Development of molecular biomarkers for monitoring of arable crops colonization with Methylobacterium symbioticum SB0023/3, a methylotrophic bacterium commonly used as a biostimulant in agriculture

Rolf Allner^1,2Przemyslaw Decewicz¹Thomas Allner²Agata Bluszcz¹Lukasz Dziewit^1,3*

• ¹University of Warsaw, Faculty of Biology, Warsaw, Poland
• ²GOBIO GmbH - Institute for Aquatic Ecology and Applied Biology, Aarbergen, Germany
• ³University of Warsaw, Warsaw, Poland

Introduction: The intensive use of synthetic fertilizers has led to increased nitrous oxide emissions, declining soil fertility, and reduced biodiversity. Biological alternatives, such as the use of endophytic bacteria to improve plant growth, are promising alternatives but require reliable monitoring tools to assess colonization success and biological effectiveness under field conditions. One of the most commonly used microbial biostimulants is Methylobacterium symbioticum SB0023/3; thus, monitoring the efficacy of inoculation and maintenance of this strain is required for adequate evaluation of fertilization practices. Methodology: The resequencing of the genome of M. symbioticum SB0023/3, followed by comparative genomics and functional annotation were performed. Specific real-time PCR primers were developed and validated for strain-specific detection. The colonization of various crops (wheat, corn, rapeseed, peas, and tomatoes) was tested under controlled conditions using developed molecular markers. Results: The resequencing of the SB0023/3 genome revealed novel genetic content and updated previous records. The resequenced genome showed 121 novel regions with 165 protein-coding genes and five tRNA. Based on the newly obtained genome, two highly specific biomarker genes (copG and ubik) were identified and, together with the (Methylobacterium spp./methylotrophs-specific) xoxF gene, validated for their strain/genus-specificity. The developed real-time PCR assays using copG and ubik biomarkers demonstrated high specificity for M. symbioticum SB0023/3, distinguishing it from related species. In contrast, the xoxF gene showed relaxed specificity and cannot be used for SB0023/3 detection. Successful endophytic colonization was confirmed in all tested crops, with high detection rates exceeding 80% in tomatoes. Classical culturing on a novel nitrogen-free medium additionally confirmed colonization, with the same validating the real-time PCR assays. Discussion: This study provides a robust, genome-informed molecular detection system for monitoring M. symbioticum SB0023/3 in crops. The presented approach enables direct detection from plant tissues, facilitating studies on colonization dynamics and biosafety. This methodology can be extended to other microbial biostimulants, supporting sustainable agricultural practices.