The use of various substances in plant cultivation, targeting different purposes such as nutrition, disease protection, pest control, etc., is widespread in agriculture worldwide.^16,17 However, alongside the positive impact on the growth and development of crops, such substances can also have negative effects, particularly on the microbial communities in both soil and plant endophytes.^18,19 Given that IGSA, a chelated fertilizer, is a new product and there is insufficient data on its effects on microbial communities, this study was conducted.

Influence of IGSA on bacterial communities of substrates – hydroponics and soil
In the first stage, we assessed whether the application of IGSA affected the number of operational taxonomic units (OTUs) and their diversity in the substrate and the endosphere of lettuce. In hydroponic cultivation of lettuce, the control samples (without IGSA) had OTU counts in filtrate, leaves, and roots of 449, 51, and 178, respectively. The Simpson and Shannon indices, characterizing alpha diversity of microbial communities in the control filtrate samples, were 0.96 and 4.03, in leaves – 0.95 and 3.53, and in roots – 0.9 and 3.1. Figure 2 shows the OTU counts and the Simpson and Shannon indices expressed as percentages of the control. The application of IGSA did not significantly affect these parameters.

In the soil experiment control samples, the OTU count in soil was 1595, 3.5 times higher than in the control filtrate sample. In leaves, the OTU count was 44, not significantly different from the hydroponic experiment. In roots, the OTU count was 296, 1.7 times higher than in the hydroponic control roots. The Simpson and Shannon indices were 0.99 and 5.65 for soil, 0.75 and 1.97 for leaves, and 0.92 and 3.66 for roots. In the soil experiment, two types of IGSA treatments were used: foliar and root application. As with the hydroponic experiment, IGSA did not affect the alpha diversity of the microbial communities in soil, leaves, and roots, nor the OTU counts in samples, except for the root sample treated with IGSA by root application (sample SRIf), where the OTU count was 73.6% of the control (Figure 3).

Thus, it was determined that in the substrates used for cultivating lettuce (soil, hydroponics), the number and alpha diversity of microorganisms were higher than in the roots and leaves of the plants. Soil contained significantly more microorganisms in both quantity and diversity compared to filtrate. Soil, unlike the aqueous medium, contains a wider range of soluble nutrients. Due to its structure and the presence of grains and aggregates of various sizes and textures, soil provides bacteria with a larger number of ecological niches. The higher number and diversity of microorganisms in the roots of soil-grown lettuce are likely due to some microorganisms being introduced from the soil environment. Bacterial penetration into root tissues can occur passively through root cracks or lateral root exit points, as well as through a number of active mechanisms.^20,21 Unlike roots, no differences in the number of OTUs and alpha diversity indices were observed in the leaves of lettuce grown on different substrates (soil, hydroponics).

Next, the influence of IGSA on the composition and abundance of dominant bacterial taxa (OTU abundance over 2% in at least one sample) in hydroponic filtrate and soil of the soil experiment was evaluated (Table 1). The same bacterial taxa were present among the dominants, but their abundance varied depending on the substrate type. In the control hydroponic filtrate, the dominant bacteria were from the family Simkaniaceae (abundance 4.2) and the genera Glutamicibacter (15.02), Flavobacterium (5.5), Devosia (5.4), and Sphingomonas (6.7). In the control soil samples, the most frequently encountered bacteria were from the genera Pseudarthrobacter (abundance 7.2), Bacillus (2.9), and Sphingomonas (6.7).

Table (1): Dominant (OTU abundance over 2% in at least one sample) bacterial taxa in the microbial community of hydroponic filtrate and soil of the experiment with IGSA chelated fertilizer

Adding IGSA to the hydroponic filtrate altered the abundance of most dominant bacterial taxa. An increase in the abundance of bacteria belonging to the genus Pseudarthrobacter, order Saccharimonadales, families Rhizobiaceae and Rickettsiaceae was observed. The abundance of bacteria from the genera Glutamicibacter, Flavobacterium, Reyranella, Devosia, and Sphingomonas, and the families Saccharimonadaceae and Paracaedibacteraceae decreased.

In the soil experiment, the effect of IGSA on the abundance of bacterial taxa was less pronounced compared to the hydroponic system. This difference is likely due to the filtrate being a more homogeneous medium compared to soil, which increases IGSA bioavailability. An increase in the abundance of bacteria belonging to the genus Pseudarthrobacter was observed in the IGSA-treated variants, both foliar and root applications (samples SSIr and SSIf).

Thus, the application of IGSA leads to changes in the abundance of some dominant strains in the substrate, particularly in hydroponic solutions. However, no elimination of any OTUs or the appearance of new dominants compared to the control occurred. The method of IGSA application (root/foliar) did not affect the composition and abundance of dominant bacterial taxa in soil and filtrate.

Soil serves as a reservoir for a large number and diversity of microorganisms compared to the aquatic environment. The number of bacteria in soil varies from less than 500,000 to 3,863,000 cells/mL, while in water, these numbers are significantly lower. Soil has a high buffering capacity, consistent pH, salinity, and other physicochemical properties. Furthermore, soil is not subject to rapid temperature changes and nutrient concentration fluctuations.^22,23 In water, conditions for the microbial community are more variable. Fluctuations in salinity, thermal and oxygen stratification, and concentrations of ammonium, nitrates, total chlorine, and monochloramine can significantly affect the number of aquatic microorganisms.^24,25 Nutrient deficiency limits microbial colonization of hydroponic systems. The number of microorganisms in it before planting is low, and the microbial community consists of a small number of species. Colonization of hydroponic systems by microorganisms mainly occurs through airborne droplets. For example, when cultivating plants using hydroponic systems, the filtrate can also be colonized by plant endophytes.^26,27

Influence of IGSA on bacterial communities of Lettuce leaves and root endosphere
Next, the effect of IGSA chelated fertilizer on the endophytic microbial community of lettuce grown in soil and hydroponic systems was evaluated (Table 2). Differences in dominant bacterial taxa in the lettuce endophytic microbiome were observed depending on the substrate used (soil/hydroponics). When using soil for lettuce cultivation, bacteria Rhodococcus qingshengii sp. with an abundance of 11.5-34.6 were found in leaves and roots, absent in the endophytic microbiome of plants grown in hydroponics. Conversely, bacteria of the genus Methylophilus were present only in hydroponic experiment samples.

Table (2): Dominant (OTU abundance over 2% in at least one sample) bacterial taxa in the endophytic microbial community of lettuce grown in soil and hydroponic systems with IGSA chelated fertilizer

In the control hydroponic lettuce leaf sample (HLK), the most abundant bacteria were from the genera Sphingomonas (abundance 10.2), Pseudarthrobacter (6.7), and Reyranella (4.1), family Simkaniaceae (5.3), and order Saccharimonadales (8.8). The endophytic microbial community of the root (HRK sample) was characterized by the following dominant genera: Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium (abundance 6.7), Sphingomonas (13), Massilia (9.9), Methylophilus (18.6), and Pseudomonas (5.1).

Using IGSA in the hydroponic experiment increased the abundance of bacteria from the families Muribaculaceae and Enterobacteriaceae and the genus Lactobacillus in leaves. At the same time, the abundance of bacteria from the genus Pseudarthrobacter, family Simkaniaceae, and order Saccharimonadales decreased. In the root microbial community, an increase in the abundance of bacteria from the genera Pseudarthrobacter and Streptomyces and a decrease in the abundance of bacteria from the families Peptostreptococcaceae and genera Romboutsia, Massilia, Pseudomonas, and Allorhizobium–Neorhizobium–Pararhizobium–Rhizobium occurred.

In the control soil-grown lettuce leaf sample, the dominant bacterial taxa were Rhodococcus qingshengii sp. (abundance 31.8) and genus Pseudomonas (37.5). In the root sample, the most frequent bacteria were Rhodococcus qingshengii sp. (abundance 19.8), genera Sphingomonas (6.8), and Pseudomonas (13.5). With the application of chelated fertilizer by both methods (root/foliar), an increase in the abundance of bacteria from the genus Sphingomonas and a slight decrease in the abundance of bacteria from the genus Pseudomonas were observed in the endophytic microbial community of lettuce leaves. Interestingly, using IGSA for lettuce grown in soil had a less pronounced effect on the number of dominant endophytic bacteria compared to its cultivation in hydroponics. The heterogeneous soil structure and sorption processes could have reduced the bioavailability of IGSA. Moreover, assembling the root microbiome in hydroponics fundamentally differs from that in soil, where plants can attract microbial partners from a more diverse pool. This assembly method leads to greater stability of the root microbiome in plants grown in soil compared to hydroponics.^26,28

Overall, the results obtained for the endospheric communities of lettuce are similar to those observed for substrates: the application of chelated fertilizer led to some changes in species composition and diversity, mainly when grown in hydroponics. However, no drastic changes in communities from the original (control) state occurred. The method of IGSA application did not lead to any additional changes in community composition.

Core endophytic microbiome of lettuce plants
Next, we identified bacterial taxa that constitute the core endophytic microbial community of lettuce leaves and roots, irrespective of the growing conditions (hydroponics/soil) (Figure 4). The core microbiome of plants consists of key microorganisms that contain genes selected through evolutionary mechanisms and necessary for plant function and adaptation.^29,30

The core microorganisms for lettuce included 12 bacterial OTUs (Table 3). These microorganisms can inhabit various plants and many other ecological niches (soil, water, biofilms, aquifers, lake sediments).^31-34 Additionally, bacteria from some detected taxa (genus Ralstonia and genus Sphingomonas) are plant pathogens.^35-38

Table (3): Core bacterial OTUs in control samples of lettuce leaves and roots grown in soil and hydroponics

Next, we identified unique OTUs present in control samples of lettuce leaves and roots depending on the substrate used. In lettuce grown in hydroponics, there were 6 and 85 unique OTUs in leaves and roots, respectively. When using soil, 7 and 205 unique bacterial OTUs were found in leaves and roots, respectively. Interestingly, using different substrates did not affect the number of unique OTUs in leaves, but significant differences were found in the number of unique OTUs in lettuce roots. Root-associated microorganisms mainly come from soil. The rhizosphere microbial community strongly influences the root microbiome composition. Soil microbial community structure and environmental parameters, including the substrate, are considered more critical factors for root bacterial community structure than plant genotype or species.^39,40

Next, the microbial communities of lettuce leaves and roots grown on different substrates with and without IGSA chelated fertilizer were analyzed separately (Figure 5 a, b). Among the leaf microbial community, 9 core microorganisms were found regardless of the substrate and fertilizer treatment. More unique taxa (30 OTUs and 23 OTUs) were found in lettuce leaf samples grown in hydroponics, regardless of IGSA treatment (samples HLK and HLI). Overall, comparing microbial communities in root samples revealed more core and unique OTUs than in leaf samples. For example, 36 root core OTUs were found, three times the number of core OTUs in leaf samples. The analysis of core OTUs in microbial communities revealed 8 common endophytes for leaves and roots, present in lettuce plants regardless of growing conditions and IGSA treatment. Notably, the most significant differences in the number of unique OTUs were found between different root samples rather than leaf samples.

Beta-diversity of bacterial communities of soil, hydroponics, and Lettuce plants endosphere
Finally, non-metric multidimensional scaling (NMDS) was used to analyze the similarities and differences in microbial communities of lettuce leaves and roots, soil, and hydroponics. In Figure 6, each point represents an individual sample, and a greater distance between them indicates greater differences between the communities of the respective samples. The figure shows that points representing endophytic communities and substrate communities can be divided into two groups, H and S by substrate type. Within the S group, three subgroups of points corresponding to soil, leaf, and root communities can be distinguished.

Within each subgroup, distances between points representing communities in control and IGSA-treated variants do not differ. NMDS analysis confirms the data obtained using other sequencing analysis methods: the substrate type (soil, hydroponics) has the most significant influence on forming endophytic communities. Chelate application does not affect the composition of endophytic or substrate communities. Lettuce root and leaf communities differ significantly, especially when grown in soil, likely due to rhizosphere microorganisms migrating into plant tissues.