nitrogenase against oxygen, which include alginate formation [41]. Furthermore, phytohormone and siderophore synthesis as well as phosphate solubilization are also among the abilities of *Azotobacter* species. These properties were suggested to be directly involved in their plant growth promotion effect [42]. Considering the above facts, an *A. vinelandii* strain has also been included in the soil inoculant.

As peroxidases were shown to play an important role in the humification properties of *Streptomyces* species [43], the potential humus-producing component of the bioinoculant was selected from *Streptomyces* isolates with a peroxidase assay, which revealed a *S. albus* strain as the best peroxidase-producing isolate. The species *S. albus* is among the geographically most widely distributed members of the genus *Streptomyces*; it could be isolated from various habitats including sea sediments, sponges, and insects [44]. This species was shown to be able to biosynthesize heterologously diverse and important natural products and was suggested to encode important natural product gene

clusters [44]. Based on the genome sequence of *S. albus*, the secretion of a series of degradative enzymes (including amylases, chitinases, glucanases, proteinases/peptidases, and a cellulase) with supposed roles in breaking down heterogeneous alternative food sources in soil could be predicted [45]. The feather-degrading abilities of *S. albus* could be exploited during the development of an eco-friendly biofertilizer feather compost [46].

Several publications are available in the literature about the combined application of beneficial microorganisms for plant growth promotion and biological control. There are studies about the co-application of multiple bacteria including the combination of *Bacillus* and *Pseudomonas* for growth promotion and biological control of soil-borne diseases in pepper and tomato [47], as well as to increase rice yields [48], or the application of a mixture of fluorescent pseudomonads to suppress take-all disease of wheat [49]. There are also examples of co-application of fungi and bacteria, for instance the combination of *T. koningii* with fluorescent pseudomonads for the control of take-all disease of wheat [50], or the combination of *T. harzianum* with an *Alcaligenes* strain for the reduction of the incidence of rot disease caused by *Phytophthora capsici* in black pepper [51]. Other microbial combinations have been applied to promote the growth of tomato [52], to control tobacco diseases [53], or to improve the salinity tolerance of *Vicia faba* [54]. The combination of microorganisms could also increase the dry matter yield and nutrient uptake by wheat grown in a sandy soil [55].

#### **5. Conclusions**

The selection of the components for the composite soil inoculant assembled in this study was driven by the idea to combine various crop protective and plant growth promoting traits (biocontrol against plant pathogenic fungi, phosphorous mobilization, stem degradation, humification, and nitrogen fixation) of different microorganisms into a preparation, which may also have the potential to exert an increased consistency of field performance under various environmental conditions. The screening strategy performed during this study proved to be applicable for the assembly of a promising composite soil bioinoculant with notable application potentials.

**Author Contributions:** H.A., P.K., C.V., L.M. and L.K. contributed to the design and implementation of the research and evaluation of the results, and participated in the preparation of the manuscript; P.K. and L.K. performed the in vitro antagonism and liquid fermentation experiments; B.B. and L.M. contributed to the selection of the bacterial components of the bioinoculant; P.K. and P.P. designed and performed the experiments on tomato plants; V.D.N. and M.T. contributed to the solid-state fermentation experiments; H.A. performed and evaluated the field experiment; I.S. performed the measurements of the soil samples taken; and A.S. performed data curation and statistical analyses. The Tables and Figures were prepared by H.A., P.K., B.B., P.P., V.D.N. and C.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the Hungarian Government and the European Union within the frames of the Széchenyi 2020 Programme (GINOP-2.3.2-15-2016-00052) and the Hungary-Serbia IPA Cross-border Co-operation Programme (PLANTSVITA project, HUSRB/1602/41/0031). L.K. and M.T. are grantees of the János Bolyai and Bolyai Plus Research Scholarships.

**Acknowledgments:** The authors thank Sarolta Szabó and Gábor Tarnai (BioeGO Ltd.) for their support during the performance of the field experiment.

**Conflicts of Interest:** The authors declare no conflict of interests.

#### **References**


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*Article*
