Skip to content
1887

Microbial Genomics logo Microbial Genomics

Volume 11, Issue 4

Comprehensive genome-wide analysis for the safety assessment of microbial biostimulants in agricultural applications Open Access

Abstract

Microbial biostimulants (MBs) offer a sustainable approach to agriculture by helping to reduce reliance on synthetic fertilizers. However, as MBs are intentionally released into the environment, their safety should be rigorously assessed. While taxa with qualified presumption of safety (QPS) benefit from established safety indications, non-QPS taxa lack such guidance. To address this gap, we propose a pipeline combining whole genome sequencing (WGS) and extensive literature search (ELS) data to evaluate microbial safety. We analysed public genomes of three QPS species (, , ) and four non-QPS genera (, , , ), screening them for virulence factors (VFs), antimicrobial resistance (AMR) genes and mobile genetic elements (MGEs). Results confirmed the safety of QPS taxa, revealing no VFs and only a few intrinsic and non-clinically relevant AMRs. Among non-QPS taxa, VF hits were more prevalent in and spp., though they were mostly related to beneficial plant interactions rather than pathogenicity. AMR genes in non-QPS taxa were primarily associated with efflux pumps or were sporadically distributed. Notably, the only genus-wide pattern observed was that most and genomes harboured chromosomally encoded -lactamases sharing similar genetic structures; however, the detected -lactamase () genes were distantly related to clinically relevant variants, and the absence of MGEs suggests a low risk of horizontal gene transfer, indicating the overall safety of these genera. In general, this WGS–ELS framework provides a robust tool for assessing the safety of non-QPS MBs, supporting regulatory decision-making and ensuring their safe use in sustainable agriculture while safeguarding public health.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): antimicrobial resistance , horizontal gene transfer (HGT) , plant growth-promoting rhizobacteria (PGPR) , risk analysis , safety evaluation , sustainable agriculture and virulence factors
© 2025 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
Loading

Article metrics loading...

/content/journal/mgen/10.1099/mgen.0.001391
2025-04-28
2026-04-18

Metrics

Loading full text...

Full text loading...

/deliver/fulltext/mgen/11/4/mgen001391.html?itemId=/content/journal/mgen/10.1099/mgen.0.001391&mimeType=html&fmt=ahah

References

  1. Glick BR. The enhancement of plant growth by free-living bacteria. Can J Microbiol 1995; 41:109–117 [View Article]
     [Google Scholar]
  2. Waksman SA. Principles of Soil Microbiology Williams & Wilkins; 1927
     [Google Scholar]
  3. Berruto CA, Demirer GS. Engineering agricultural soil microbiomes and predicting plant phenotypes. Trends Microbiol 2024; 32:858–873 [View Article] [PubMed]
     [Google Scholar]
  4. Ganugi P, Caffi T, Gabrielli M, Secomandi E, Fiorini A et al. A 3-year application of different mycorrhiza-based plant biostimulants distinctively modulates photosynthetic performance, leaf metabolism, and fruit quality in grapes (Vitis vinifera L.). Front Plant Sci 2023; 14:1236199 [View Article] [PubMed]
     [Google Scholar]
  5. Giorni P, Bulla G, Bellotti G, Antinori ME, Guerrieri MC et al. In planta evaluation of different bacterial consortia for the protection of tomato plants against Alternaria spp. infection and Alternaria toxins presence in fruits. Front Hortic 2024; 3: [View Article]
     [Google Scholar]
  6. Kimotho RN, Maina S. Unraveling plant-microbe interactions: can integrated omics approaches offer concrete answers?. J Exp Bot 2024; 75:1289–1313 [View Article] [PubMed]
     [Google Scholar]
  7. Bashan Y, de-Bashan LE, Prabhu SR, Hernandez J-P. Advances in plant growth-promoting bacterial inoculant technology: formulations and practical perspectives (1998–2013). Plant Soil 2014; 378:1–33 [View Article]
     [Google Scholar]
  8. Shahwar D, Mushtaq Z, Mushtaq H, Alqarawi AA, Park Y et al. Role of microbial inoculants as bio fertilizers for improving crop productivity: a review. Heliyon 2023; 9:e16134 [View Article] [PubMed]
     [Google Scholar]
  9. Mahdi I, Fahsi N, Hijri M, Sobeh M. Antibiotic resistance in plant growth promoting bacteria: a comprehensive review and future perspectives to mitigate potential gene invasion risks. Front Microbiol 2022; 13:999988 [View Article] [PubMed]
     [Google Scholar]
  10. Pawlett M, Hannam JA, Knox JW. Redefining soil health. Microbiology 2021; 167:001030 [View Article] [PubMed]
     [Google Scholar]
  11. Banerjee S, van der Heijden MGA. Soil microbiomes and one health. Nat Rev Microbiol 2023; 21:6–20 [View Article] [PubMed]
     [Google Scholar]
  12. Baumgardner DJ. Soil-related bacterial and fungal infections. J Am Board Fam Med 2012; 25:734–744 [View Article] [PubMed]
     [Google Scholar]
  13. Fang G-Y, Liu X-Q, Jiang Y-J, Mu X-J, Huang B-W. Horizontal gene transfer in activated sludge enhances microbial antimicrobial resistance and virulence. Sci Total Environ 2024; 912:168908 [View Article] [PubMed]
     [Google Scholar]
  14. Gu X, Lu X, Lin S, Shi X, Shen Y et al. A comparative genomic approach to determine the virulence factors and horizontal gene transfer events of clinical Acanthamoeba isolates. Microbiol Spectr 2022; 10:e0002522 [View Article] [PubMed]
     [Google Scholar]
  15. Riva F, Dechesne A, Eckert EM, Riva V, Borin S et al. Conjugal plasmid transfer in the plant rhizosphere in the one health context. Front Microbiol 2024; 15:1457854 [View Article] [PubMed]
     [Google Scholar]
  16. Johler S, Kalbhenn EM, Heini N, Brodmann P, Gautsch S et al. Enterotoxin production of Bacillus thuringiensis isolates from biopesticides, foods, and outbreaks. Front Microbiol 2018; 9:1915 [View Article] [PubMed]
     [Google Scholar]
  17. EBIC The fertilising products regulation should allow microbial plant biostimulants to access the EU market in a way that fosters innovation. In The Fertilising Products Regulation Should Allow Microbial Plant Biostimulants to Access the EU Market in a Way That Fosters Innovation 2022
     [Google Scholar]
  18. EFSA On a generic approach to the safety assessment of micro-organisms used in feed/food and feed/food production. In On a Generic Approach to the Safety Assessment of Micro-Organisms Used in Feed/Food and Feed/Food Production 2003
     [Google Scholar]
  19. Lüth S, Deneke C, Kleta S, Al Dahouk S. Translatability of WGS typing results can simplify data exchange for surveillance and control of Listeria monocytogenes. Microb Genom 2021; 7:000491 [View Article] [PubMed]
     [Google Scholar]
  20. Biggel M, Etter D, Corti S, Brodmann P, Stephan R et al. Whole genome sequencing reveals biopesticidal origin of Bacillus thuringiensis in foods. Front Microbiol 2021; 12:775669 [View Article] [PubMed]
     [Google Scholar]
  21. EFSA Update of the list of QPS-recommended biological agentsintentionally added to food or feed as notified to EFSA 11: suitability of taxonomic units notified to EFSA untilseptember 2019. In Update of the List of QPS-Recommended Biological Agentsintentionally Added to Food or Feed as Notified to EFSA 11: Suitability of Taxonomic Units Notified to EFSA untilSeptember 2019 2019
     [Google Scholar]
  22. Maroniche GA, Puente ML, García JE, Mongiardini E, Coniglio A et al. Phenogenetic profile and agronomic contribution of Azospirillum argentinense Az39T, a reference strain for the South American inoculant industry. Microbiol Res 2024; 283:127650 [View Article] [PubMed]
     [Google Scholar]
  23. Monteiro RA, Balsanelli E, Wassem R, Marin AM, Brusamarello-Santos LCC et al. Herbaspirillum-plant interactions: microscopical, histological and molecular aspects. Plant Soil 2012; 356:175–196 [View Article]
     [Google Scholar]
  24. Benmrid B, Ghoulam C, Ammar I, Nkir D, Saidi R et al. Drought-tolerant rhizobacteria with predicted functional traits enhanced wheat growth and P uptake under moderate drought and low P-availability. Microbiol Res 2024; 285:127795 [View Article] [PubMed]
     [Google Scholar]
  25. Rodriguez-R LM, Conrad RE, Viver T, Feistel DJ, Lindner BG et al. An ANI gap within bacterial species that advances the definitions of intra-species units. mBio 2024; 15:e0269623 [View Article] [PubMed]
     [Google Scholar]
  26. Jain C, Rodriguez-R LM, Phillippy AM, Konstantinidis KT, Aluru S. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat Commun 2018; 9:5114 [View Article] [PubMed]
     [Google Scholar]
  27. Meier-Kolthoff JP, Göker M. TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat Commun 2019; 10:2182 [View Article] [PubMed]
     [Google Scholar]
  28. Auch AF, Klenk H-P, Göker M. Standard operating procedure for calculating genome-to-genome distances based on high-scoring segment pairs. Stand Genomic Sci 2010; 2:142–148 [View Article] [PubMed]
     [Google Scholar]
  29. Duchêne DA. Phylogenomics. Curr Biol 2021; 31:R1177–R1181 [View Article] [PubMed]
     [Google Scholar]
  30. Wattam AR, Davis JJ, Assaf R, Boisvert S, Brettin T et al. Improvements to PATRIC, the all-bacterial bioinformatics database and analysis resource center. Nucleic Acids Res 2017; 45:D535–D542 [View Article] [PubMed]
     [Google Scholar]
  31. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 2004; 32:1792–1797 [View Article] [PubMed]
     [Google Scholar]
  32. Cock PJA, Antao T, Chang JT, Chapman BA, Cox CJ et al. Biopython: freely available Python tools for computational molecular biology and bioinformatics. Bioinformatics 2009; 25:1422–1423 [View Article] [PubMed]
     [Google Scholar]
  33. Letunic I, Bork P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res 2021; 49:W293–W296 [View Article] [PubMed]
     [Google Scholar]
  34. Koutsoumanis K, Allende A, Alvarez-Ordóñez A, Bolton D et al.Hazards (BIOHAZ), E. P. on B Update of the list of qualified presumption of safety (QPS) recommended microbiological agents intentionally added to food or feed as notified to EFSA 20: suitability of taxonomic units notified to EFSA until March 2024. EFSA J 2024; 22:e8882 [View Article] [PubMed]
     [Google Scholar]
  35. Liu B, Zheng D, Jin Q, Chen L, Yang J. VFDB 2019: a comparative pathogenomic platform with an interactive web interface. Nucleic Acids Res 2019; 47:D687–D692 [View Article]
     [Google Scholar]
  36. Bortolaia V, Kaas RS, Ruppe E, Roberts MC, Schwarz S et al. ResFinder 4.0 for predictions of phenotypes from genotypes. J Antimicrob Chemother 2020; 75:3491–3500 [View Article] [PubMed]
     [Google Scholar]
  37. Alcock BP, Raphenya AR, Lau TTY, Tsang KK, Bouchard M et al. CARD 2020: antibiotic resistome surveillance with the comprehensive antibiotic resistance database. Nucleic Acids Res 2019; gkz935: [View Article]
     [Google Scholar]
  38. Peluso S, Aguilera‐Gómez M, Bortolaia V, Catania F, Cocconcelli PS et al. Catalogue of antimicrobial resistance genes in species of Bacillus used to produce food enzymes and feed additives. EFS3 2024; 21:8931E [View Article]
     [Google Scholar]
  39. Ggplot2—Wickham—2011—WIREs Computational Statistics—Wiley Online Library n.d https://wires.onlinelibrary.wiley.com/doi/full/10.1002/wics.147 accessed 12 November 2024
  40. Team, R. C R A language and environment for statistical computing, R Foundation for Statistical. In Computing 2020 https://cir.nii.ac.jp/crid/1370298755636824325
     [Google Scholar]
  41. Price MN, Dehal PS, Arkin AP. FastTree 2--approximately maximum-likelihood trees for large alignments. PLoS One 2010; 5:e9490 [View Article] [PubMed]
     [Google Scholar]
  42. Allen HK, Moe LA, Rodbumrer J, Gaarder A, Handelsman J. Functional metagenomics reveals diverse beta-lactamases in a remote Alaskan soil. ISME J 2009; 3:243–251 [View Article] [PubMed]
     [Google Scholar]
  43. Castanheira M, Simner PJ, Bradford PA. Extended-spectrum β-lactamases: an update on their characteristics, epidemiology and detection. JAC Antimicrob Resist 2021; 3:dlab092 [View Article] [PubMed]
     [Google Scholar]
  44. Wang M, Liu G, Liu M, Tai C, Deng Z et al. ICEberg 3.0: functional categorization and analysis of the integrative and conjugative elements in bacteria. Nucleic Acids Res 2024; 52:D732–D737 [View Article] [PubMed]
     [Google Scholar]
  45. Russell SJ, Garcia AK, Kaçar B. A CRISPR interference system for the nitrogen-fixing bacterium Azotobacter vinelandii. Microbiology 2023 [View Article]
     [Google Scholar]
  46. Matte LM, Genal AV, Landolt EF, Danka ES. T6SS in plant pathogens: unique mechanisms in complex hosts. Infect Immun 2024; 92:e0050023 [View Article] [PubMed]
     [Google Scholar]
  47. Lopez-Reyes L, Soto-Urzua L, Mascarua-Esparza MA, Herrera-Camacho I, Caballero-Mellado J. Antibiotic resistance and β-lactamase activity in Azospirillum. Soil Biol Biochem 1989; 21:651–655 [View Article]
     [Google Scholar]
  48. Palmer M, Steenkamp ET, Blom J, Hedlund BP, Venter SN. All ANIs are not created equal: implications for prokaryotic species boundaries and integration of ANIs into polyphasic taxonomy. Int J Syst Evol Microbiol 2020; 70:2937–2948 [View Article] [PubMed]
     [Google Scholar]
  49. Gushgari-Doyle S, Lui LM, Nielsen TN, Wu X, Malana RG et al. Genotype to ecotype in niche environments: adaptation of Arthrobacter to carbon availability and environmental conditions. ISME Commun 2022; 2:32 [View Article] [PubMed]
     [Google Scholar]
  50. Xin X-F, Kvitko B, He SY. Pseudomonas syringae: what it takes to be a pathogen. Nat Rev Microbiol 2018; 16:316–328 [View Article] [PubMed]
     [Google Scholar]
  51. Herman L, Chemaly M, Cocconcelli PS, Fernandez P, Klein G et al. The qualified presumption of safety assessment and its role in EFSA risk evaluations: 15 years past. FEMS Microbiol Lett 2019; 366:fny260 [View Article] [PubMed]
     [Google Scholar]
  52. Pierson LS, Pierson EA. Metabolism and function of phenazines in bacteria: impacts on the behavior of bacteria in the environment and biotechnological processes. Appl Microbiol Biotechnol 2010; 86:1659–1670 [View Article] [PubMed]
     [Google Scholar]
  53. Hansen LH, Planellas MH, Long KS, Vester B. The order Bacillales hosts functional homologs of the worrisome cfr antibiotic resistance gene. Antimicrob Agents Chemother 2012; 56:3563–3567 [View Article]
     [Google Scholar]
  54. Nøhr-Meldgaard K, Struve C, Ingmer H, Agersø Y. Intrinsic tet(L) sub-class in Bacillus velezensis and Bacillus amyloliquefaciens is associated with a reduced susceptibility toward tetracycline. Front Microbiol 2022; 13: [View Article]
     [Google Scholar]
  55. Magome TG, Ochai SO, Hassim A, Bezuidenhout CC, van Heerden H et al. A genome-based investigation of the Priestia species isolated from anthrax endemic regions in Kruger National Park. Infect Genet Evol 2024; 123:105649 [View Article] [PubMed]
     [Google Scholar]
  56. Liao C, Huang X, Wang Q, Yao D, Lu W. Virulence factors of Pseudomonas aeruginosa and antivirulence strategies to combat its drug resistance. Front Cell Infect Microbiol 2022; 12:926758 [View Article] [PubMed]
     [Google Scholar]
  57. Shao X, Zhang X, Zhang Y, Zhu M, Yang P et al. RpoN-dependent direct regulation of quorum sensing and the type VI secretion system in Pseudomonas aeruginosa PAO1. J Bacteriol 2018; 200:10 [View Article] [PubMed]
     [Google Scholar]
  58. Chepsergon J, Moleleki LN. Rhizosphere bacterial interactions and impact on plant health. Curr Opin Microbiol 2023; 73:102297 [View Article] [PubMed]
     [Google Scholar]
  59. Qin S, Xiao W, Zhou C, Pu Q, Deng X et al. Pseudomonas aeruginosa: pathogenesis, virulence factors, antibiotic resistance, interaction with host, technology advances and emerging therapeutics. Signal Transduct Target Ther 2022; 7:199 [View Article] [PubMed]
     [Google Scholar]
  60. Vandana, Das S Genetic regulation, biosynthesis and applications of extracellular polysaccharides of the biofilm matrix of bacteria. Carbohydr Polym 2022; 291:119536 [View Article] [PubMed]
     [Google Scholar]
  61. VanOtterloo LM, Trent MS. Microbial primer: Lipopolysaccharide - a remarkable component of the Gram-negative bacterial surface. Microbiology 2024; 170:001439 [View Article] [PubMed]
     [Google Scholar]
  62. Héloir M-C, Adrian M, Brulé D, Claverie J, Cordelier S et al. Recognition of elicitors in grapevine: From MAMP and DAMP perception to induced resistance. Front Plant Sci 2019; 10:1117 [View Article] [PubMed]
     [Google Scholar]
  63. Boak EN, Kirolos S, Pan H, Pierson LS, Pierson EA. The type VI secretion systems in plant-beneficial bacteria modulate prokaryotic and eukaryotic interactions in the rhizosphere. Front Microbiol 2022; 13:843092 [View Article] [PubMed]
     [Google Scholar]
  64. Barret M, Morrissey JP, O’Gara F. Functional genomics analysis of plant growth-promoting rhizobacterial traits involved in rhizosphere competence. Biol Fertil Soils 2011; 47:729–743 [View Article]
     [Google Scholar]
  65. Boukhatem ZF, Merabet C, Tsaki H. Plant growth promoting actinobacteria, the most promising candidates as bioinoculants?. Front Agron 2022; 4: [View Article]
     [Google Scholar]
  66. Xu X, Xu M, Zhao Q, Xia Y, Chen C et al. Complete genome sequence of Cd(II)-resistant Arthrobacter sp. PGP41, a plant growth-promoting bacterium with potential in microbe-assisted phytoremediation. Curr Microbiol 2018; 75:1231–1239 [View Article] [PubMed]
     [Google Scholar]
  67. Horna G, López M, Guerra H, Saénz Y, Ruiz J. Interplay between MexAB-OprM and MexEF-OprN in clinical isolates of Pseudomonas aeruginosa. Sci Rep 2018; 8:16463 [View Article] [PubMed]
     [Google Scholar]
  68. Castillo P, Molina R, Andrade A, Vigliocco A, Alemano S et al. Phytohormones and other plant growth regulators produced by PGPR: the genus Azospirillum. In Cassán FD, Okon Y, Creus CM. eds Handbook for Azospirillum: Technical Issues and Protocols Springer International Publishing; 2015 pp 115–138 https://doi.org/10.1007/978-3-319-06542-7_7
     [Google Scholar]
  69. Mattos MLT, Valgas RA, Martins JF da S. Evaluation of the agronomic efficiency of Azospirillum brasilense strains Ab-V5 and Ab-V6 in flood-irrigated rice. Agronomy 2022; 12:3047 [View Article]
     [Google Scholar]
  70. Boggio SB, Roveri OA. Catalytic properties of an endogenous beta-lactamase responsible for the resistance of Azospirillum lipoferum to beta-lactam antibiotics. Microbiology 2003; 149:445–450 [View Article] [PubMed]
     [Google Scholar]
  71. Pandey P, Dubey AP, Mishra S, Singh VS, Singh C et al. β-lactam resistance in Azospirillum baldaniorum Sp245 is mediated by lytic transglycosylase and β-lactamase and regulated by a cascade of RpoE7→RpoH3 sigma factors. J Bacteriol 2022; 204:e0001022 [View Article] [PubMed]
     [Google Scholar]
  72. Kunhikannan S, Thomas CJ, Franks AE, Mahadevaiah S, Kumar S et al. Environmental hotspots for antibiotic resistance genes. Microbiologyopen 2021; 10:e1197 [View Article] [PubMed]
     [Google Scholar]
/content/journal/mgen/10.1099/mgen.0.001391
Loading
Comprehensive genome-wide analysis for the safety assessment of microbial biostimulants in agricultural applications
M Gen 11, 001391 (2025); https://doi.org/10.1099/mgen.0.001391
/content/journal/mgen/10.1099/mgen.0.001391
/content/journal/mgen/10.1099/mgen.0.001391
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Supplementary material 2

EXCEL

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An error occurred
Approval was partially successful, following selected items could not be processed due to error