Multi-strain consortia are expected to protect plants more reliably than single strains, yet this is rarely tested directly, and the behavior of introduced strains in the field is difficult to follow. We assembled a consortium of Stenotrophomonas rhizophila MGMM118, Bacillus halotolerans MGMM119, Pseudomonas grimontii MGMM120 and Pseudomonas viciae MGMM121 strains isolated from the winter wheat rhizosphere and characterized their hydrolytic activities, auxin production, and analyzed their genomes. The consortium increased shoot and root length of wheat. In biocontrol experiments on barley, the consortium reduced the root rot disease index caused by Fusarium culmorum by approximately ten-fold (5.6% vs. 59.7%), outperforming each individual strain (11.9–17.9%). The consortium also reduced Fusarium root rot in tomato by two-fold. In a field experiment on wheat, strain-specific qPCR detected all introduced microorganisms in the wheat rhizosphere at up to 106 CFU per gram of soil, demonstrating different dynamics for each individual strain. Metabarcoding showed that community structure depended on plant developmental stage rather than on the consortium application. Since metabarcoding could not reliably identify the introduced strains, we suppose that strain-specific qPCR is a more appropriate tool for monitoring of inoculants, particularly in complex soil microbial communities.

Monitoring individual strains in complex microbial communities remains a fundamental challenge in microbial ecology and biotechnology. Here, we present an integrated pipeline for identifying and validating strain-specific loci (SSL) in four biotechnologically relevant plant growth promoting strains from three genera (Stenotrophomonas, Bacillus, and Pseudomonas). The pipeline applies a two-round specificity-filtering strategy combining whole-genome comparison and high-sensitivity BLASTn validation of revealed strain-specific loci (SSL) against the NCBI nucleotide database. SSL count decreased with increasing Average nucleotide identity (ANIb) of the strains used for the analysis, ranging from one locus in B. halotolerans (ANIb = 98.91%) to 15 loci in S. rhizophila (ANIb = 86.49%). All 25 SSL were universally AT-rich, mainly accessory-genome-associated, with flanking regions enriched in genes of unknown function (34.6%) and mobile genetic elements (19.2%). TaqMan qPCR assays targeting SSL demonstrated high specificity—no target sequences were detected across ten geographically distinct soil samples, nor in a native rhizosphere metagenome—and sensitivity, with limits of detection of 0.01–0.1 pg of genomic DNA. Spike-in experiments in soil yielded method detection limits (MDL) of 850–15,000 CFU/g. All four strains were detected in the wheat rhizosphere seven days after consortium application in a field experiment, validating the pipeline for multi-strain field monitoring.

Monitoring individual strains in complex microbial communities remains a fundamental challenge in microbial ecology and biotechnology. Here, we present an integrated pipeline for identifying and validating strain-specific loci (SSL) in four biotechnologically relevant strains of Stenotrophomonas, Bacillus, and Pseudomonas genera. The pipeline applies a two-round specificity-filtering strategy combining whole-genome comparison and high-sensitivity BLASTn validation of revealed strain specific loci (SSL) against the NCBI nucleotide database. SSL count inversely correlated with Average nucleotide identity (ANIb) of the strains used for the analysis, ranging from one locus in B. halotolerans (ANIb = 98.91%) to 15 loci in S. rhizophila (ANIb = 86.49%). All 25 SSL were universally AT-rich, mainly accessory-genome-associated, with flanking regions enriched in genes of unknown function (34.6%) and mobile genetic elements (19.2%). TaqMan qPCR assays targeting SSL demonstrated high specificity – no target sequences were detected across ten geographically distinct soil samples – and sensitivity, with limits of detection of 0.01–0.1 pg of genomic DNA. Spike‑in experiments in soil yielded method detection limits (MDL) of 850 – 15000 CFU/g. All four strains were detected in the wheat rhizosphere seven days after consortium application in a field experiment, validating the pipeline for multi-strain field monitoring.