Cyclic lipopeptides constitute are an important group of secreted secondary metabolites from Bacillus bacteria, as they not only have antimicrobial properties, but also benefit plants by improving soil water status (Fechtner et al. 2011) and inducing systemic resistance through the activation of jasmonic acid/ethylene and salicylic acid (Penha et al. 2020). Moreover, the recent studies highlight the ecological role of CLPs production in soil multispecies community (Chevrette et al. 2022), for example, surfactin enables Bacillus to reduce the toxicity of lipopeptides produced by Pseudomonas (Chevrette et al. 2022). The study by Luzzatto-Knaan et al. (2019) demonstrated the role of surfactins as interspecies recruitment factor – where surfactins recruited Paenibacillus dendritiformis into its ecological niche. Another study revealed the changes in B. subtilis proteome under the surfactin treatment (Luzzatto-Knaan et al. 2019). Thus, Bacillus secondary metabolites shape the microbiome composition, potentially modulating plant health and productivity.
In our work, we investigated the ability of cyclic lipopeptides produced by Bacillus velezensis to modulate the structure of soil and potato rhizosphere bacterial and fungal communities in relation to soil functionality and plant productivity. Existing studies on the impact of antibiotics on soil functionality do not provide a clear pattern (Cycoń et al. 2019; Pino-Otín et al. 2022). The specific effect of antibiotics depends on their effective concentration and the duration of exposure. These parameters are in turn determined by the physico-chemical properties of the antibiotic and the soils themselves (Jia et al. 2023; Rigolet et al. 2024). It is known that lipopeptides in the soil environment are biodegraded by the microbiota present, and although biodestructor strains have been isolated, this aspect has generally not been sufficiently studied (Lima et al. 2011; Habe et al. 2021). Asaka & Shoda (1996) found that surfactant levels remained stable in sterile soil for 25 days, whereas in non-sterile soil, stability may decline more rapidly (Raaijmakers et al. 2010). It can therefore be assumed that CLPs, when added to the soil, acted for 1–2 weeks, after which an indirect post-antibiotic effect developed.
In our study, it was found that CLPs stimulated the metabolic activity of the soil microbiome; the release of carbon dioxide and the accumulation of microbial biomass were several times more active compared to the background level. Activation of soil microbiota was accompanied by accumulation of microbial enzymes. Extracellular enzymes are the main functional indicator of soil health and fertility, playing a role in soil nutrient transformation, energy metabolism, and the degradation of various compounds (Uwituze et al. 2022).
In general, the pattern of activity under the influence of CLPs was as follows: enzymes were activated as early as the 7th day of exposure, and the effect persisted up to 28 days, after which activity decreased to control levels or lower. Compared to other enzymes, LAP stands out, which was activated on day 28 in the control and on day 56 in the CLPs-treated microcosms.
The relationship between soil enzymatic activity and microbiome structure can be achieved through the identification of resistant and susceptible taxa in the soil community. We found that the addition of CLPs reduced the alpha diversity of the bacterial community only by the 56th day of exposure, and the diversity of the fungal community, on the contrary, increased gradually with exposure throughout the entire 56 days of the experiment. We identified 150 bacterial ASVs and 133 fungal ASVs which constitute the dominant parts of classified community. At the genus level, exposure to the CLPs led to changes in the abundance of 26 bacterial and 29 fungal ASVs. Among these, 20 bacterial and 21 fungal genera increased in the relative abundance, while nine bacterial and eight fungal genera decreased.Thus, exposure to CLPs affected the relative abundance of approximately 19% of ASVs in the bacterial-fungal community, with a similar effect on both the bacterial and fungal components.
Analysis of existing works does not provide a clear answer to how the structure of the soil microbial community is modulated under the influence of CLPs. The invasion of exogenous Bacillus has a positive, neutral and negative effects on microbiomes in terms of biodiversity and richness dependent from soil properties, plants seeded (Mahapatra et al. 2022). More detailed experiments under controlled conditions showed that that introduction of surfactin-produced Bacillus into the soil-derived semisynthetic communities contained 13 genera, reduced only two bacterial genera (Kiesewalter et al. 2020). The closest design to our study is the work of Yuan et al. (2017) where the purified CLPs mixture, consisting of iturin A, fengycins and bacillomycins, was added to soil (the authors do not specify the soil type) for four weeks. The results of 18S rRNA amplicon sequencing revealed a decrease in alpha diversity and a shift in the abundance of 31 fungal genera, among them, 12 increased and 19 decreased in the CLPs-treated samples (Yuan J. et al. 2017). Other work describes the effects on the bacterial and fungal community the treatments with purified fingering or iturin. They found increase in fungal Shannon`s index and no changes for bacterial community (Xiao et al. 2021).
Thus, we suggest that the effects on the microbial community may vary due to differences in soil physicochemical properties and experimental design. Our study includes a longer exposure period and a more detailed analysis of the microbial community and its changes as a result of exposure to CLPs.
Analyzing the contribution of microorganisms to enzymatic activity by calculating Spearman coefficients, we determined that the greatest modulating effect on enzyme activity occurs when exposed to 10 mg of CLPs during the first 28 days. The strongest correlations (Rs > 0.8) between enzyme activity and microbial abundance were established for the follow bacterial genera: Methylocapsa, Gaiella (AP); Hyphomycrobium (NAG); Reyranella, Gaiella, o_GP3 (CBH); Gaiella, Reyranella, Baekduia (BG). For the fungal genera: Geminibasidium (NAG); Leptosphaeria, Fusarium, Penicillium (Xylo); Fusarium, Penicillium (BG); Fusarium, Penicillium, Plectosphaerella (CBH); Fusarium, Penicillium (AP); Leptosphaeria, Penicillium (LAP). The LAP activity stands apart, the contribution to the activity of this enzyme on day 56 comes exclusively from the fungal community, which includes 10 genera. The mathematical processing allow us to made the regression model that can predict the enzymatic activity of soil depending on the presence and the abundance of specific soil microorganismsallowing us to create a regression model for them. Interestingly, for all the enzymes except LAP, Hyphomicrobium provides possible contribution to the activity, whereas the contribution of Methylocapsa is negative (and of a smaller or a comparable degree). In the case of LAP, the situation is inverse and the negative effect of Hyphomicrobium is much stronger than the positive contribution of Methylocapsa (moreover, this is the only case where none of the experiment conditions is statistically significant). Another interesting feature of the models for the rest five enzymes is that as soon as Hyphomicrobium is a predictor, the factor of the experiment duration plays statistically significant role in the activity of the enzymes.
Existing studies assess the contribution of bacteria and fungi to enzymatic activity differently. Ullah et al. (2023) found that fungal communities were significantly linked to C-cycling enzymes but not N-cycling enzymes. This is confirmed by a number of works in which, for example, NAGase activity is constitutive for a diverse group of fungi and correlated with soil fungal biomass (Miller et al. 1998). Yanyan Liu et al. (2023) revealed that enzymatic activities of soils were associated mostly with bacteria than fungi and showed that BG and AP activities is related with bacterial community composition, while LAP and polyphenolic oxidase activities determined by fungi.
We mentioned that the enzymatic activity of soils is an indicator of its fertility. We tried to establish the relationship between the activation of the soil microbial community and plant productivity in a field experiment. It was found that the treatment of potato planting material with lipopeptides leads to a more pronounced rate of plant growth, photosynthesis processes and has a positive effect on yield. We suggest that there are two possible explanations for this result. Firstly, lipopeptides can directly activate the processes of plant growth and development. This is illustrated by the following examples: treatment of plants during the first 15–20 days of their development, or seeds before they are sown, with certain biologically active substances, causes certain metabolic changes (Kisil et al. 2023). In addition, applying surfactin A (20 mg/mL), purified from Bacillus subtilis, to soil containing rice seedlings two days after planting, increased the growth and development of Super Basmati rice, resulting in an 80% yield increase (Sarwar et al. 2018).
The second mechanism potentially determining the plant-stimulating activity of lipopeptides is the effect on the microbiome of the potato rhizosphere, as well as on the soil root microbiome. It is known that the production of surfactins is necessary for the successful colonization of the rhizosphere zone by PGP-bacteria (Bais et al. 2004). Thus, in the work of Sarwar et al. (2018) showed that rice roots inoculated with B. subtilis NH100 on day 18 contained up to 200 mg/l surfactin. On tomato roots, a rhizosphere strain of Bacillus produced up to 0.3 µg of surfactin and 0.02 fengycin per gram of roots (Debois et al. 2015). Direct measurements of lipopeptides amount produced by B. subtilis growing on root of cucumber revealed 59 µg surfactin and 1 mg of iturin per gram of roots (Kinsella et al. 2009).
It is possible that the artificial application of cyclic lipopeptides to plant roots stimulates the formation of a stable symbiosis between beneficial microbiota and the plant immediately from the moment of planting, which distinguishes these plants from the control group (when colonization of the rhizosphere by soil indigenous microbiota requires time and certain environmental conditions). Profiling of the soil microbial community of the root zone showed that experimental exposure led to an increase in the proportion of plant-beneficial microorganisms (Hyphomicrobium, Rhizobium, Bradyrhizobium, Mortierella, etc.).