Biological Systems: Theory and Innovation

Identification of lipopeptide synthesis genes by RT-qPCR in endophytic strains Bacillus pumilus and Bacillus amyloliquefaciens

Viacheslav Shopinskyi*, Liudmyla Butsenko, Oleksandr Subin, Yuliia Kolomyets

chemslava@gmail.com

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Abstract

Endophytic bacteria of the genus Bacillus are promising agents for biological plant protection due to their broad spectrum of antimicrobial metabolites, in particular cyclic lipopeptides (CLPs). The endophytic strains Bacillus pumilus E11 and Bacillus amyloliquefaciens E12, isolated from Pinus sylvestris L. seeds, exhibited antagonistic activity against phytopathogenic microorganisms; however, the molecular-genetic basis of this activity remained unestablished. The aim of the study was to characterise the transcriptional activity of CLPs synthesis genes (fengycin, surfactin, iturin A) in B. pumilus E11 and B. amyloliquefaciens E12 using RT-qPCR with specificity verification via melting curve analysis. Total RNA was isolated from pure cultures in the stationary growth phase using GuSCN buffer with DTT and proteinase K; DNA was eliminated by treatment with DNase I; reverse transcription was performed using the RevertAid First Strand cDNA Synthesis Kit, and RT-qPCR analysis was carried out on a CFX96 Opus (Bio-Rad) amplifier with SYBR Green. Relative expression levels were calculated using the 2-ΔΔСt method with the 16S rRNA reference gene. During the study, the fenD gene of the fengycin operon was identified and its specificity was confirmed by a melting peak (74.0-74.5°C) in both strains studied. The mean Cq value for fenD amplification was 30.3 for B. pumilus E11 and 29.8 for B. amyloliquefaciens E12, and the mean Cq values for 16S rRNA were 21.8 and 23.8 respectively. The relative transcriptional activity of the fengycin biosynthesis gene in B. amyloliquefaciens E12 exceeds that of B. pumilus E11 by a factor of 5.7 (ΔΔCt = -2.5). The surfactin (srfA) and iturin A (ituA) synthesis genes were not detected in any of the strains. Thus, fengycin proved to be the only transcriptionally confirmed antifungal CLP among the studied endophytic strains, confirming their activity against the microfungi Fusarium oxysporum and Alternaria alternata, as determined in previous studies. The high level of fen operon transcription in B. amyloliquefaciens E12 confirms the potential of this strain as a producer of antifungal metabolites for the biocontrol of plant pathogens

Keywords

microorganisms; fengycin; biosynthesis; operon; biocontrol; antifungal activity; Pinus sylvestris

Suggested citation
Shopinskyi, V., Butsenko, L., Subin, O., & Kolomyets, Yu. (2026). Identification of lipopeptide synthesis genes by RT-qPCR in endophytic strains Bacillus pumilus and Bacillus amyloliquefaciens. Biological Systems: Theory and Innovation, 17(3), 22-37. https://doi.org/10.31548/biologiya/3.2026.22
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