Evaluating response mechanisms of soil microbiomes and metabolomes to Bt toxin additions

https://doi.org/10.1016/j.jhazmat.2023.130904Get rights and content

Highlights

  • Higher level additions of Bt toxins increased soil NH+4-N, NO2--N, and SOM.

  • Longer incubation times led to higher levels of soil NH+4-N, NO2--N, and SOM.

  • Addition of higher Bt toxin altered soil C, N, and P cycling.

  • Bt toxin addition did not adversely affect soil microbial diversity and stability.

  • Putative mechanistic relations of Bt toxins, soils, and microbes were characterized.

Abstract

The accumulation and persistence of Bt toxins in soils from Bt plants and Bt biopesticides may result in environmental hazards such as adverse impacts on soil microorganisms. However, the dynamic relationships among exogenous Bt toxins, soil characteristics, and soil microorganisms are not well understood. Cry1Ab is one of the most commonly used Bt toxins and was added to soils in this study to evaluate subsequent changes in soil physiochemical properties, microbial taxa, microbial functional genes, and metabolites profiles via 16S rRNA gene pyrosequencing, high-throughput qPCR, metagenomic shotgun sequencing, and untargeted metabolomics. Higher additions of Bt toxins led to higher concentrations of soil organic matter (SOM), ammonium (NH+4-N), and nitrite (NO2--N) compared against controls without addition after 100 days of soil incubation. High-throughput qPCR analysis and shotgun metagenomic sequencing analysis revealed that the 500 ng/g Bt toxin addition significantly affected profiles of soil microbial functional genes involved in soil carbon (C), nitrogen (N), and phosphorus (P) cycling after 100 days of incubation. Furthermore, combined metagenomic and metabolomic analyses indicated that the 500 ng/g Bt toxin addition significantly altered low molecular weight metabolite profiles of soils. Importantly, some of these altered metabolites are involved in soil nutrient cycling, and robust associations were identified among differentially abundant metabolites and microorganisms due to Bt toxin addition treatments. Taken together, these results suggest that higher levels of Bt toxin addition can alter soil nutrients, probably by affecting the activities of Bt toxin-degrading microorganisms. These dynamics would then activate other microorganisms involved in nutrient cycling, finally leading to broad changes in metabolite profiles. Notably, the addition of Bt toxins did not cause the accumulation of potential microbial pathogens in soils, nor did it adversely affect the diversity and stability of microbial communities. This study provides new insights into the putative mechanistic associations among Bt toxins, soil characteristics, and microorganisms, providing new understanding into the ecological impacts of Bt toxins on soil ecosystems.

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,

References (59)

  • H. Tapp et al.

    Persistence of the insecticidal toxin from Bacillus thuringiensis subsp kurstaki in soil

    Soil Biol Biochem

    (1998)
  • M. Xiao et al.

    Effects of water management practices on residue decomposition and degradation of Cry1Ac protein from crop-wild Bt rice hybrids and parental lines during winter fallow season

    Ecotoxicol Environ Saf

    (2015)
  • N. Xu et al.

    Effect of biochar additions to soil on nitrogen leaching, microbial biomass and bacterial community structure

    Eur J Soil Biol

    (2016)
  • D. Alok et al.

    Symbiotic nitrogen fixation and endophytic bacterial community structure in Bt-transgenic chickpea (Cicer arietinum L)

    Sci Rep

    (2020)
  • S. Baumgarte et al.

    Field studies on the environmental fate of the Cry1Ab Bt-toxin produced by transgenic maize (MON810) and its effect on bacterial communities in the maize rhizosphere

    Mol Ecol

    (2005)
  • M. Bahram et al.

    Structure and function of the global topsoil microbiome

    Nature

    (2018)
  • J.G. Caporaso et al.

    QIIME allows analysis of high throughput community sequencing data

    Nat Methods

    (2010)
  • D.A. Cataldu et al.

    Rapid colorimetric determination of nitrate in plant tissue by nitrification of salicylic acid

    Comm Soil Sci Plant Anal

    (1975)
  • D. Chourasiya et al.

    Microbial diversity and soil health in tropical agroecosystems (Chapter 2)

  • M.J. Claesson et al.

    Comparative analysis of pyrosequencing and a phylogenetic microarray for exploring microbial community structures in the human distal intestine

    PLOS One

    (2009)
  • S.R. Cotta et al.

    Different effects of transgenic maize and nontransgenic maize on nitrogen-transforming archaea and bacteria in tropical soils

    Appl Environ Microbiol

    (2014)
  • K.Z. Coyte et al.

    The ecology of the microbiome: networks, competition, and stability

    Science

    (2015)
  • F.T. De Vries et al.

    Soil bacterial networks are less stable under drought than fungal networks

    Nat Commun

    (2018)
  • Y. Deng et al.

    Molecular ecological network analyses

    BMC Bioinforma

    (2012)
  • A.B. Dohrmann et al.

    Importance of rare taxa for bacterial diversity in the rhizosphere of Bt- and conventional maize

    ISME J

    (2013)
  • K.K. Fan et al.

    Biodiversity of key-stone phylotypes detennines crop production in a4-decade fertilization experiment

    ISME

    (2021)
  • E.A. Franzosa et al.

    Gut microbiome structure and metabolic activity in inflammatory bowel disease

    Nat Microbiol

    (2019)
  • K. Faust et al.

    Microbial interactions: from networks to models

    Nat Rev Microbiol

    (2012)
  • H. Gruber et al.

    Fate of Cry1Ab protein in agricultural systems under slurry management of cows fed genetically modified maize (Zea mays L.) MON810: a quantitative assessment

    J Agr Food Chem

    (2011)
  • Cited by (0)

    1

    These authors contributed equally to this work.

    View full text