Evaluating response mechanisms of soil microbiomes and metabolomes to Bt toxin additions
Graphical Abstract
Introduction
Bt toxins are crystal proteins produced by Bacillus thuringiensis (Bt) in the early stage of spore formation that exhibit highly specific insecticidal activities [30], [38]. Bt toxin applications primarily include the use of Bt transgenic plants and Bt biopesticides. Indeed, transgenic crops with insect resistance via Bt comprise the largest global fraction of agriculturally planted crop areas [21], [32]. Artificial modification leads to the release of Bt toxins into soils from roots during Bt crop growth that then contributes to toxin levels introduced into soils from pollen during tasseling and from plant residues after harvesting crops [41]. The total amount of Bt remaining in agricultural field soils due to Bt crops could amount to approximately 165 g Bt toxins ha−1 when considering only the root biomass [49]. Furthermore, Bt biopesticides account for about 90% of the production of microbially produced pesticides and have become the most widely used microbial pesticides globally [43]. Consequently, the construction of Bt-recombinant engineered strains has become a major focus in Bt biopesticide research, especially because their expressed recombinant Bt insecticidal proteins remain in soil environments.
Bt toxins expressed and released by Bt transgenic plants or Bt recombinant strains are structurally and functionally very different from Bt prototoxins naturally expressed by B. thuringiensis in soils. The former can consequently be considered an environmentally exogenous compound with insecticidal toxicity that may also have potential ecological risks [52]. In particular, Bt toxins can be closely absorbed onto soil particles and humic acids, forming bound Bt toxins. Bound Bt toxins are more difficult to biodegrade compared to water-dissolved Bt toxins and can persistently retain biocidal activity, resulting in longer-term environmental hazards [10], [41]. Hence, the environmental behaviors of Bt insecticidal toxins and their effects on soil ecology require additional investigation.
Soils harbor vast populations of soil organisms, including microorganisms that play fundamental roles in nutrient cycling via soil carbon (C), nitrogen (N), and phosphorus (P) cycles [29], [3]. Because of these important ecological functions, soil microorganisms serve as sensitive indicators for environmental changes Chourasiya et al., 2017. Moreover, soil microbial communities play critical roles in maintaining ecosystem stability and sustainability [13]. Nevertheless, the responses of co-occurrence networks within these communities to Bt toxin additions remain unknown. Thus, identifying complex patterns of microbial networks to detect and investigate complex microbial interaction webs [18], [57] can contribute to a more comprehensive understanding of the stability and vulnerability of communities to Bt toxin additions. Numerous previous studies have evaluated the effects of Bt toxins on soil microbial activity [47], microbial functional groups [40], and microbial community diversity characteristics [31], [32]. However, modeling the co-occurrence of microorganisms, identifying microbial relationships essential for community stability, and detecting responses of various interactions to Bt toxin addition requires further investigation.
Proteins are sources of organic C and N that are degraded into amino acids, from which inorganic C and N can be finally released. Thus, microbial functional groups involved in these nutrient cycling pathways can be affected by the addition of proteins into soils. Consequently, Bt toxin expression may strongly influence soil microbial community structures. Likewise, microbial community composition can shape environments with Bt toxin presence via microbially-produced metabolites involved in soil C, N, and P cycling [17]. However, studies have thus far indicated that soil microbial communities are negligibly affected by Bt toxin addition [31], [40], [47], although these studies were based on culture-dependent and genetic fingerprinting approaches. Thus, the lack of detected responses may be related to the relatively low sensitivity of these analytical methods. Consequently, the dynamic relationships among soil characteristics with microbial taxa, functional genes, and metabolic pathways in response to Bt toxin exposure should be investigated.
New approaches are needed to help associate soil microbial taxa distributions to metabolites in the presence of Bt toxins, in addition to developing a more comprehensive understanding of the dynamic relationships among exogenous Bt toxins, soil characteristics, and native microorganisms. Deep shotgun metagenomic sequencing and community-wide association studies have enabled more in-depth characterization and insights into the taxonomic and functional diversity of soil microbiomes compared to traditional methods such as cultivation-based, fluorescence in situ hybridization, microarray, or 16 S rRNA gene amplicon sequencing methods [17], [33], [56]. Moreover, metabolomics enables the direct investigation of how microbial taxa transform small molecule metabolites within their environments, thereby helping to reveal the mechanisms underpinning the interplay between microbial communities and environments [11], [33]. Different types of meta-omics analyses can complement and mutually support each other. Thus, integrated meta-omics datasets can yield more in-depth and thorough understandings of microbial communities beyond the sum of each individual dataset [58]. Nevertheless, the integrative analysis of various meta-omics datasets remains limited and has not yet been used to study the responses of microbial taxa, functional potentials, and metabolic pathways to Bt toxin addition into soil environments.
In this study, soil microbial community succession was evaluated after exposure to different amounts of Bt toxins over 100 days using high-throughput sequencing of 16 S rRNA genes. Furthermore, the abundances of microbial functional genes responsible for C fixation, C degradation, N fixation, ammoxidation, nitrification, denitrification, and P utilization were quantified using high-throughput qPCR. Moreover, shotgun metagenomic sequencing was concomitantly used alongside untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomic profiling to identify microorganisms, microbial enzymes, and metabolites of soils that were differentially abundant in Bt toxin addition soils compared to non-Bt addition soils. Finally, the relationships among exogenous Bt toxins, soil characteristics, and native microbial taxa were evaluated using combined multi-omics analyses to better inform environmental risk assessments of Bt toxins.
Section snippets
Soil sampling and incubation
Soil samples were collected from a vegetable field at the Shanghai Academy of Agricultural Sciences (31°13′18″ N, 121°19′10″ E) in Shanghai beginning on May 16, 2021. The field was traditionally rotated with pakchoi, and no Bt crops were previously planted there. All soils in the area have been identified as loam that comprise the most fertile growing conditions globally. Soil sample collection was performed as described by Jiao et al. [22], and soil characteristics are summarized in
Addition of high levels of Bt toxins and longer incubation times altered soil physiochemical properties
An initial application concentration of 500 ng Cry1Ab toxins g−1 to soils led to ELISA-based detection of only 873.10 pg g−1 soil of extracted water-dissolved Cry1Ab after one day of incubation. The concentrations of water-dissolved Cry1Ab toxins in different soil samples were consistent with the initial addition levels (Supplementary Table S1). The measured levels of Bt toxins gradually decreased with incubation time, although Bt toxin concentrations did not significantly differ between Bt
Remaining dynamics of exogenous Bt toxins in soils in addition to their effects on soil physicochemical properties and microbial communities
The experiments of this study demonstrated that the concentrations of water-dissolved Cry1Ab in soils after 1 day of incubation (initial application of 5 or 10 ng g−1) plummeted to levels observed in control soils (Supplementary Table 1). In particular, the extracted amount of Cry1Ab represented less than 1% of the total 500 ng g−1 Bt toxin that were initially applied, consistent with the results of Valldor et al. [49]. These results could be caused by rapid adsorption of Bt toxins onto soil
Conclusions
In this study, a multi-omics framework was used to demonstrate that soil nutrients (NH4+-N, NO2-N, and SOM) and low molecular weight metabolites involved in the cycling of the above nutrients, in addition to associated microbial functional genes, significantly changed with increasing Bt toxin addition and time. Importantly, Bt toxin addition did not significantly alter the abundances of potential microbial phytopathogenic taxa and did not reduce soil microbiome diversity and stability,
Environmental implications
Bt toxins released from Bt plants and Bt biopesticides can intimately associate with soil particles, thereby persistently retaining their biocidal activity and potentially resulting in longer-term environmental hazards such as adverse impacts on soil microorganisms. The effects of Bt toxins on soil microbial ecology are consequently important environmental risk assessments. In this study, changesin soil physiochemical properties, microbial taxa, microbial functional genes, and metabolic
CRediT authorship contribution statement
Li P designed and supervised this work; Ge L, Song LL, Wang LY, Li YJ, Sun Y, Wang C, Wu GG, Chen J, and Pan AH performed this work; Ge L and Song LL wrote the manuscript. Wu YF, and Quan ZZ revised this work. All authors read and approved the manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript. We gratefully acknowledge Prof. Haiyan Chu, Dr. Yu Shi, Dr. Kunkun Fan and Dr. Xu Liu (Institute of Soil Science, Chinese Academy of Sciences, China) for their genuine help for statistical analyses of high-throughput sequencing data. We also sincerely thank Prof. Yuji Jiang (Institute of Soil Science, Chinese Academy of Sciences, China), Dr Mengting (Maggie) Yuan (University of California,
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These authors contributed equally to this work.