Tomato Seed Inoculation with Bacillus subtilis Biofilm Mitigates Toxic Effects of Excessive Copper in the Substrate

Abstract

Accumulation of copper (Cu) in soils devoted to intensive agriculture due to anthropogenic additions is becoming a significant threat to plant productivity. Biological inoculants may play an important role in alleviating toxic effects of heavy metals on plants. The plant-growth-promoting rhizobacteria (PGPR) Bacillus subtilis subsp. spizizenii has demonstrated the ability to reduce harmful impacts of heavy metals on crops. This study aimed to evaluate the suitability of seed inoculation with biofilm produced by this bacterium to mitigate the severity of Cu toxicity on tomato. In the laboratory, first, B. subtilis was cultivated under increased Cu concentrations. Then, germination of inoculated and non-inoculated tomato seeds was tested for Cu concentrations of 0, 50, 100, 150, and 200 ppm. Next, a greenhouse experiment was conducted for four months to assess the effects of both inoculation and excess 150 ppm Cu in the substrate. The studied treatments included control, no inoculation and Cu surplus, inoculation and no Cu surplus, and inoculation plus Cu surplus. In the laboratory, first, the bacterium’s ability to grow in a liquid medium containing Cu was confirmed. Thereafter, we verified that the germination of non-inoculated seeds was negatively affected by Cu, with higher concentrations leading to a more detrimental effect. However, seed inoculation with biofilm mitigated the adverse impact of Cu on germination. Under greenhouse conditions, excess Cu significantly reduced root dry weight, tomato number, and tomato yield compared with the control, whereas shoot dry weight, plant height, leaf area, and soluble solid concentration (Brix index) did not experience significant changes (p < 0.05). However, seed inoculation mitigated the toxic effects of excess Cu, significantly enhancing all the aforementioned plant parameters, except plant height. Seed inoculation also significantly reduced the Cu contents in the fruits of tomato plants growing in the metal contaminated substrate. The biofilm of the B. subtilis strain used demonstrated its effectiveness as a bioinoculant, attenuating the detrimental effects induced by a substrate with excess Cu.

1. Introduction

Horticultural belts, often acting as green belts, are typically established around urban centers, worldwide for cultivation of fruits, vegetables, flowers, and ornamental plants [1,2,3]. They can provide space for food production and support local agricultural communities. Farming in these areas presents a main competitive advantage, because of their proximity to the consumer market. The immediacy to the end user also allows intensive production of a large number of vegetable species, even if climatic and soil conditions are not the most suitable for some crops [4]. While peri-urban soils can be used for horticulture, they are not always ideal. Despite successful cultivation of certain species, these soils may present challenges. For example, physical factors like poor structure or layers of different textures can hinder water and nutrient movement [5], while chemical constraints such as excessive pesticide or heavy metal contamination can negatively impact soil health and plant growth [6,7,8]. Thus, from an ecotoxicological point of view, green belt soils are considered a subject of “sensitive use”, which means that the accumulation of heavy metals requires attention to prevent leaching to groundwater and minimize risks to excess plant uptake and accumulation, ultimately affecting human health [9,10]. The world market for fertilizers and plant protection products is dominated by inputs of synthetic origin, which increase the productivity of crops. However, the irrational use of such chemicals reduces the quality of soil in terms of fertility, heavy metal accumulation, etc., and, in general, causes significant toxic effects on the environment [5,8,11]. To cope with metal toxicity, in sustainable farming systems, there has been a marked growing interest in the adoption of new environmentally friendly production technologies, among which biological inoculants are an increasingly accepted option. More specifically, research on plant-growth-promoting rhizobacteria (PGPRs) able to resist the presence of toxic metals, while limiting their bioavailability in roots and accumulation in plants has become relevant for enhancing plant growth under excessive metal concentrations [11,12].

Heavy metals cannot be biologically or chemically degraded in nature, so once released, they accumulate in different environmental compartments (air, water, soil, biota) to hazardous levels. Moreover, heavy metals frequently undergo chemical and physical transformations that may generate more toxic and bioavailable species [13,14]. In general, heavy metal accumulation in agricultural soils enables root accessibility and uptake, which may lead ultimately to translocation into the plant parts suitable for human consumption, thus becoming a source of toxic effects. Potential heavy metal enrichment in the topsoil of peri-urban areas has been associated with various diffuse or point sources [5,6,7,8]. Copper is one of the most common metals contaminating peri-urban soils. The deposition of Cu into topsoil is mainly caused by the application of inorganic and organic fertilizers, insecticides, and fungicides, which has been steadily increasing. Other sources of excess Cu are mining and industry. Contamination of agricultural soils by Cu, especially vineyards and orchards, has been widely reported. Soil Cu acts as an essential nutrient at an optimum level and as an extremely toxic agent at high amounts [14,15,16]. As an essential micronutrient for plants, Cu is a cofactor for a variety of enzymes, and it plays an important role in photosynthesis, respiration, the antioxidant system, and signal transduction [14,17,18,19]. Excess Cu inhibits growth and causes oxidative stress. Symptoms produced by high Cu concentrations are chlorosis and necrosis, stunting, leaf discoloration, and inhibition of root growth [16,20,21,22,23,24]. Several bacterial genera are known to act as PGPRs, colonizing plant roots and enhancing plant growth through various mechanisms [25,26]. The genus Bacillus has demonstrated high resistance to toxic metals [27,28], which is promising for the development of bioinoculants that could be applied in contaminated agricultural soils. Moreover, several authors have shown the ability of bacteria within this genus to act as PGPRs through various mechanisms including phosphorus solubilization, synthesizing plant-growth-regulating hormones, etc. [29,30,31,32]. In particular, Bacillus subtilis subsp. spizizenii has demonstrated beneficial effects on the development of horticultural species [33,34]. Microorganisms can be formulated as either a planktonic inoculum or a biofilm for different applications. Bacteria in a biofilm provides a more secure path to reproduction and survival, while planktonic bacteria have a lower chance of survival. Therefore, planktonic formulation has been demonstrated to be less efficient, while the application of microbial biofilms as bioinoculants has become more relevant [33,34]. A biofilm is made up of a matrix of exopolysaccharides, with an elevated water content and minor amounts of proteins and DNA. It contains cells in different stages of differentiation, proteins, DNA, lipopeptides, and cell lysis products [35,36,37]. In nature, biofilms are protected environments that shield cells from physical, chemical, and biological changes, increasing their survival. So, biofilms constitute a protected mode of life in which bacteria ensure their survival. [34] Specifically, it has been demonstrated that inoculating tomato seeds using biofilm from B. subtilis subsp. spizizenii promoted plant growth more efficiently than the conventional method of planktonic application of this bacterium [34].

Argentina does have a large territory with an uneven distribution of population that is heavily concentrated around Buenos Aires. As a consequence, a large part of horticulture is being developed in areas surrounding main cities that provide a buffer zone between rural and urban environments. Horticultural belts stand out at La Plata, Mar del Plata, and Buenos Aires cities, serving to buffer urban and rural areas. Specifically, alarming levels of Cu have been reported in soils from the surrounding area of Buenos Aires city, with maximum values of 688 ppm Cu in north areas [38] and 3500 ppm Cu in south areas [5]. Moreover, the eastern part of the city is situated within the Matanza-Riachuelo River basin, which is known for its high level of pollution streaming from industrial and domestic sources. De Cabo et al. [39] reported high concentrations of heavy metals both in riverbank soils and sediments sampled within different stretches of this river, where maximum Cu concentrations reached 108 ppm. In Argentina, the maximum permitted levels of available Cu to maintain agricultural soil quality have been established at 150 ppm (Law 24051). However, other regulations have established stricter limits, such as the ICRLC Guidance note 59/83 (1987), which considers maximum levels of 130 ppm Cu, or the Dutch soil quality standards (2003), which propose levels of 36 ppm Cu [5].

Tomato (Solanum lycopersicum L.) is a globally significant horticultural crop, widely cultivated and with a large production in green belts [40,41]. The nutritional importance of tomato fruit lies in its high content of bioactive components (vitamins, antioxidants), minerals, and fiber, which make it an important functional food [42,43]. The surface area cropped to tomato in the abovementioned green belts of Buenos Aires Province has been estimated at approximately 3 × 10³ hectares of tomato, accounting for approximately one-fourth of the total country surface devoted to this crop [41].

The fate of trace metal levels in tomato, including Cu, has been previously studied. Tomatoes can accumulate trace metals from the soil or from irrigation water or fertilizers [1]. However, the effect of seed inoculation with biofilm in mitigating excess Cu levels of the substrate has not been addressed until now. In light of the above rationale, the objectives of this study are (1) to analyze the growth promoter effect of the biofilm obtained from Bacillus subtilis subsp. spizizenii when inoculating tomato seeds, (2) to evaluate the effect of seed inoculation on alleviating toxic effects resulting from tomato cultivation on a substrate with excessive Cu, and (3) to assess the impact of both biofilm seed inoculation and substrate excess Cu on the fruit concentration of this metal.

2. Materials and Methods

2.1. Experimental Site

Laboratory tests and greenhouse experiments were conducted at the Faculty of Agronomy, University of Buenos Aires (FAUBA), Argentina (34°45′ S latitude and 60°31′ W longitude, 25 m altitude). The site has a humid subtropical climate, classified as Cfa according to Köppen. Experiments were conducted using seeds of the Rio Grande variety of S. lycopersicum L. This variety is known for its high yield, disease resistance, and suitability for processing among which biological inoculants are an increasingly accepted option. It is considered a determinate variety, meaning the plants display a concentrated fruit ripening, stopping growth when they reach a certain size.

2.2. Bacterial Activation and Biofilm Preparation

The biofilm inoculation experiments were carried out using the strain of Bacillus subtilis subsp. spizizenii retrieved from the culture collection AGRAL, which was kept at the Faculty of Agronomy, Buenos Aires University (FAUBA). This B. subtilis strain was originally purchased at the American Type Culture Collection (ATCC), Accession No. 6633. Colonies of B. subtilis from the stock culture were inoculated in Petri dishes containing nutrient agar [34]. The plates were then incubated at 30 °C for 24 h to achieve bacterium reactivation.

Biofilm production was performed by inoculating B. subtilis into the culture medium and subsequent incubation at 30 °C for 96 h [34]. The culture medium was a liquid minimal salt medium (MSM) with 1% glycerol and 55 mM L-glutamic acid as a carbon source, whose composition has been quoted before [34]. Erlenmeyer flasks (500 mL) containing 150 mL of culture medium were used to carry out incubation under static conditions. The biofilm was produced at the air-liquid interface and extracted with a glass rod. Afterwards it was used for in vitro germination assays and for greenhouse trials. A more detailed description has been provided in previous work [33,34]. To avoid challenges by mixing biofilm and seeds, the biofilm was placed in a sterile crystallizing dish, onto which S. Lycopersicum seeds were deposited. The biofilm was then carefully incorporated and mixed with the seeds using a glass road to ensure complete impregnation.

2.3. Laboratory Tests

2.3.1. Effect of Cu Concentration on Bacterial Growth

Prior to germination assays and greenhouse trials, the tolerance of the bacterium to increasing Cu concentrations in the culture medium was determined. Colonies of B. subtilis were cultivated in the MSM mentioned before with Cu concentrations of 0, 50, 100, 150, and 200 ppm, each in triplicate. Copper chloride (CuCl₂) was added as a Cu source. Bacteria incubation was implemented under rotary shaking at 150 rpm and 30 °C. Bacterial growth was determined by spectrophotometric measurement of the absorbance at 610 nm (OD_610nm).

2.3.2. In Vitro Seed Germination

The germination test aimed to evaluate the impact of Cu concentration and biofilm inoculation. For this test a randomized block design was adopted with ten treatments, including four different Cu concentrations (50, 100, 150, and 200 ppm) in the growing medium, both with and without seed inoculation, plus two control treatments (no Cu and no Cu plus inoculation). In all the treatments, germination rate and radicle length were measured.

The seeds of the Rio Grande variety were superficially disinfected by immersion in a 70% alcohol solution. Subsequently, the seeds were washed three times in sterile distilled water and laid on sterile filter paper for drying. The inoculated treatments were prepared by thoroughly mixing tomato seeds with the biofilm because of the strong adhesion between biofilm and seeds. For the non-inoculation treatments, the seeds were soaked in distilled water under the same conditions.

For in vitro germination tests, inoculated and non-inoculated seeds were placed in sterile Petri dishes, which contained a layer of sterile cotton wool topped with Whatman qualitative filter paper Grade 3. Seeds were moisturized by adding 5 mL of sterile distilled water (control) or Cu solutions of 50, 100, 150, or 200 ppm, depending on the treatment. Fifty seeds per treatment were evaluated.

After moistening, seeds were incubated in darkness at 22 °C for 10 days. The indicator of seed germination was the visible appearance of a 2 mm long radicle at the end of the 10-day incubation period [44]. Thereafter, young sprouts keep growing with a photoperiod, i.e., daylight hours, for 5 days, so that 15 days after the start of the experiment the radicle length of seedlings was determined.

Three germination parameters or indices were quantified from the seed germination rate and the radicle elongation, measured at 10 and 15 days since experiment kick-off, respectively [44]. These indices were designed as relative germination percentage (RG, %), relative root elongation (RRE), and relative percentage germination index (GI). They were calculated as follows:

$$RG (\%)=\left({\displaystyle \frac{number of germinated seeds in a treatment}{number of germinated seed in control treatment}}\right) \times 100$$

(1)

$$RRE (\%)=\left({\displaystyle \frac{radicle length in a treatment}{radicle length in control treatment}}\right)\times 100$$

(2)

$$GI (\%)=\left({\displaystyle \frac{RG\times RRE}{100}}\right)$$

(3)

Results are portrayed based on the relative toxicity criteria proposed by Emino et al. [45], which consider three levels of toxicity (high, moderate, and no toxicity) depending on the germination index (

Table 1. Phytotoxicity criteria established according to Emino et al. (2004) [45].

Germination Index (GI%)	Toxicity Level
<50%	High
50–80%	Moderate
>80%	No toxicity

).

2.4. Greenhouse Cultivation

Greenhouse experiments were initiated to analyze the effects of excess Cu in the substrate and seed inoculation on tomato plant growth and fruit production and quality. On the basis of the results obtained from the seed germination tests, it was determined to prepare treatments with metal contamination by adding 150 Cu ppm to a commercial substrate. A randomized block design was adopted with four treatments consisting of (1) control with no seed inoculation and no contaminated substrate, (2) no seed inoculation and substrate added with 150 ppm Cu (Cu), (3) seed inoculation and no contaminated substrate (I), and (4) seed inoculation and substrate with 150 ppm Cu (I+Cu).

2.4.1. Provision of a Cu-Rich Crop Substrate

Under greenhouse conditions tomato seeds were sown and raised in a medium composed of a mixture of commercial substrate and compost at a 3:1 ratio. The commercial substrate had a pH of 5.8 and it was humus- and ash-rich, with 55% organic matter and 45% ash on a dry basis. The C/N ratio was 30%, while moisture content was 50%.

Metal-rich substrates are prepared by incorporating a metal salt, which is used as a precursor of ions. Initially, the salt added is totally bioavailable, but over time the metal may react with the substrate components, producing no bioavailable forms. For this reason, the proportion between the total amount of Cu in the substrate and Cu forms accessible to the plant had to be first established. To quantify the required amount of total Cu, aliquots of substrates homogeneously added with different metal amounts were allowed to stabilize for one month. Thereafter, the bioavailable Cu concentration was determined via atomic spectrophotometry, using the metal chelating agent diethylenetriaminepentacetic acid (DTPA), as described before [46]. Following this procedure, it was estimated that, to obtain a concentration of 150 ppm bioavailable Cu, an initial amount of 300 ppm of this metal should added to the substrate.

2.4.2. Inoculation, Sowing, and Growth Conditions

Tomato seeds designated for the “I” and “I + Cu” treatments were inoculated with biofilm, while seeds used for the “control” and “Cu” treatments were not inoculated. After that, sowing was carried out in germination trays consisting of 7.5 cm tall × 5 cm diameter cells (approximately 0.15 L). For each treatment, 25 individual seeds were planted, with each seed placed into a separate cell of the tray. Sowing cells contained the growing substrate described above. At 30 days after sowing, the seedlings were replanted into 7 L containers filled with the same substrate. After transplanting, seedlings were grown for 4 months, which allowed the plants to develop from seedling to maturity.

The experiments were conducted under natural light, in the period from November to February, i.e., austral late spring and summer. The average greenhouse temperature was 30 ± 5 °C. The tomato plants were irrigated two or three times per week to replenish the water volume required to maintain substrate moisture near field capacity.

2.4.3. Plant and Fruit Analysis at Harvest

At harvest, morphometric analysis was conducted to evaluate above- and belowground biomass production, plant height (PH), and leaf area (LA). These parameters were measured for each of the 25 plants across the four studied treatments. These data were then averaged to obtain a mean value for each specific treatment. Freshly collected plants were kept at 70 °C to a constant weight to determine root and shoot biomass. A caliper was used to measure PH. To determine LA, ten entire and fresh leaves were sampled from the middle part of the plant canopy. No more than one leaf of medium size per branch was collected. Single leaves were placed over a white background and fully expanded; then, they were photographed. The area of each leaf was estimated via IMAGE J software 1.8.0 for scientific image analysis [47].

Tomato fruits from each of the plants across the four treatments were counted and weighed to obtain the number of fruits and the average fruit weight. Fruit quality was evaluated in the fruit juice via total soluble solids content (°Brix) determination. Measurements were carried out in triplicate, using a handheld refractometer.

2.5. Analysis of Cu on Tomato Fruit

Manually collected tomato fruits were carefully washed with tap water to remove surface particles. Next, they were first air dried and kept in an oven at 70 °C until constant weight. Thereafter, they were crushed and grinded to a fine powder using a pestle and mortar.

Total Cu concentrations were quantified via inductively coupled plasma-mass spectrometry (ICP-MS), using an ELEMT XR instrument (Thermo-Finnigan, Waltham, MA, USA) after sample digestion. Digestion was performed following Method 3051A [48], using nitric acid. Briefly, a 500 mg sample was placed in a Teflon PFA digestion vessel, and 10 mL of concentrated nitric acid was added. The vessels were capped and put inside a microwave oven (CEM model MDS-2000). After digestion, the samples were dissolved and filtered through a 0.45 µm pore size cellulose nitrate membrane filter (Millipore Iberica, Barcelone, Spain).

2.6. Statistical Analysis

One way ANOVA was used to determine significant differences between the means of the different treatments studied. The number of treatments considered were ten and four, in the germination tests and the greenhouse experiments, respectively. Post hoc Tukey’s test was used to determine which specific groups differed significantly from each other at the p < 0.05 probability level. Analyses were conducted using the InfoStat version 2008 software package.

3. Results

3.1. Bacterial Tolerance to Increased Cu Concentrations in the Culture Medium

The efficiency of applying a seed bioinoculant to increase plant biomass and fruit yield and to alleviate metal toxic effects mainly depends on the viability of the inoculant components over time. Bacterial viability is particularly important when bioinoculants are designed to be applied to contaminated soils or substrates. Therefore, the ability of the B. subtilis strain to grow in the presence of Cu was first tested. Figure 1 shows the growth curves of the studied bacteria, measured as OD_610nm, corresponding to a control treatment without Cu and for treatments with increasing Cu concentrations added to a liquid minimum salt medium (MSM) supplemented with 55 mM L-glutamic acid and 1% glycerol at 30 °C for 96 h.

Although the B. subtilis strain considered here was able to grow on Cu-containing media, the higher the heavy metal concentration in the growth medium, the more pronounced the inhibitory effect on bacterial growth becomes. Therefore, adding 50, 100 or 150 ppm Cu to the culture medium led to reduced growth rates of 30, 46, and 60%, respectively, relative to those of the control treatment without Cu, whereas after the addition of 200 ppm Cu, the growth rate was very poor.

Therefore, the bacterial growth rate tends to decrease as the copper concentration increases. For all the Cu concentrations tested, the time points of the logarithmic phase (LogF), the latency phase (LF), and the stationary phase (SF) of the growth curves were identified. Regardless of the Cu concentration in the culture media, at 30 °C, the B. subtilis strain reached the midpoint of LF after approximately 7 h of incubation, while it reached the SF after approximately 60 h.

3.2. Impacts of Inoculation and Cu Levels on Seed Germination

The seed germination process is a fundamental aspect of food production and is affected by various environmental factors, including excess concentrations of heavy metals in the soil. The results of the germination tests carried out on Petri dishes are presented below. The treatments considered included both inoculated and non-inoculated tomato seeds germinating with distilled water (control treatment) and in solutions with four different Cu concentrations, namely, 500, 100, 150, and 200 ppm. After seedling emergence, the relative germination percentage and relative root elongation were determined, from which the germination index was estimated.

3.2.1. Relative Germination Percentage

Figure 2 shows the impact on the relative germination percentage of both seeds inoculated with B. subtilis biofilms and the increasing concentrations of Cu.

In the control treatment, seed inoculation with biofilm did not influence the RG percentage. Conversely, the presence of Cu had an inhibitory effect on the RG of non-inoculated seeds and significantly (p < 0.05) reduced it from 50 ppm Cu onwards. Therefore, without inoculation, the RG percentage was 20% and 30% lower than that of the control treatment at 150 and 200 ppm, respectively.

However, inoculation with B. subtilis biofilm prevented this inhibitory effect, so no significant differences from the control were found up to a concentration of 150 ppm Cu. Although, in the treatment with 200 ppm Cu, germination of the biofilm-inoculated seeds was significantly reduced (p < 0.05), the bioinoculant had a positive effect, increasing the RG percentage by 24% compared with that in the non-inoculated treatment.

3.2.2. Relative Root Elongation

A key aspect of seed germination is seedling development, as assessed by relative root elongation. The effects of seed inoculation and increasing Cu concentration in the solution on the RRE of tomato are shown in Figure 3.

The impact of Cu concentration on the decline of relative root elongation mainly depended on its concentration. Solutions with 50 and 100 ppm of Cu showed significantly lower values of RRE compared to the control. The greatest negative effects occurred at Cu concentrations of 150 and 200 ppm, which triggered 50% and 65% RRE decreases, respectively, in the absence of biofilm inoculation of tomato seeds.

No effect of seed inoculation on RRE was detected in the control treatment. Moreover, inoculation of seeds with biofilms did not prevent the negative effects of the metal at 50 and 100 ppm Cu. However, for the highest Cu concentrations, the inoculated treatments showed significant (p < 0.05) increases in RRE, compared with the non-inoculated treatments. So, RRE increases of 24 and 40% were recorded for 150 and 200 ppm, respectively.

3.2.3. Germination Index

The relative germination index takes into account the joint effects of germination and seedling development and provides a quantification of heavy metal toxicity. The effects of seed inoculation with biofilm and increasing Cu concentrations in the solution on the germination index of tomato are shown in Figure 4.

The results obtained for GI are in line with those previously described for RG and RRE. Compared with the control treatment, adding Cu concentrations of 50 and 100 ppm significantly (p < 0.05) reduced GI with respect to the control; notwithstanding, the GI values of both the inoculated and non-inoculated treatments were still higher than 50%, which indicates moderate toxicity. However, for 150 and 200 ppm Cu in the medium, the GI values were 40% and 24% for 150 and 200 ppm Cu, respectively, which is considered high toxicity. The beneficial effect of biofilm inoculation during germination was also corroborated by GI results, since bioinoculant application increased the GI from 40% to 63% at 150 ppm Cu and from 24% to 60% at 200 ppm Cu, whereby the toxicity was reduced from high to moderate in this range of high Cu concentrations.

3.3. Inoculation and Excess Substrate Cu Impacts on Plants and Fruits

Next, results of plant growth, fruit yield, and quality of the tomatoes at harvest, obtained under greenhouse conditions, are presented. So, the impacts of seed inoculation versus no inoculation and the presence or absence of excess Cu in the substrate are compared. The Cu concentration added to the substrate was 150 ppm of available Cu, on the basis of the maximum permitted values according to Argentinean legislation for agricultural soils, as previously stated; this Cu level had evident negative effects on tomato seed germination, as mentioned before. As previously stated, the four treatments studied were as follows: control, I, Cu, and I + Cu.

3.3.1. Plant Variables

Root and Shoot Biomass

Figure 5 shows the mean root and shoot biomass of tomato plants at harvest measured in the studied treatments.

Adding 150 ppm Cu to the substrate negatively affected root development, resulting in a 50% decrease in the root dry weight of tomato plants. Biofilm inoculation of tomato seeds, however, had positive effects on root growth in both the treatments without and with the addition of Cu to the substrate. Therefore, relative to the non-inoculated treatments, inoculation promoted increases in the root dry weight of 37% when Cu was not incorporated into the substrate and of 107% when the substrate contained 150 ppm extra Cu. Moreover, root growth at harvest was not significantly different (p < 0.05) between the Cu-free treatment and the 150 ppm Cu treatment. Therefore, seed inoculation protected tomato roots from the deleterious effects of the metal.

Excess Cu in the substrate did not have a significant (p < 0.05) effect on tomato shoot dry weight. However, seed inoculation with biofilm was beneficial for tomato shoots of plants growing either in substrates with no extra Cu or in substrates with an additional 150 ppm Cu; 47% and 40% increases in shoot biomass for the former and latter Cu doses, respectively, were recorded. Therefore, shoot growth was significantly greater in the inoculated treatments than in the non-inoculated treatments (p < 0.05).

Height and Leaf Area

The effects of extra Cu in the substrate and seed inoculation with biofilm on tomato plant height and leaf area are shown in Figure 6. The 150 ppm Cu added to the substrate had no statistically significant impact (p < 0.05) on the height and the surface area of the tomato plants; note that this lack of excess Cu effect was also observed in the shoot biomass (Figure 5). Seed inoculation with biofilm significantly p < 0.05) increased the height of plants grown in substrates with no extra Cu added, but the beneficial impact on plant height was not observed in plants grown in substrates with no extra Cu. Thus, the increases in height of the tomato plants induced by inoculation were 20% and 12% for the treatments with no Cu and 150 ppm extra Cu, respectively.

In the absence of seed inoculation, leaf area showed no significant difference (p < 0.05) between the two substrate Cu levels compared. However, the positive effects of seed inoculation with biofilms on leaf area were more important than those on plant height. Therefore, seed inoculation with biofilm significantly (p < 0.05) increased the leaf area, triggering increases of 67% and 64% for substrates without and with Cu addition, respectively.

3.3.2. Fruit Yield and Quality

Once harvested, tomato fruit may be either freshly consumed or preserved long-term via various methods. Therefore, compared with plant biomass production, ripe tomato yield, and quality are of greater interest. Figure 7 shows the variables recorded for assessing the yield and quality of the edible part of the plant: the mean number of fruits per plant, mean fruit weight, and fruit quality as assessed by the Brix index.

The addition of Cu to the substrate had a strong negative effect on fruit number, triggering a 50% reduction so that the mean number of tomatoes per plant decreased from 6 to 3. Seed inoculation with biofilms significantly (p < 0.05) increased the number of tomatoes growing both in the treatment with no added Cu and in the treatment with 150 ppm extra available Cu. Therefore, on average, the fruit number increased from 6 to 9 in the Cu-free treatment and from 3 to 5 in the treatment with excess Cu. Biofilm inoculation partially reversed the deleterious effect of excess Cu in the substrate, so the fruit numbers in the control treatment (i.e., no inoculation and no Cu addition) and I+Cu treatment (i.e., inoculation plus excess substrate Cu) were not significantly different (p < 0.05).

In contrast, adding Cu to the substrate in the absence of inoculation affected neither the mean tomato weight nor the total soluble solids value (Brix), which is considered a quality index. On the other hand, seed inoculation with biofilm significantly (p < 0.05) increased the mean fruit weight and Brix, the positive effects of inoculation resulted in values of these parameters being 40% greater than those of the control treatment. Notably, the Brix value of the inoculated treatment with no excess Cu reached 3.9, approaching the optimum Brix value of 4.0, which is considered a reference for fresh tomato consumption [49]. In the presence of Cu, the positive effect of inoculation was maintained in the case of Brix value but not for the fruit weight. Notably, the Brix value of the inoculated treatment with excess Cu increased by 17% compared to the control without Cu.

On average, the tomato yields for the studied treatments were 228 ± 16 g (control), 477 ± 24 g (I), 114 ± 12 g (Cu), and 215 ± 19 g (I + Cu). Comparing the I with the I + Cu treatment on the one hand and the control with the Cu treatment, on the other hand, excess Cu in the substrate significantly (p < 0.001) decreased tomato yield. The decline of yield was about 50% when the control treatment was compared with the Cu treatments, and even more than 45% when I and I+Cu treatments were compared. Conversely, inoculation very significantly increased tomato yield when both the absence and excess of Cu were considered.

3.4. Impact of Inoculation and Cu Substrate Excess on Cu Fruit Concentration

Overall, regardless of treatment, the Cu fruit concentration ranged from 5.11 to 10.38 mg/kg. The lowest value corresponded to the treatments with no Cu addition to the substrate, while the highest value corresponded to the treatments with Cu excess (Figure 8).

Excess Cu in the substrate caused significant (p < 0.05) increases in the levels of this metal in fruits (Figure 8). In the absence of inoculation, tomato plants growing in substrates with 150 ppm excess Cu presented increases in the fruit concentrations of this metal relative to those in the control treatment of 23.9%. Conversely, in the inoculated treatments with excess Cu (I+Cu), the fruit Cu levels increased by 57.7%, relative to those in the I treatment. Finally, seed inoculation significantly reduced the Cu level of fruits; the extent of this reduction was 29% in the treatments with no added Cu and 64% in the treatments with excess Cu.

4. Discussion

Bacillus subtilis subsp. spizizenii inoculated on the horticultural species Lactuca sativa L., Solanum lycopersicum L. and the legume Glycine max L. (MERR) has been shown to be a potent biostimulant [33], biofertilizer [50], and biopesticide [51]. Positive effects were observed when the PGPRs were applied either in the traditional formulation, e.g., the planktonic state, or as a biofilm. However, the biofilm formulation produced the best results. These previous studies motivated our work, which aimed to evaluate whether biofilm seed inoculation could act as an efficient bioinoculant when Solanum lycopersicum L. was growing on a substrate with high Cu content.

The B. subtilis strain used in our work was able to grow in liquid medium containing Cu, even at a concentration of 150 ppm, without reducing the percentage of germination. At a concentration of 200 ppm copper, there was almost no bacterial growth. This finding is consistent with previous evidence that the use of Cu as a bactericide at a concentration of 200 ppm led to almost no bacterial growth, both for Gram-positive (including Bacillus) and Gram-negative bacteria; this finding is consistent with the high toxicity level found in our work when a solution with 200 ppm Cu was used. Additionally, recently, the critical toxicity level of 100 ppm Cu in soils has been documented [52]. In our study, as previously indicated, 150 ppm of available Cu was used as a reference for agricultural soil quality in accordance with Argentinean legislation (Law 24051), but this value differs from other standards.

Germination and the first stage of seedling development are phases of great sensitivity to changing environmental conditions and adverse external factors, such as the abundance of heavy metals in the soil [53]. In our work, germination tests effectively evaluated the toxic effects of Cu on the seeds of the Rio Grande tomato variety. A marked inhibition of germination, which increased with increasing Cu concentration, was observed. This result agrees with the findings of [54], who noted 38.3% inhibition of tomato seed germination at 175 ppm Cu. [23] reported that deleterious effects on germination are activated by the inhibitory action of heavy metals on enzymes involved in the mobilization of seed carbon reserves. Thus, following this author, the presence of metal hinders the nutrition of the embryo. In contrast, in our study, biofilm-inoculated seeds were partially protected from the toxic action of Cu, so their germination power decreased significantly only when they were grown under the highest imposed Cu concentration, namely, 200 ppm. Even at this concentration, B. subtilis somewhat protected the seeds, as the germination percentage of the inoculated seeds was greater than that of the non-inoculated seeds. This finding indicates that the biofilm formulation of B. subtilis was able to withstand the toxic action of heavy metals, promoting germination. As indicated above, the planktonic formulation of this bacterium did not grow at 200 ppm.

Toxic effects of Cu were also observed on root elongation at all the concentrations tested. Moreover, in our work, biofilm seed inoculation was able to partially alleviate the negative effects of high Cu concentrations. El-Tayeb et al. [55] reported that increasing the Cu concentration in the germination solution generally has a severe negative effect on the radicle. The benefits of Bacillus inoculation have also been reported by Pandey et al. [56]; these authors reported increases in the percentage of germination and root elongation in rice seeds when a liquid formulation was used.

The toxicity criterion used in this work is the germination index, which takes into account three phases, namely, imbibition, emergence, and radicle elongation. In other words, this criterion is based on both the germination capacity of the seed and the subsequent development of the seedling [45,57]. A Cu concentration of 150 ppm induced high toxicity in non-inoculated seeds; however, seed inoculation reduced the toxicity from high to moderate, even at the highest Cu concentration tested, namely, 200 ppm. Therefore, seed inoculation with the B. subtilis biofilm allowed the bacterium to utilize its growth-promoting activity, even in a highly Cu-contaminated environment, during germination and seedling emergence. One of the reasons for biofilm effectiveness lies in its own structure, since it contains exopolysaccharides, which facilitate intimate contact between seeds and bacteria, providing greater opportunities for colonization. This point is supported by previous work, where seed biofilm inoculation of the Rio Grande variety of tomato showed much greater results than did conventional liquid inoculation, e.g., a planktonic formulation [34].

After 4 months of tomato growth in the greenhouse, excess Cu in the substrate affected root growth but not shoot growth, plant height or leaf area at harvest. However, plants developed from biofilm-inoculated seeds presented a significant (p < 0.05) increase in all of these parameters, even those growing in a substrate with excess Cu (I+Cu treatment). The beneficial effects of inoculation on tomato growth parameters could be due to the ability of B. subtilis subsp. spizizenii to act as a biofertilizer and phytostimulant by releasing indole acetic acid (IAA) and cytokinins, which have been shown to modify cell morphogenesis and proliferation. Most likely, this results in increased development of the main root, lateral roots, and absorbing hairs, allowing the root to increase its area of exploration in the soil and increasing its ability to take up nutrients. In addition, seed inoculation can benefit plant nutrition via the ability of the inoculant to solubilize phosphorus [33,58,59]. Moreover, it has been determined in the laboratory that this inoculant persists inside the roots of tomato plants from the time of seed inoculation to harvest so that its positive effects can occur throughout the entire crop [34]. Another advantage of Bacillus sp. as a biofertilizer is its ability to fix nitrogen [60] and release siderophores, which make iron available for plant use [61].

The presence of Cu severely affected plant yield in terms of the number of tomatoes harvested. This is consistent with the abovementioned decline in root biomass, which prevents the absorption of sufficient nutrients to enable the plants to produce as many fruits as those in the control treatment. The toxic effect of Cu, however, was avoided by inoculation of the seeds with biofilm, which resulted in yields of similar magnitude to those of plants growing on metal-free substrates. These results could be considered the biofertilization mechanisms described above, which would provide the plant with more nutrients and stimulants to produce a large number of fruits. Seed inoculation was also beneficial in terms of tomato quality (Brix, e.g., soluble solids content), both in the Cu-free and excess Cu treatments in the substrate. Notably, a Brix value near the optimum value (ºBrix 4.0) was reached in the inoculated treatment with no excess substrate Cu.

The concentrations of Cu in the tomato fruits of the Rio Grande variety ranged from 5.11 to 10.38 mg/kg. Although relative difference between treatments as a function of excess Cu in the substrate and inoculation, absolute differences of the fruit Cu levels showed small variations. Concentration of Cu in the edible parts of tomatoes cultivated on contaminated and non-contaminated soils and substrates have been reported by several authors [1,62,63,64,65,66]. The factors studied included growth media contamination, fertilization, organic or conventional cultivation, substrate depth, cultivar type, etc. The tomato fruit Cu concentrations previously reported are highly variable. For example, Ahmed et al. [1] in a field experiment involving contaminated and non-contaminated farms found Cu concentration ranging from 0.26 to 0.63 mg/kg Cu. However, Khan et al. [62] found Cu levels ranging from 27.8 to 35.7 mg/kg in a greenhouse experiment in which the effects of CO₂ concentration on two different tomato varieties were analyzed.

Plants growing in a substrate with excess Cu, showed an increase in fruit metal levels, both under no inoculation and inoculation. Trebolazabala et al. [66] reported that in tomato Cu is stored mainly in the root cortex and in the secondary veins of leaves, whereas in fruits, its concentration is very low. The accumulation of Cu in the cell walls of roots and leaves, as well as its storage inside the cell in the vacuolar compartment, is one of the mechanisms by which plants protect against toxic metals by moving them away from metal-sensitive sites [23].

Tomato plants growing under excess Cu and inoculation (treatment Cu+I) presented a lower metal concentration in fruits than those with extra Cu and no inoculation (treatment Cu). It has been shown that B. subtilis associates or adheres to the root surface to develop a biofilm [67,68]. Thus, a part of the Cu accumulated in the roots could move from the exopolysaccharides and proteins of the biofilm, which immobilize this metal [69]. Notably, Cu adsorption to biofilms results in a protective mechanism for bacteria, allowing them to tolerate toxic metal concentrations [70,71].

Several authors have reported that various bacteria with PGPR attributes can affect the mobility and availability of heavy metals through different mechanisms involving biosorption processes, intracellular accumulation, precipitation, redox processes, and methylation, among others [72,73,74,75]. Similarly, Cu adsorption on the components of the bacterial biofilm associated with the roots could decrease the heavy metal concentration in direct contact with the root, thus enhancing the protective effect of the inoculant. Therefore, the low Cu levels of tomato fruit could also be a result of the decrease in the amount of Cu in contact with the roots.

For regulatory purposes aimed at ensuring consumer safety, the FAO/WHO recommend a maximum permissible limit of 73 mg/kg for copper in vegetables like tomatoes [76]. Permissible limits can vary depending on the specific guidelines they follow, and the value of 40 mg/kg also has been considered as safe for the population [77]. Although great variations in fruit Cu concentrations have been documented [1,62,63,64,65,66,77], our results indicate that Cu levels in the fruits of tomato Río Grande variety are within the acceptable range for safe consumption.

In summary, bioinoculants, such as biofilm produced by Bacillus subtilis subsp. spizizenii, offer promising opportunities to reduce the dependency on chemical fertilizers and pesticides for farmers. These inoculants can enhance nutrient availability, improve plant growth, and offer some protection against pathogens, potentially reducing the need for synthetic fertilizers and pesticides. However, more research is needed to address the challenges regarding environmental variability, scalability, and other knowledge gaps before realizing the full potential of these bioproducts.

5. Conclusions

Bacillus subtilis subsp. spizizenii showed high resistance to the presence of Cu, tolerating concentrations up to 150 ppm of this metal. Moreover, inoculation of S. lycopersicum var. Rio Grande seeds with biofilm produced by B. subtilis protected the seeds from the toxic effects of high Cu levels in the substrate (150 or 200 ppm) during germination, decreasing the toxicity of the metal from high to moderate. At the end of the growth cycle of tomato cropped in a substrate with excess Cu, plants treated with seed inoculation of biofilm exhibited notable improvements in root and shoot biomass, plant height, leaf area, fruit yield, and fruit quality, as assessed by Brix index. While excess Cu in the substrate increased the Cu content in tomato (S. lycopersicum) fruit, seed inoculation with B. subtilis biofilm reduced this elevated metal concentration, and importantly, the final fruit Cu levels did not exceed safe thresholds for human consumption.

Inoculating seeds with biofilms produced by Bacillus subtilis subsp. spizizenii can help prevent or lessen the harmful effects of copper (Cu) on plant growth at the end of the growth cycle. This beneficial effect is likely due to the biofilm’s ability to promote plant growth and potentially reduce the toxicity of soil heavy metals. Our work also confirms that biofilm inoculation is an optimal application method. In addition, bioinoculants potentially reduce the need for synthetic fertilizers and pesticides. However, more research is essential for their widespread adoption under field conditions.