**Impact of Sodium Hypochlorite Applied as Nutrient Solution Disinfectant on Growth, Nutritional Status, Yield, and Consumer Safety of Tomato (***Solanum lycopersicum* **L.) Fruit Produced in a Soilless Cultivation**

**Maira Lykogianni 1,2, Eleftheria Bempelou 3, Ioannis Karavidas 4, Christos Anagnostopoulos 3, Konstantinos A. Aliferis 1,5,\* and Dimitrios Savvas 4,\***


**Abstract:** Soilless crop production is spread worldwide. It is a cultivating technique that enhances yield quality and quantity, thus contributing to both food safety and food security. However, in closed-loop soilless crops, the risk of spreading soil-borne pathogens through the recycled nutrient solution makes the establishment of a disinfection strategy necessary. In the current study, sodium hypochlorite was applied to the recycled nutrient solution as a chemical disinfectant to assess its impact on plant growth, leaf gas exchange, fruit yield, tissue mineral composition, and possible accumulation of chlorate and perchlorate residues in tomato fruits. The application of 2.5, 5, and 7.5 mg L−<sup>1</sup> of chlorine three times at fortnightly intervals during the cropping period had no impact on plant growth or gas exchange parameters. Furthermore, the application of 2.5 mg L−<sup>1</sup> of chlorine led to a significant increase in the total production of marketable fruits (total fruit weight per plant). No consistent differences in nutrient concentrations were recorded between the treatments. Moreover, neither chlorate nor perchlorate residues were detected in tomato fruits, even though chlorate residues were present in the nutrient solution. Therefore, the obtained tomatoes were safe for consumption. Further research is needed to test the application of chlorine in combination with crop inoculation with pathogens to test the efficiency of chlorine as a disinfectant in soilless nutrient solutions.

**Keywords:** soilless; disinfection of nutrient solution; chlorates; perchlorates; tomato classes

#### **1. Introduction**

Independence from the soil as a means of rooting allows the optimization of both physical and chemical characteristics in the root environment, as well as a more effective control of the phytopathogenic microorganisms [1]. These characteristics result in higher crop yields with usually lower production costs, combined with reduced pesticide use and high product quality [2]. Given these shortcomings of soil-based production systems, crop production worldwide has shifted to soilless culture in greenhouses, and one of the main reasons for this development is the more efficient control of soil-borne pathogens [3]. Nevertheless, soilless cultivation provides a free start from pathogens but cannot exclude the incidence of a pathogen infection during the cropping period. Especially in closed soilless systems, there are reports on the spread of phytopathogens through the recycled nutrient solution (NS), leading to complete crop failure [4,5]. In closed-loop soilless cultivation systems, the spread of plant diseases associated with the recycling of the NS has been

**Citation:** Lykogianni, M.; Bempelou, E.; Karavidas, I.; Anagnostopoulos, C.; Aliferis, K.A.; Savvas, D. Impact of Sodium Hypochlorite Applied as Nutrient Solution Disinfectant on Growth, Nutritional Status, Yield, and Consumer Safety of Tomato (*Solanum lycopersicum* L.) Fruit Produced in a Soilless Cultivation. *Horticulturae* **2023**, *9*, 352. https:// doi.org/10.3390/horticulturae9030352

Academic Editor: Francesco Giuffrida

Received: 31 January 2023 Revised: 25 February 2023 Accepted: 3 March 2023 Published: 7 March 2023

**Copyright:** © 2023 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 (https:// creativecommons.org/licenses/by/ 4.0/).

suggested to be among the most important problems for growers [6]. Hence, disinfection of the NS by applying chlorine is a common method of NS disinfection in closed-loop soilless crops. However, in the Mediterranean greenhouses, periodic application of chlorine as a NS disinfectant is a common practice also in open soilless crops, which constitute the standard type of soilless systems in the region. Nevertheless, the safety of chlorine application is still an open question, as there are limited data about the possible accumulation of chlorate/perchlorate residues in harvested vegetables originating from soilless cultivations treated with chlorine [7].

Soilless culture is a cultivation technique that can increase the quantity and quality of production while reducing water use [8], thus contributing to the requirements for food security and sustainability [9]. Moreover, many governmental and non-governmental organizations highlight its benefits for food security [10–13]. In fact, the imperative need to meet the increasing nutritional needs of the growing population has upgraded the importance of soilless cultivation as an efficient crop production system [14].

In addition, soilless cultivation is a controlled environment system of agricultural production that could enhance food safety in a crop, thanks to the lower need for application of agrochemicals against soil-borne plant diseases [1] and the much lower risk of contamination with heavy metals originating from polluted soils [13,15]. Nevertheless, it has been pointed out in the literature that there are safety risks also in soilless grown vegetables [16–18], although there are approaches towards resolving such issues [19]. Nonetheless, although soilless culture provides a free start from soil-borne pathogens [8], various soil pathogens have been detected in soilless crops, e.g., fungi, bacteria, viruses, and nematodes [4]. Hence, the need to install disinfection systems in soilless cultivation systems is imperative. The chemical methods of NS disinfection in a soilless culture include the use of chlorine (Cl), chlorine dioxide, bromine, ozone, hydrogen peroxide, etc. [4]. Regarding chlorine, it can be applied in liquid, solid, or gaseous form [20]. The most common method, especially in the Mediterranean basin [21], is the application of Cl in its liquid form as sodium hypochlorite, namely bleach [4,7]. In general, the recommended Cl concentration after the addition of sodium hypochlorite is between 2.5 and 5 mg L−<sup>1</sup> for the treatment of various fungi and oomycetes, such as species of the genus *Pythium* sp. [22,23], *Phytophthora* sp. [24], *Fusarium oxysporum* f. sp. *dianthii* [25], bacteria such as *Agrobacterium tumefaciens* [26], *CLSV* (cucumber leaf spot virus) [27] and root-knot nematodes (*Meloidogyne javanica*) [28], so that no phytotoxicity is expected. Nevertheless, further research is necessary about the effect of bleach on cultivated plants when used as an NS disinfectant, as there are concerns for sodium (Na+) and Cl<sup>−</sup> accumulation [21] as well as the presence of residues and byproducts toxic to humans in the edible products [4].

Regarding disinfection with sodium hypochlorite, it has been reported that it is likely to lead to the formation of derivatives such as chloramines, organic halogens, and trihalomethanes (chloroform, dichlorobromomethane, bichromobromomethane, and bromoform) [4]. In addition, Bull et al. [29] highlighted that the use of the hypochlorite anion (ClO−) in the disinfection of NS may be associated with the production of chlorates that can cause acute toxicity in humans [29]. Nevertheless, this risk is associated with the dosage and the frequency of application, as well as the plant part consumed as an edible product.

Exposure of humans to chlorates (ClO3 −) and perchlorates (ClO4 −) is expected from residues occurring as by-products of the use of chlorinated disinfectants in food processing and water treatment [30]. From monitoring data collected between 2014 and 2018 [31], residues at quantitative levels were found in various commodities, leading to the establishment of maximum residue levels (MRLs) for chlorates, which were currently set to 0.05–0.7 mg kg−<sup>1</sup> depending on the commodity [32]. These MRLs are tentative, and the discussion at EU level is still ongoing [33–35]. For tomatoes, the current MRL is set at 0.1 mg kg<sup>−</sup>1. In a previous investigation [7], the use of potassium hypochlorite (KClO) for disinfection of the NS supplied to a soilless tomato cultivation led to the accumulation of chlorate residues in fruits.

Globally, the tomato is one of the most important vegetables [36]. Additionally, it serves as an important model-organism in plant research [37]. In 2019, tomato was cultivated on 6,117,242.00 hectares with a production of 243,635,433.00 tons worldwide, and the countries with the highest production were China, India, and Turkey [38]. Within this context, the scope of the current work was to investigate the effect of sodium hypochlorite application in the NS supplied to an open soilless cultivation system of tomato on plant growth and total yield and detect possible safety risks for the consumer. Therefore, in addition to the agronomic parameters, residue analysis of chlorates and perchlorates in tomato fruits was performed.

#### **2. Results and Discussion**

#### *2.1. Chloride Concentrations in the Drainage Solution*

As shown in Table 1, the chloride concentration in the drainage solution was significantly higher in the treatment group of 7.5 mg L−<sup>1</sup> compared to the control group and the 2.5 mg L−<sup>1</sup> treatment group at 30 days after the first application (DAFA). This is due to the addition of sodium hypochlorite, which was not accompanied by a commensurate increase in its uptake by the plants after its application. This hypothesis is supported by measurements of the chloride concentration in leaf and root samples as well as fruit samples (results in Section 2.3), which were similar before and after sodium hypochlorite application to the NS.

**Table 1.** Chloride concentration in the drainage solution samples collected at 30, 37, and 48 DAFA of sodium hypochlorite in a soilless cultivation of tomato. Sodium hypochlorite was applied at concentrations of 2.5, 5.0, and 7.5 mg L−<sup>1</sup> of chlorine, while in the control treatment no sodium hypochlorite was added.


Values (means of 4 replications), followed by different letter in each column indicate significant differences according to Duncan's multiple range test (*p* < 0.05). NS = Not Significant, \* = Significant (*p* ≤ 0.05).

Although chlorination is a popular method of disinfecting NS in soilless cultures, it is not considered to be used to its full potential, mainly due to technical issues related to the monitoring of free available chlorine [39]. Most phytopathogens are controlled by chlorine concentrations of 1–3 mg L−1, while higher initial concentrations (5–10 mg L−1) are required as chlorine reacts with various other substances in NS [20,39]. Other important factors affecting the success of chlorination are pH, temperature, and organic matter content in the NS, as well as pathogen type and pathogen load [4].

As shown in Table 1, the highest chloride concentration in the drainage (9.05 mg L<sup>−</sup>1) was detected in the treatment with a 7.5 mg L−<sup>1</sup> chlorine application one day after the third application of sodium hypochlorite (30 DAFA). However, the difference was significant only in comparison with the control treatment and the treatment with the addition of sodium hypochlorite at 2.5 mg L−<sup>1</sup> of chlorine.

#### *2.2. Growth of Plant and Gas Exchange*

The disinfection of the NS by applying sodium hypochlorite did not significantly affect the growth of the plants, as indicated by the absence of any statistical differences in the leaf biomass and total leaf area (Table 2). Furthermore, no phytotoxicity symptoms were observed in any of the treatments.

**Table 2.** Estimation of fresh weight (fw), dry weight (dw), dry matter content (DMC, %), specific leaf area (SLA) and leaf area (cm2) in the collected leaf samples.


The numbers represent mean values of 4 replications. NS = not significant based on one-way ANOVA (*p* < 0.05).

Generally, many studies have reported on the effect of chlorine on various plant species by applying different forms of chlorine (gas, chlorine dioxide, etc.) through different disinfection protocols [6,25,26,40–44]. Therefore, a direct comparison of their results is not possible.

In general, chlorine at concentrations higher than 5 mg L−<sup>1</sup> can cause phytotoxicity in many plants, while some plants are sensitive even at much lower concentrations (0.05 mg L<sup>−</sup>1) [43]. In this study, disinfection with sodium hypochlorite was selected because it is economically affordable for producers, effective against important plant pathogens, and very common in agricultural practice. Unlike other chemical methods of disinfection, sodium hypochlorite does not degrade quickly, thereby resulting in a longer disinfection capacity. The results of the present study concerning the disinfection effect on tomato growth coincide with the 5 mg L−<sup>1</sup> rule [43]. Nevertheless, chlorine application following the current disinfection protocol with sodium hypochlorite did not affect the growth of tomato plants cv. 'ELPIDA' even at 7.5 mg L−<sup>1</sup> chlorine.

Gas exchange was not affected by the application of chlorine up to a concentration of 7.5 mg L−1, as indicated by the absence of any significant differences in the net photosynthetic rates, transpiration rate, stomatal conductance, intercellular CO2, and water use efficiency (Table 3). Nevertheless, there are examples in which chlorine, applied in gaseous form, affected the photosynthesis of *Pinus* plants by reducing their photosynthetic capacity [40]. Chlorine, as an anion, can be beneficial to plants by substituting for nitrates in vacuoles and positively impacting photosynthesis [44]. Therefore, some researchers used

chlorine to replace part of the nitrates in the NS in tomato [45] and tobacco [46] and found that the stomatal conductivity was affected. However, the concentrations of chlorine tested in this experiment were lower than those in the above-mentioned studies and did not result in any significant differences.

**Table 3.** Net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, transpiration rate and water use efficiency in leaves (3rd or 4th fully developed leaf from the top) of tomato plants at 0, 31, and 43 DAFA of sodium hypochlorite at three different concentrations of chlorine in the supplied nutrient solution.


The numbers represent mean values of 4 replications ± standard error. NS = Not Significant based on one-way ANOVA (*p* < 0.05).

#### *2.3. Fruit Yield and Fruit Mineral Composition*

Analysis of variance showed that the disinfection of NS using sodium hypochlorite at a concentration of 2.5 mg L−<sup>1</sup> of chlorine significantly boosted the total production of marketable fruits when considering total fruit weight per plant (Figure 1A). Nonetheless, it should be noted that the total production for the control treatment was relatively low due to a lower average weight per fruit than the typical weight for this variety. This is possible due to the small scale of this experiment compared to a commercial production.

**Figure 1.** (**A**) Total and (**B**) Extra class production of tomato fruit after disinfection of the nutrient solution using sodium hypochlorite in an open soilless system applying chlorine concentrations of 2.5, 5 and 7.5 mg L<sup>−</sup>1. Values (means of four replications, bars = SE), followed by different letter in each column indicate significant differences according to Duncan's multiple range test (*p* < 0.05).

To the best of our knowledge, this is the first study in the relevant scientific literature indicating an enhancement of tomato yield after the addition of chlorine in the NS. The increased yield in the treatment of 2.5 mg L−<sup>1</sup> of chlorine was not accompanied by statistical differences between the treatments regarding gas exchange. Hence, it cannot be ascribed to an enhancement of anabolic functions. However, considering that the dose of chlorine that led to this yield increase was low, it could be attributed to hormetic effects [47,48]. An alternative explanation is that the presence of chlorine in the NS protected plants from low-impact infections of the root system, which did not cause symptoms detectable by visual observation, thus improving their performance compared to plants not treated with chlorine.

The mineral analysis of tomato fruits revealed a significantly higher Mg concentration when chlorine was applied at a concentration of 2.5 mg L−1, which correlates well with the higher yield in this treatment (Table 4). The chloride content in the fruit was increased by chlorine application only in the second harvest (43 DAFA) and only in the 7.5 mg L−<sup>1</sup> treatment, and the difference was significant compared not only with the control but also with the other two chlorine treatments. In general, the concentration of nutrients in the fruits ranged between 2.4–4.1 mg g−<sup>1</sup> for phosphorus, 41.5–98.0 mg g−<sup>1</sup> for potassium, 2.5–3.5 mg g−<sup>1</sup> for chlorine, 0.16–0.21 mg g−<sup>1</sup> for calcium, and 1.5–1.8 mg g−<sup>1</sup> for magnesium.

**Table 4.** Mineral nutrient analysis in tomato fruit (mg g−<sup>1</sup> dw) harvested 31 and 43 DAFA of sodium hypochlorite in an open soilless system at chlorine concentrations of 2.5, 5.0 and 7.5 mg L<sup>−</sup>1.


Values (means of four replications), followed by the same letter in each column indicate significant differences according to Duncan's multiple range test (*p* < 0.05). NS = Not Significant, \* significant (*p* ≤ 0.05), \*\* = significant (*p* ≤ 0.001).

Chlorine is usually reported for its phytotoxic action when absorbed by the roots at excessively high rates in the form of chloride ions [49]. However, chlorine is an essential trace element in chloroplasts, where it participates in the oxygen evolution process in photosystem II [50] and possibly controls the function of some enzymes involved in cell division [51]. Chlorine is absorbed by plants as the chloride ion at concentrations ranging from low [0.1–0.2 mg g−<sup>1</sup> [44]] to high [2–20 mg g−<sup>1</sup> [51]]. At higher concentrations than those required for its essential functions, chloride may have beneficial functions such as osmoregulation [52,53]. However, high concentrations of chloride in the plant tissues are considered to limit the growth of plants [54,55]. The reasons behind this phenomenon are not clear [52] and probably differ in the various susceptible plants such as *Lotus corniculatus* [56], citrus [57] and grapevine [54], which show phytotoxicity at concentrations of 4–7 mg g−<sup>1</sup> dw [58]. Tomatoes have a high tolerance to chloride concentration compared to other vegetables grown in greenhouses [45]. More specifically, in a closed soilless system, when part of the nitrogen was replaced by chloride, no effect on total yield was reported [45]. Nonetheless, reduction of physiological abnormalities in tomato fruits (white and green spots, blossom end rot) has been indicated when chloride levels are high (8–10 mM) [59,60]. Finally, it should be noted that high chloride concentrations under stress (NaCl) could increase soluble solids and dry matter content and also affect fruit firmness [61].

#### *2.4. Chlorates and Perchlorates in the Nutrient Solution*

#### 2.4.1. Stability of Chlorate and Perchlorate Residues in the Nutrient Solution

Perchlorate residues were not detected in any of the fruit samples. Regarding chlorate, which was formed at the beginning (0 day), residues were found at 0.45 and 1.45 mg L−<sup>1</sup> in the solutions with 2.5 and 5% ammonia, respectively. On the first day, a recovery of 62% compared to day 0 was observed in the 5% ammonia solution, but in the next sample points, recoveries were all above 70%, indicating that no degradation above 30% or formation of perchlorate was observed during the 28-day period of the study (Table 5).

**Table 5.** Summary results of the 28-day storage stability study of chlorate residues in the nutrient solutions with 2.5 and 5% ammonia concentration. The nutrient solution was composed to support the fruiting of an open soilless system cultivation of tomato.


<sup>1</sup> initial concentration of chlorate residues detected in each NS. <sup>2</sup> SD: Standard deviation. <sup>3</sup> Recoveries above 100% indicate that a degradation did on occur, but the higher concentration compared to the initial are observed due to measurement uncertainty which in our case is 50% [62].

#### 2.4.2. Detection of Chlorate Residues in the Nutrient Solution

Chlorate residues were detected in the NS of all treatments (Figures 2 and S1), whilst no perchlorate residues were found in any treatment. Furthermore, the concentration of chlorate residues was increasing in the different treatments following the increased sodium hypochlorite addition in the NS. More specifically, the highest concentration of chlorates determined was 0.325 mg L−<sup>1</sup> in the treatment of 7.5 mg L−<sup>1</sup> of chlorine.

Industry quality controls and official food safety controls have detected chlorate residues in various vegetables. Relatively high concentrations have been found in tomato (0.2 mg kg−1) and carrot (0.3 mg kg−1) samples, which exceeded the MRLs set by Reg. 2020/749 (0.1 mg kg−<sup>1</sup> and 0.15 mg kg−1, respectively) [30]. Nonetheless, it has been suggested that these high levels derive from post-harvest handling where bleach is used as a disinfectant [63–65].

#### *2.5. Chlorates and Perchlorates in Tomato Fruit*

After the fortification and analysis of QC samples in the LC/MS/MS, recovery values and relative standard deviations for each substance and charge level were calculated. The accuracy of the method was expressed as a percent recovery. Chlorate recovery values ranged from 111 to 119%, while for perchlorates the range was from 116 to 119%. Repeatability was expressed as % RSD, which ranged from 7–19% for chlorates and from 2–7% for perchlorates. The results are considered acceptable and prove that the method was reliable.

**Figure 2.** Chlorate residues detected in the nutrient solution of an open soilless cultivation of tomato after application of chlorine at concentrations of 2.5, 5 and 7.5 mg L<sup>−</sup>1. The bars represent standard deviations.

Chlorate and perchlorate residues were not detected in tomato fruit samples of the extra class grade (Figure S2). These results indicate that the fruits were safe for consumption. Chlorates are generally an important inorganic derivative of chlorine used for disinfection, and the key question is if they can be absorbed and accumulated in edible parts during plant production [66]. Here, in all analyzed fruit samples, the residues were lower than the limit of quantification (LOQ = 0.01 mg kg−1). In contrast, Dannehl et al. (2016) found that chlorate residues were detected after disinfection of the NS with KClO in a closed soilless tomato growing system [7]. Furthermore, chlorate residues have been recorded in "baby" lettuce and spinach samples when the crops were irrigated with chlorinated water [67,68]. However, chlorates and perchlorates do not coexist [7]. Finally, Lonigro et al. (2017) suggested that organochlorine compounds in soil, roots, and leaves are linked to the chlorine concentration in the irrigation water or nutrient solution [42].

Disinfection of vegetables using chlorine has many applications, e.g., production, harvest, and post-harvest handling of fresh fruits and vegetables for many decades [69–71]. In the past, high concentrations of chlorine were applied since there was no awareness about possible toxic residues in the final products [72,73]. Nevertheless, it is now clear that chlorine can react with organic matter, leading to derivatives such as chloroform (CHCl3) or trihalomethanes that are carcinogenic at high doses [74]. Therefore, low doses of chlorine, when using bleach, are recommended for disinfection of NSs in soilless crops to produce safe products free from chlorine byproducts, in addition to avoiding possible symptoms of phytotoxicity [66].

#### **3. Conclusions**

Disinfection of the nutrient solution in open soilless cropping systems by applying chlorine up to a dose of 7.5 mg L−<sup>1</sup> three times at fortnightly intervals did not lead to phytotoxicity in tomato. However, the application of chlorine at a concentration of 2.5 mg L−<sup>1</sup> increased the total fruit production, due mainly to an increase in the number of fruits per plant. Additionally, no residues of chlorates or perchlorates were detected in tomato fruits. Nonetheless, further research is necessary to address the gap in the literature regarding the disinfection methods applied and their efficiency in controlling economically important pathogens.

#### **4. Materials and Methods**

#### *4.1. Experimental Design, Biological Material and Cropping Conditions*

4.1.1. Greenhouse Cultivation

An experiment with tomato (*Solanum lycopersicum* cv. "ELPIDA") cultivated in bags containing perlite was carried out in a glasshouse at the Agricultural University of Athens in Greece (37◦59 10 N, 23◦42 29 E, altitude 24 m) from May to July 2019.

The soilless cultivation system comprised 10 channels, eight of which were used to apply the experimental treatments (2 channels per treatment), while the other two were used as border lines (Figure 3). A completely randomized design was followed. Six bags of perlite (33 L, Perlite Hellas, Athens, Greece) were placed in each channel, and two tomato plants were planted in each bag. Prior to transplanting, the perlite bags were irrigated with NS up to saturation, and subsequently their bottoms were slit to allow for free drainage of the NS from the root zone. The composition of the NS was computed using the algorithm developed by Savvas and Adamidis (1999) [75] after setting as target nutrient values those suggested by Savvas et al. (2013) [76] for open soilless cultivations of tomato. The pH of the supplied NS was adjusted to 5.6 using nitric acid. The nutrient solution compositions applied at different cropping stages are shown in Table S1. One day after wetting the substrate (10 May 2019), the tomato seedlings, which were at the 8-leaf stage, were transplanted. The channels had a slope of 1–2% to facilitate drainage, and every two days the values of pH and electrical conductivity (EC) were recorded. The NS collected at the lower end of each channel was discharged. To aid fruit setting, a bumblebee hive (*Bombus terrestris*) (Bio Insecta, Thessaloniki, Greece) was placed inside the experimental greenhouse compartment, and the usual pruning [2,77] and plant protection practices were followed [78,79]. More specifically regarding plant protection, integrated pest management (IPM) principles were applied. Protective nets were put on the greenhouse openings as well as adhesive traps to prevent infestation by insects. In addition, seven days after transplanting the plants, the beneficial insect *Macrolophus pygmaeus* was released as a plant protection measure against *Tuta absoluta*.

**Figure 3.** Experimental pipeline for the study of the impact of sodium hypochlorite applied as a nutrient solution disinfectant on growth, nutritional status, yield, and consumer safety of tomato fruit produced in a soilless cultivation.

Corrective recipes were also used during the cropping period whenever they were necessary. The irrigation frequency was automatically adjusted using a heliometer to measure the solar radiation intensity and an irrigation controller, aiming to achieve a drainage percentage of about 30%.

#### 4.1.2. Disinfection Methodology

Sodium hypochlorite was applied through the last irrigation cycle of the day, while no irrigation was applied during the night. During the cropping period, sodium hypochlorite was applied to the tomato crop three times at fortnightly intervals. Common bleach containing 4.5% *w*/*v* sodium hypochlorite (Ostria Ultra Power, Lamia, Greece) was used. More specifically, the amounts of bleach needed to apply three different chlorine concentrations, namely 2.5, 5, and 7.5 mg L−1, were calculated and added to the NS supplied. These chlorine concentrations were selected to range around the average concentration of chlorine that is not supposed to negatively affect the plants according to the EPA (5 mg L−1) [43]. The final concentration of chloride was not tested in the NS after the application of sodium hypochlorite, but it was measured at the drainage the following day (Table 1). Furthermore, the pH was tested on site in the NS after the addition of sodium hypochlorite and in the drainage solutions. The first application took place on 11 June 2019, i.e., about one month after the beginning of fruit set (16 May 2019). All applications of sodium hypochlorite are shown in detail in Table S2.

#### 4.1.3. Sample Collection

In order to estimate the impact of the treatments on plant growth, nutrient concentrations and the possible formation of chlorates and/or perchlorates in plant tissues, including leaves, fruits, and roots, were sampled. Leaf samples (3rd and 4th fully developed leaves from the top) were collected 0, 31, and 43 days after the first application of sodium hypochlorite (DAFA—days after first application). Fruit samples were collected at DAFA 31 and 43. The time points of sampling were chosen in an effort to discover the effect of sodium hypochlorite on tomato plants after the third chlorine application (31 DAFA) and almost ten days later, namely slightly before the end of the culture. At the end of the cultivation (48 DAFA), the whole root system was also selected, separated from perlite grains, and used as root samples. A total of two plant tissue samples were collected from two different plants in each channel, thereby obtaining four replicate samples per treatment. Prior to the nutrient analysis, the fresh weight of each sample was recorded, and then all samples were placed in an oven at 65◦C for drying until their weight was stabilized. Finally, the dry matter content (DMC) and specific leaf area (SLA, on fresh leaves) were calculated [80].

In addition, samples of NS were collected from the drainage solution before the first harvest (30 DAFA), 7 days later (37 DAFA), and at the end of the cultivation (48 DAFA) to determine the level of chloride and nutrients. Two drainage solution samples were collected from each channel, thereby obtaining four replicates per treatment.

#### *4.2. Gas Exchange Measurements*

The treatment impact on net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate were determined using a Li-6400 instrument (Li-Cor, Inc., Lincoln, NE, USA) [81]. The measurements were conducted in leaves of the same physiological stage (the most recent fully expanded leaf) at the same time of the day (between 09:00 am and 12:00 pm) prior to their sampling, as described in Section 4.1.3. All measurements were conducted under natural light conditions and ambient CO2 atmospheric concentration on sunny days to exclude light intensity effects. Furthermore, water use efficiency was estimated as previously described [82].

#### *4.3. Plant Mineral Status*

Initially, the dried plant samples were ground using a ball mill and sieved (40-mesh) until they obtained the texture of powder. The procedure was followed by dry burning at 500 ◦C for 8 h and the extraction of minerals using 10 mL of 1 M HCl to determine tissue P, K, Ca, and Mg concentrations. The extracts were filtered and stored at 4 ◦C until further processing. Phosphorus was estimated photometrically (Anthos Zenyth 200, Biochrom, Holliston, MA, USA) by applying the ammonium phosphomolybdate method [83]. Potassium was estimated through flame photometry using a Sherwood Model 410 (Sherwood Scientific Ltd., Cambridge, UK). Ca and Mg were measured by employing atom absorption spectrophotometry using a Shimadzu AA-7000 instrument (Shimadzu Europa GmbH, Duisburg, Germany). Chloride was measured using a chloridometer (Thermo Scientific Orion Star A214, Thermo Fisher Scientific, Waltham, MA, USA) in aqueous extracts of the powdered leaf samples after filtering.

#### *4.4. Estimation of Total Fruit Production*

Fruit was harvested twice, on 12 July 2019 and 24 July 2019, i.e., 2 and 14 days after the last application of sodium hypochlorite, respectively. At each harvest date, all ripe fruits were collected and graded into extra class, class I, class II, and non-marketable produce in accordance with the EU Regulation (543/2011) [84]. The fruit yield was estimated by recording the total number of fruits per plant (for both time points together and for each tomato class separately) and the total fruit weight per plant, while calculating the average fruit weight.

#### *4.5. Residue Determinations*

In order to investigate the possible contamination of harvested tomato fruits with chlorates (ClO3 −) and perchlorates (ClO4 −) due to disinfection of the NS with sodium hypochlorite, residue analysis was performed as described below:

#### 4.5.1. Chemicals and Reagents

Chlorate, perchlorate, and internal standards (IS) (perchlorate 18O4 and chlorate 18O3) stock solutions at 80 mg L−<sup>1</sup> in MeOH were obtained from the EU-Reference Laboratory for pesticides requiring single residue methods. Methanol (HPLC gradient grade) and water (LC-MS grade) were bought from Fischer chemicals. Acetic acid (99% for analysis) and formic acid (99%, for analysis) were bought from Carlo-Erba reagents.

#### 4.5.2. Investigation of the Stability of Chlorates and Perchlorates in the NS Samples

During the final application of sodium hypochlorite in the different treatments, two 0.5 L samples of NS were collected from the drippers, and their chlorate and perchlorate concentrations were determined.

The nutrient solution, which had previously been analyzed to confirm that no detectable residues of chlorates and perchlorates were present, served as a control to investigate the stability of chlorate and perchlorate residues in NS under freezing conditions. Two solutions with 2.5 and 5% ammonia concentrations were prepared and stored at 4 ◦C. During this time, successive sampling of the fortified NS was performed at days 0, 1, 2, 4, 6, 8, 11, 21, 22, and 28 and was followed by injection into the LC/MS/MS chromatographic system.

#### 4.5.3. Extraction Procedure of Tomato Fruit Samples

A laboratory sample of 2 kg of the extra class tomato fruits was collected and homogenized 8 h after harvest, after removal of the calyx. The homogenized samples were stored at −20 ◦C until further processing.

Residues analysis was performed following the in-house laboratory method M17, which is based on the QuPPe protocol [85]. A brief description of the procedures is described below:

An aliquot of 10 ± 1 g of homogenized sample was weighted, and 10 mL of the extraction solvent methanol (acidified 1% HCOOH) was added. The mixture was allowed to soak for 30 min before the addition of the extraction solvent. The mixture was shaken by hand for 1 min and centrifuged at 4000 rpm (1792× *g*) for 5 min. An aliquot of the extract was transferred into a screw cup-top storage vial. Before injecting it into the chromatographic system, the final solution was filtered through a 0.45 μm disposable cellulose syringe filter.

#### 4.5.4. Chromatographic Analysis of Fruit Samples—Instrumentation

For the chromatographic analysis of the samples, a Varian liquid chromatography LC-MS/MS system consisting of two Varian Prostar 210 pumps and a Prostar 420 autosampler using a 100 mL syringe was used, combined with a triple quadrupole mass spectrometer (Varian model 1200 L) and equipped with an electrospray ionization (ESI) interface, operating in the negative mode. Separation was performed on a Hypercarb 2.1 × 100 mm 5 <sup>μ</sup><sup>m</sup> at a flow rate of 0.4 mL min−1. The column was at room temperature. Eluent A consisted of an aqueous solution of 1% CH3COOH, while eluent B was MeOH with 1% CH3COOH. The LC gradient started at 100% A and was linearly decreased to 70% B, over 10 min. Finally, the gradient was instantly switched to 100% A and equilibrated for 5 min before the next injection took place. The injection volume was 10 mL.

The following instrumental settings were used: the source temperature was set at 50 ◦C and the drying gas temperature at 300 ◦C. Drying gas and nebulizing gas were nitrogen generated from a high purity generator at 18 psi and synthetic air (purity > = 99.99%) at 40 psi, respectively. For the operation in MS/MS mode, Argon 99.999% was used as a collision gas with a pressure of 1.8 mTorr. The scanning of the transitions was conducted with a dwell time of 50 ms per transition. The number of data points across the peaks was at least ten. Capillary voltage (CV) and collision energy (CE) varied depending on the precursor ion and product ion and are presented in Table S3.

#### 4.5.5. Method Performance

The analytical method applied to determine chlorates and perchlorates in tomato fruit samples of extra class has been proposed by the EURLs (European Reference Laboratories for Single Residue Methods), hence method validation data are available. In order to ensure the performance of the present analysis and verify the obtained results, a brief validation of the method was performed. Considering that an internal standard was used to quantify the results, calibration standards were applied to the solvent. Specifically, quality control samples containing 0.01 mg kg−<sup>1</sup> (in 2 replicates, n = 3), 0.1 mg kg−<sup>1</sup> (in 2 replicates, n=3), and 1 mg kg−<sup>1</sup> (in 2 replicates, n = 2) in chlorates—perchlorates were prepared by fortifying the corresponding concentration in control tomato samples to determine the accuracy of the method. The samples used as controls had previously been analyzed, and it was confirmed that they had no residues from or interferences with the substances under investigation. Accuracy was checked by assessing the recovery for the selected concentrations, and repeatability was verified by measuring the relative standard deviation (RSD).

#### *4.6. Statistical Analysis*

The experiment was set up as a completely randomized design examining one factor (sodium hypochlorite concentration) with four levels and four replicates per treatment. The statistical analysis was performed using the Rstudio program (Version: 1.3.1093, Boston, MA, USA) and the Agricolae package [86]. First, box plots were made to ensure the lack of outliers in the various parameters studied for each treatment separately. The normality of the data was checked in terms of skewness and kurtosis [87], and Levene's test was employed to test the equality of the variances. When an ANOVA analysis rendered a significant difference at the significance level of 95%, post-hoc comparisons using the Duncan's Multiple Range Test with *p* < 0.05 were performed [88].

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/horticulturae9030352/s1, Figure S1: Chlorate residue analysis in a nutrient solution sample (red) of an open soilless system cultivation of tomato and in HPLC water (green). The nutrient solution sample corresponds to the 7.5 mg L−<sup>1</sup> of chlorine treatment after the application of sodium hypochlorite in which chlorates residues were detected; Figure S2: LC-MS/MS scan data of chlorate (red) and perchlorate (green) residue analysis in tomato fruit samples

collected from the 5 mg L−<sup>1</sup> chlorine treatment; Table S1: Recipes of nutrient solutions used in tomato cultivated in an open soilless system; Table S2: Details about the three disinfection applications using sodium hypochlorite in the nutrient solution supplied to a tomato cultivated in an open soilless system; Table S3: Precursor ion, product ion, Capillary Voltage (CV) and Collision Energy (CE) for the chlorates and perchlorates analytes examined with LC/MS/MS for the determination of their residues in tomato fruits and nutrient solution samples of an open soilless system cultivation.

**Author Contributions:** Conceptualization: D.S. and E.B.; funding acquisition: D.S.; investigation: M.L., E.B. and I.K.; methodology: M.L., E.B., I.K., C.A. and D.S.; project administration: D.S.; Supervision: D.S. and K.A.A.; writing—original draft; M.L. and E.B.; writing—review and editing: M.L., E.B., I.K., C.A., K.A.A. and D.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the Hellenic Foundation for Research and Innovation (H.F.R.I.) under the "First Call for H.F.R.I. Research Projects to support Faculty members and Researchers and the procurement of high-cost research equipment grant" (Project Number: HFRI-FM17- 3196—NUTRISENSE).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We would like to thank the students Maria Theodorakopoulou, Nikos Andreou, and Athanasia Papakosta for their significant contribution in the field work and analysis of the nutrients.

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

#### **References**


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## *Article* **Bio-Fertilizers Reduced the Need for Mineral Fertilizers in Soilless-Grown Capia Pepper**

**Hayriye Yildiz Dasgan 1,\*, Mehmet Yilmaz 1, Sultan Dere 1,2, Boran Ikiz <sup>1</sup> and Nazim S. Gruda 3,\***


**Abstract:** Soilless cultivation is extensively used in the greenhouse industry. Recently, hydroponic cultivation of capia pepper has become popular among growers. Capia pepper is harvested at the red maturity stage, and intensive mineral fertilizers are usually used for soilless cultivation. This study was performed in a greenhouse during spring under Mediterranean climatic conditions. The effects of bacteria and mycorrhiza on capia pepper plant growth, yield, fruit quality, and nutrition were investigated. Furthermore, the synergistic effects of these two bio-fertilizers were investigated. Our objective was to replace 20% of mineral fertilizers with bio-fertilizers in a soilless culture system. The use of 80% mineral fertilizers, in combination with mycorrhiza and bacteria, provided a 32.4% higher yield than the control (100% mineral fertilizer without bio-fertilizers). Moreover, the concentrations of N, P, K, Ca, Mg, Fe, Mn, Zn, and Cu in the leaves of pepper plants fed with the reduced mineral fertilizers combined with bio-fertilizers were higher than that of the control. In addition, fruit parameters, such as fruit weight, diameter, volume, the electric conductivity of the fruit juice, and total soluble solids, were significantly higher in this treatment compared to the control. Using 80% mineral fertilizer with only bacteria provided a 24.2% higher yield than the control. In conclusion, mineral fertilizers were successfully reduced by 20% using bacteria and mycorrhiza. These results provide an eco-friendly approach to a sustainable environment.

**Keywords:** bacteria; *Capsicum annuum* L.; coco pith; mycorrhiza; synergistic effects; yield

#### **1. Introduction**

Intense monoculture vegetable growing in conventional soil greenhouses increases the risk of disease and pest outbreaks. Consequently, high amounts of pesticides and herbicides are needed, increasing environmental pollution. On the other hand, this cultivation method can reduce certain nutrients' availability, leading to soil exhaustion. Our endeavors to compensate for the situation could, in turn, increase the level of unbalanced nutrients and negatively affects soil fertility. For instance, long-term chili monoculture generated significant changes in soil nutrients, aggregates and enzymes [1]. All these factors severely limit crop productivity over the years.

In recent years, the gradual decrease in agricultural lands, the effects of climate change, soil-borne problems, food security, and environmental issues have increased the tendency towards soilless cultivation as controlled agriculture [2,3]. A soilless culture system (SCS) is considered one of the most promising approaches, combining increased production without damaging its supporting ecosystem [3]. Likewise, soilless culture systems have been adopted in many countries for a long time. Plant nutrition management is a crucial factor in the success of soilless cultivation systems. The right nutrient solution will increase the complete fulfillment of plants' requirements for optimal growth and development. However, although soilless cultivation in the modern greenhouse business is the favorite

**Citation:** Dasgan, H.Y.; Yilmaz, M.; Dere, S.; Ikiz, B.; Gruda, N.S. Bio-Fertilizers Reduced the Need for Mineral Fertilizers in Soilless-Grown Capia Pepper. *Horticulturae* **2023**, *9*, 188. https://doi.org/10.3390/ horticulturae9020188

Academic Editor: Silvana Nicola

Received: 4 December 2022 Revised: 27 January 2023 Accepted: 30 January 2023 Published: 2 February 2023

**Copyright:** © 2023 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 (https:// creativecommons.org/licenses/by/ 4.0/).

choice among growers, it brings intensive nutrition to the plants through mineral fertilizers. Coco pith is an organic substrate produced from the wastes of coco fruits. It has a high water-holding capacity, cation-exchange capacity, aeration, a light bulk density, an ideal pH and EC, and is free of pathogens. These properties have made it one of the most preferred soilless growing media in the greenhouse industry in recent years [3]. Therefore, coco pith was chosen for capia pepper cultivation in this study.

In recent years, improvements in beneficial microorganisms have increased the tendency to use bio-fertilizers as valuable tools in sustainable agriculture. Unlike in soil, in hydroponic growing soilless systems there are no beneficial microorganisms in the root environment, and plants cannot benefit from these microorganisms. Therefore, bio-fertilizers such as mycorrhiza fungi, bacteria, and microalgae are commonly adapted to soilless growing systems [4–7]. Bio-fertilizers are effective strains of microorganisms that help crop plants' nutrition by enhancing growth, yield, and crop quality in soilless culture systems [8]. At the same time, they provide environmentally friendly agriculture by reducing the use of mineral fertilizers. Inoculation of arbuscular mycorrhizal fungi (AMF) and plant-growthpromoting rhizobacteria (PGPR) become an alternative solution to increase the efficiency of nutrient usage as well as absorption of nutrients by plants [5,9]. The use of bio-fertilizers in the soilless growing of several vegetable species was previously reported in related literature [10–13].

Similarly, studies also acknowledged the beneficial effects of bio-fertilizers on pH and electrical conductivity (EC) of the nutrient solution regulations, chelator secretion in the rootzone, increased uptake of nutrients, plant growth, yield, and crop quality [7,14]. In an SCS, the nutrient solution that consists of mineral fertilizers is used in every irrigation. An open system means the nutrient solution is not recyclable, and the excess drainage solution (about 20–25% of the applied nutrient solution) can pollute the environment and groundwater resources. In the greenhouse sector, it is noteworthy that a significant majority of SCSs produce crops in an open system. For this reason, less mineral fertilizer means environmental protection. Bio-fertilizers improve, in addition, the pH and EC of the nutrient solution in the root zone [7]. Therefore, they provide a high intake of nutrients from plants. In addition, biofertilizers decrease nitrate and increase the antioxidants and minerals of vegetables [13,15]. Hence, biofertilizers provide positive contributions to human nutrition. However, studies on bio-fertilizers used in reducing mineral fertilizers are quite limited. On the other hand, farmers' and consumers' awareness of environmental and ecological concepts has recently increased. As a result, some farmers who want to produce according to a sustainable model prefer to use organic fertilizers and/or bio-fertilizers in soilless cultivation systems [16].

Capia (*Capsicum annuum* L.) is a type of pepper with a long conical shape, red color in maturity, and a sweet taste; it is rich in vitamins C and A, folic acid, potassium, mineral substances, phenolic compounds, carotene, and antioxidant compounds [17,18]. It has a dense juicy pulp with a rich aroma in the red maturity stage. Capia pepper fruit at the red ripeness stage has the following morphological properties: 80–125 g weight, 15–20 cm length, 40–55 mm width, 150–250 cm3 volume, and 3.5–4.0 mm flesh thickness. Capia pepper seeds germinate in 7–10 days. The seedlings grow in around 30–40 days. The plant grows best between 22–25 ◦C during the day and 15–18 ◦C at night. An increase in daytime temperature to 28 ◦C accelerates red ripening. Red ripe capia fruits are harvested in approximately 90 days from seedling planting. Capia pepper, very popular in the Balkan countries and Turkey, is commonly used for pepper paste, pepper juice, pickles, frozen products, frying, and peppery sauce in summer field production [18].

On the contrary, greenhouse capia is used for table consumption during the cold winter season. The capia pepper's soilless cultivation in the greenhouse has recently become popular among growers. Approximately 3,018,775 tons of pepper are produced in Turkey, and 49% of this is capia pepper [19]. The consumer preference in Turkey is primarily capia pepper. Recently, capia cultivation has increased with soilless techniques in the greenhouse. Greenhouse producers search for environmentally friendly fertilizers that increase product yield and quality.

To our knowledge, reducing mineral fertilizers and substituting them with biofertilizers has not been previously investigated in SCSs for capia pepper. Therefore, this study aimed to reduce the intensive use of mineral fertilizers by inoculating with beneficial microorganisms such as AMF and PGPR. Moreover, bio-fertilizers' effects on plant growth, yield and fruit quality of soilless-grown capia pepper were investigated in this study.

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

#### *2.1. Plant and Bio-Fertilizer Materials and Experimental Conditions*

This study was conducted in a glasshouse at 36◦59 N, 35◦18 E, and 23 m above the Mediterranean Sea level in the early spring growing season (February-July). Climatic conditions inside the glasshouse were 23–25 ◦C during the day and 15–20 ◦C at night, with 50–60% relative humidity and natural sunlight conditions. Trademarked Lale F1 capia pepper of the Istanbul Tarim company was used. We used coco pith slabs as a soilless cultivation medium, cultivating four plants in every slab, with four slabs in each replication. The size of the coco pith slab was 100 cm long, 20 cm wide, and 10 cm deep. For the randomized complete block experimental design with seven treatments and four replicates, 16 plants were used in each replicate. Pepper seedlings 35 days old were transplanted into the coco pith slabs 25 cm above the row and 80 cm between the rows Figure S1.

The mycorrhiza bio-fertilizer Endo Roots Soluble (ERS), a cocktail from nine different mycorrhiza species: Glomus intraradices, Glomus aggregatum, Glomus mosseae, Glomus clarum, Glomus monosporus, Glomus deserticola, Glomus brasilianum, Glomus etunicatum, and Gigaspora margarita, was used. The liquid bacteria Medbio bio-fertilizer used in the experiment contained four different bacteria species: Bacillus subtilis (1 × <sup>10</sup>9), Bacillus licheniformis (2 × 106), Bacillus megaterium (1 × 109) and Pseudomonas putita (1 × <sup>10</sup>10). Pepper seedlings were inoculated only once during transplanting, with approximately 2000 mycorrhizae spores per plant; with the growth of the root system, mycorrhiza spores multiply in a symbiosis relationship. Meanwhile, bacteria were applied every ten days to the roots during growing. PGPR was applied at 50 mL per plant from the 1 mL Metbio in 1 L nutrient solution. Repeated use of PGPR can stabilize the number of bacteria in the rootzone. Preliminary trials were carried out to determine the bacteria application method. Very successful results were obtained from these preliminary trials. The method used did not ever lead to uneven application or other causes of error. Soilless pepper plants, provided with a nutrient solution with 100% mineral fertilizers, served as a control (Table 1) [20]. Moreover, we substituted 20% and 40% of the mineral fertilizers with mycorrhiza, bacteria, and their combination. In the study, seven treatments were applied.

**Table 1.** The nutrient solution used in the control treatment (100% mineral fertilization) (mg L<sup>−</sup>1).


1. Common nutrient solution, 100% mineral fertilization (as control) (Table 1),

2. 60% mineral fertilization (MF) + PGPR,

3. 60% MF + AMF


#### *2.2. Nutrient Solution and Irrigation*

The amount of nutrient solution applied to the plants was determined based on the daily drainage ratio (DR) from the base of the coco pith slabs. The drainage ratio was approximately 20% ± 5 [21]. The pH and EC of the nutrient solution during the cultivation period were maintained within the range of 6.0–6.5 and 2.0–2.8 dS m<sup>−</sup>1, respectively.

DR = drainage solution (mL) ÷ applied nutrient solution (mL) × 100

#### *2.3. Parameters Examined in the Experiment*

Plant growth parameters, such as plant height, stem diameter and the number of branches, were measured 80 days after seedling transplanting (DAT) (Table 2). In addition, shoot and leaf fresh weights and leaf area per plant were recorded at 164 DAT at the end of the experiment. The leaf area was determined by a leaf area meter (Li-3100, LICOR, Lincoln, NE, USA) and indicated as cm<sup>2</sup> plant−1. Ten plants per plot were used for the measurements.


**Table 2.** Effects of the bio-fertilizers on plant height, diameter and branches, 80 DAT.

DAT: days after transplanting; MF: mineral fertilizer; PGPR: plant-growth-promoting rhizobacteria; AMF: arbuscular mycorrhizal fungi; NS: not significant. Different letters within a column indicate significant differences.

Pepper fruits were harvested weekly when they reached the red maturity stage (Figure 1). The cumulative yield of pepper fruit is expressed as kg m−<sup>2</sup> for the total harvest. Red ripe pepper fruit sampling, 15 fruits per replication, was used for fruit quality measurements. The pH, EC, total soluble solids (TSS), and titratable acidity were measured in the capia pepper fruit.

2.3.1. Determination of Leaf Potassium (K), Calcium (Ca), Magnesium (Mg), Iron (Fe), Zinc (Zn), Manganese (Mn), and Copper (Cu) by Atomic Absorption Spectrophotometry

Leaf samples, 20 fully mature leaves of 10 plants per replicate, were collected at 80 DAT for mineral nutrient analysis. Leaves were dried in a forced-air oven at 65 ◦C for 48 h and ground through a 40-mesh sieve for elemental analysis [21]. The samples were dry-ashed in a muffle furnace at 550 ◦C for six hours. The ash was then dissolved in 0.1 M hydrochloric acid (HCl). K, Ca, Mg, Fe, Mn, Zn, and Cu concentrations were determined using an atomic absorption spectrophotometer [22].

**Figure 1.** Soilless-grown capia peppers were harvested weekly when they reached the red maturity stage. MF: mineral fertilizer.

#### 2.3.2. Determination of Leaf Total Nitrogen (N) by the Kjeldahl Method

Dry-ground leaf samples weighing 1 gm were weighed out; 5 mL of concentrated H2SO4 and a selenium tablet were placed on them; there were burned in the combustion unit of a Kjeldahl apparatus at 400 ◦C for 1 h until the color turned pale. Then, distillation was performed with 28% NaOH in a Kjeldahl tube distillation apparatus. Boric acid and the indicator solution were added to the ammonia released during distillation, and then, titration was performed with 0.01 N HCl. The total nitrogen of the leaf was calculated with the amount of HCl consumed in the titration (modified from [22]).

#### 2.3.3. Determination of Leaf Phosphorus (P) by the Barton Method

The dry-ashed, furnaced and dissolved leaf samples (as mentioned above) were reacted with Barton's solution. The phosphorus concentration was determined at a wavelength of 430 nm in the spectrophotometer (modified from [22]).

#### *2.4. Statistical Analysis*

Data were analyzed using one-way analysis of variance (ANOVA) with the SAS-JMP/7 statistical program. The averages of the treatments were compared with the least significant difference (LSD) test at *p* ≤ 0.05 level.

#### **3. Results**

#### *3.1. Effects of Bio-Fertilizers on Plant Growth*

The synergistic effect of mycorrhiza and bacteria with 80% mineral fertilizer increased pepper plant height in our experiment. They were approximately 21% taller than the control. The tallest pepper plants were grown by using both bio-fertilizers (Table 2). Pepper plants grown in 60% MF + PGPR + AMF were as tall as the control plants. There was no statistically significant difference between treatments for plant stem diameter and number of branches (Table 2). Lucas et al. [23] reported a positive enhancing effect of the beneficial bacteria *Bacillus licheniformis* on tomato and pepper plant height and stem thickness. Moreover, *Bacillus licheniformis* increased auxins and gibberellins in the pepper and tomato plants.

AMF are beneficial to plants by mobilizing nutrients in the root zone, production of siderophores, improving nutrient and water use efficiencies, promoting nutrient uptake of roots, protecting plants from pathogens, and increasing plants' tolerance for abiotic stresses [24]. PGPR are also beneficial to plants by increasing photosynthesis and stimulating the production of phytohormones (indole acetic acid-IAA, cytokinin, gibberellin), secondary metabolic products such as vitamins, and amino acids [25,26]. At the end of the experiment, the heaviest shoot and leaf weights after the control were 545.0 and 262.3 g plant−1, using bacteria and mycorrhiza with 80% mineral fertilizer (Table 3). Similarly, a positive effect of bacteria on tomato and pepper leaf area was found [23]. However, the combination of bacteria and mycorrhiza induced a synergistic effect in our experiment. The photosynthetic performance of the plants largely determines biomass. Kandiannan et al. [27] investigated two bacteria and one mycorrhiza, applied in single, double, and triple combinations to the black pepper plant (*Piper nigrum*) grown in containers. The double and triple combinations significantly increased plant height, leaf area, and biomass production compared to the control plants.


**Table 3.** Effects of the bio-fertilizers on shoot and leaf weights 164 DAT at the end of the cultivation.

DAT: days after transplanting; MF: mineral fertilizer; PGPR: plant growth promoting rhizobacteria, AMF: arbuscular mycorrhizal fungi. Different letters within a column indicate significant differences.

The leaf area of pepper plants increased by using biofertilizers. The leaf is the major photosynthetic apparatus of plants. A synergistic effect of microorganisms was observed for this parameter. AMF, PGPR and 80% mineral fertilizer resulted in a leaf area close to the control (Figure 2). The increase in leaf area might be due to increased nutrient availability due to the production of phytohormones. This, in turn, caused an enhancement in plant growth and fruit yield. As an efficient photosynthetic organ, the leaf area most likely induced the building of more plant carbohydrates [13].

**Figure 2.** Effects of biofertilizers on pepper plant leaf area 164 DAT at the end of cultivation. DAT: day after transplanting. 1:100% MF, 2:60% MF + PGPR, 3:60% MF + AMF, 4:60% MF + PGPR + AMF, 5: 80% MF + PGPR, 6:80% MF + AMF, 7:80% MF + PGPR + AMF. MF: mineral fertilizer; PGPR: plant-growth-promoting rhizobacteria; AMF: arbuscular mycorrhizal fungi. Different letters within a column indicate significant differences, LSD0.05:1413, *p* < 0.0001.

#### *3.2. Effects of Bio-Fertilizers on Total Fruit Yield and Fruit Number*

Total pepper fruit yield ranged from 3.30 to 5.80 kg m−2. The application of bacteria and mycorrhiza to 80% mineral fertilizer induced the highest yield, 32.4% higher than the control (4.38 kg m−2). The second- and third-highest yields were 24.2% and 11.2% for the 80% MF + PGPR and 80% MF + AMF treatments, respectively (Figure 3). In bio-fertilized plants with the consortium of PGPR + AMF, better plant growth and biomass production could promote photosynthesis more effectively. Therefore, accumulated supply facilitates fruit development and contributes to a higher total yield. According to Dere et al. [2], bacteria and mycorrhiza can enhance plant nutrient uptake and, in turn, photosynthesis. The lowest yield was obtained from 60% MF + AMF with 3.30 kg m−<sup>2</sup> with a 16.4% yield decrease compared to the control. Maboko et al. [28] grew soilless tomato plants at 25% and 50% low nutrient levels with mycorrhiza in heated and unheated tunnels. Mycorrhiza worked more effectively in heated tunnels and increased tomato yields at the reduced nutritional treatments. Perhaps the temperatures in heated tunnels contributed to betterestablishing mycorrhiza fungus. Baum et al. [29] reported that mycorrhizal inoculation increased pepper plant growth, fresh biomass, and total yield. Aini et al. [5] found that soilless-grown lettuce associated with PGPR + AMF increased the synthesis of growthpromoting plant hormones, primarily cytokines, which enhances leaf growth. While leaf growth contributes to canopy development, a greater photosynthetically active surface area becomes available, improving plant growth and yield. El-Tohamy et al. [9] reported similar results. Bio-fertilization resulted in higher N, P, and K contents of tomato leaves and higher indole acetic acid, gibberellins, and cytokines. Backer et al. [16] reported that mixing bacteria with mycorrhizal fungi improved corn, tomato, and soybean yields.

**Figure 3.** Effects of the bio-fertilizers on total fruit yield of soilless-grown capia pepper under reduced mineral fertilizers in the Mediterranean climate in spring greenhouse conditions. 1:100% MF, 2:60% MF + PGPR, 3:60% MF + AMF, 4:60% MF + PGPR + AMF, 5:80% MF + PGPR, 6:80% MF + AMF, 7:80% MF + PGPR + AMF. MF: mineral fertilizer; PGPR: plant-growth-promoting rhizobacteria; AMF: arbuscular mycorrhizal fungi. Different letters within a column indicate significant differences, LSD0.05: 1.134, *p*: 0.0023.

The number of pepper fruit harvested per m2 in our experiment varied from 35 to 48 fruit m2. The highest number of fruits was obtained with the treatment using 80% mineral fertilizer combined with bacteria (Figure 4). The presence of 80% mineral fertilizer with bacteria provided better fruit set and development. The number of fruits per unit area of 80% MF + AMF is lower than 80% MF + PGPR and equal to 100% MF (Figure 4). Since the yields of the 80% MF + AMF and 80% MF + PGPR per unit area were in the same significance level and higher than the 100% MF (Figure 3), single-fruit weight of 80% MF + AMF was higher than that of the 80% MF + PGPR and 100% MF (Table 4). It seems that biofertilizer ingenuity induced better plant nutrition and promoted photosynthesis and fruiting.

**Figure 4.** Effects of the bio-fertilizers on total fruit number of soilless-grown capia pepper under reduced mineral fertilizers in the Mediterranean climate in spring greenhouse conditions. 1:100% MF, 2:60% MF + PGPR, 3:60% MF + AMF, 4:60% MF + PGPR + AMF, 5:80% MF + PGPR, 6:80% MF + AMF, 7:80% MF + PGPR + AMF. MF: mineral fertilizer; PGPR: plant-growth-promoting rhizobacteria; AMF: arbuscular mycorrhizal fungi. Different letters within a column indicate significant differences, LSD0.05: 4.119, *p*: <0.001.

#### *3.3. Effects of Bio-Fertilizers on Capia Pepper Fruit Quality Properties*

Using soilless culture systems to control nutrients and temperature in the rootzone and managing environmental and agronomic factors can improve product quality [28,30,31]. Dasgan et al. [13] showed that biofertilizers could affect the leaf yield, nitrate amount, and mineral and antioxidant content of basil (*Ocimum basilicum* L.) in a floating culture. In addition, Baum et al. [29] summarized that many research results prove the positive effects of AMF on plant growth, P crop physical and chemical characteristics, and produce quality. Our study's average pepper fruit weight ranged from 91.96 to 125.16 g. The treatment containing 80% mineral fertilizers with mycorrhiza and bacteria produced 19% heavier fruits than the control. As in the case of the total yield, the combined use of bacteria and mycorrhiza showed synergistic effects on fruit growth and physical properties, e.g., firmness and flesh thickness (Table 4). The second-heaviest fruit was from the 80% mineral fertilizers with mycorrhiza treatment (118.86 g), which was 13% heavier than the control. Effects of bio-fertilizers on pepper fruit height and diameter also increasingly affected the fruit weight. Dasgan et al. [15] reported that when the mineral fertilizers were reduced

by 20% and 40%, mycorrhiza, bacteria, and microalgae increased soilless-grown squash size. Although the effects of the treatments on pepper firmness and flesh thickness were insignificant, the effects on fruit volume were remarkable (Table 4). The maximum fruit volume was obtained from the 80% MF + PGPR + AMF treatment, with 246 cm3 (Table 4).


**Table 4.** Effects of the treatments on the physical properties of capia pepper fruit.

MF: mineral fertilizer; PGPR: plant-growth-promoting rhizobacteria; AMF: arbuscular mycorrhizal fungi; NS: not significant. Different letters within a column indicate significant difference.

Since mycorrhiza and bacteria increased the production of carbohydrates by enhanced photosynthesis and nutrient uptake, it may also have supported significantly increased electrical conductivity (EC) and total soluble solids of the pepper fruit (Table 5). The effects of the treatments on pH and titratable acidity were similar, although there were no statistically significant differences among the treatments (Table 5). Maboko et al. [28] found that inoculating mycorrhiza into soilless-grown tomatoes increased fruit quality, especially total soluble solids and total dry matter. Baum et al. [29] reported that mycorrhiza increased ascorbic acid in chili pepper, beta carotene in potatoes, and carotenoids, phenolics, anthocyanins, chlorophyll and some mineral nutrients in lettuce. In conclusion, bio-fertilizer affects the product quality of vegetables. Michałoj´c et al. [30] investigated the effect of mycorrhiza in two nutrient solutions with EC 2600 and 1900 μS cm−<sup>1</sup> on the quality of soilless-grown tomatoes. Tomato fruits produced with mycorrhiza contained significantly more total soluble solids than non-mycorrhizal ones.

**Table 5.** Effects of the treatments on chemical properties of capia pepper fruit.


MF: mineral fertilizer; PGPR: plant-growth-promoting rhizobacteria; AMF: arbuscular mycorrhizal fungi; NS: not significant; EC: electrical conductivity; TSS: total soluble solids; TA: titratable acidity. Different letters within a column indicate significant differences.

A sensory panel test was conducted by untrained amateur groups and reports showed that there was no significant difference among the treatments for the taste of the pepper such as spiciness, texture and flavor (unpublished data).

#### *3.4. Effects of Bio-Fertilizers on Mineral Nutrient Concentration*

Several studies reported that bacteria and mycorrhizae contribute to plant growth by increasing mineral nutrient uptake [25,30,32,33]. The concentrations of macro elements N, P, K, Ca, Mg, and microelements Fe, Mn, Zn, and Cu in the leaves of pepper plants provided with bio-fertilizers were higher than that of the leaves of the control treatment supplied with 100% mineral fertilizers (Tables 6 and 7). The bacteria and mycorrhiza used in this experiment improved plant nutrition by supplying and facilitating nutrient uptake. According to the pepper plant tissue analysis and interpretation of Hochmuth et al. [34], the mineral nutrition status of the plants fed with the biofertilizers was determined to be sufficient.


**Table 6.** Effects of the bio-fertilizers on pepper leaf macronutrient concentrations (%).

MF: mineral fertilizer; PGPR: plant-growth-promoting rhizobacteria; AMF: arbuscular mycorrhizal fungi. Different letters within a column indicate significant differences.


**Table 7.** Effects of the bio-fertilizers on leaf micronutrient concentrations (mg kg<sup>−</sup>1).

MF: mineral fertilizer; PGPR: Plant-growth-promoting rhizobacteria; AMF: arbuscular mycorrhizal fungi. Different letters within a column indicate significant differences.

Baum et al. [29], reported that the arbuscular mycorrhizal fungi with plant-growthpromoting bacteria, with a 50% reduction of P fertilizer during seedling transplanting, increased the growth and yield of pepper plants. Thus, biofertilizers could substitute P fertilizer in pepper cultivation. Ortas [35] showed that mycorrhizal application enhanced pepper plants' P and Zn content. Bio-fertilizers enrich the root zone with plant nutrients through N fixation, P, and K mineralization and stimulate plant growth regulators [24]. In chili pepper cultivation, mycorrhiza and bacteria have reported excellent synergistic effects on pepper plant nutrition by providing significant advantages to the uptake of P, Zn, Cu, Mn, and Fe nutrients [36]. In addition, some studies [37–39] noted that co-inoculation of mycorrhiza and bacteria improves nutrient uptake. Although the mechanism is not well known, Bharadwaj et al. [40] stated that the AMF secrete carbohydrates, amino acids, and unidentified compounds that could make the environment favorable for the growth of AMF-associated bacterial growth.

#### **4. Conclusions**

Bacteria and mycorrhiza reduced the need for mineral fertilizers used in soillessgrown capia pepper by 20%. Furthermore, combining mycorrhiza and bacteria was more effective than their individual use. Thus, we observed a synergistic effect between the two biofertilizers. The application of the 80% MF + PGPR + AMF induced the highest yield of 5.80 kg m−2, which is 32.4% higher than the 100% MF control yield (4.38 kg m−2). The second- and third-highest yields were 24.2% and 11.2% higher than that of the control for the 80% MF + PGPR and 80% MF + AMF treatments, respectively. The results obtained from the study showed that the use of biofertilizers increased the yield. Therefore, using bacteria and mycorrhiza in soilless-grown capia pepper is an eco-friendly approach to a sustainable environment that reduces synthetic mineral fertilizers and protects the environment.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/horticulturae9020188/s1, Figure S1: Views of the capia red peppers on the plants in greenhouse (a,b) and harvested in the lab for fruit analysis (c).

**Author Contributions:** All the authors contributed to this research. H.Y.D. and M.Y. designed the experiment. Conceptualization; data curation; formal analysis; investigation; resources; funding acquisition: H.Y.D., M.Y., S.D., B.I. Supervision; writing—review, and editing: H.Y.D. and N.S.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Cukurova University Research Foundation (BAP) under project number FYL-2018-10403.

**Data Availability Statement:** The data presented in this study are available in the article.

**Acknowledgments:** We thank the Research Foundation Office of Cukurova University (BAP).

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

#### **References**


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