Agnes Belak

The increasing demand for raw and minimally processed agricultural products has highlighted the necessity for natural strategies that enhance crop performance and reduce microbial risks along the food production chain. The objective of this study was to select and characterise bacterial strains having biocontrol and plant growth-promoting (PGP) traits from putative endophytes of surface sterilised sweet pepper ( Capsicum annuum L var. grossum ). Amongst the 42 bacterial strains examined, seven exhibited antagonistic activity against either Escherichia coli ATTC 8739 or Listeria innocua CCM 4030 ^T , with two of these bacteria inhibiting both surrogate food-borne pathogens. The inhibitory strains were identified by sequencing the 16S rRNA and rpoB genes, revealing five Pseudomonas , one Enterobacter and one Bacillus species. In tests of PGP traits, such as the indole-3-acetic acid (IAA) production, phosphate and zinc solubilisation, and cellulase activity, Pseudomonas protegens SESK2 exhibited the strongest potential. These findings demonstrate the dual functionality of the selected putative endophytic bacteria, and highlight their potential application in sustainable agri-food systems.

Research background. Several loop-mediated isothermal amplification (LAMP) assays with good performance characteristics have been developed for the detection of Listeria monocytogenes in food; however, there are only a few cases in which DNA extraction, amplification and sensing have been performed in a single-tube system. Experimental approach. The efficiency of DNA extraction by lysis buffers was tested using LAMP. New primer sets for LAMP assays were designed using PrimerExplorer V5 software. The sensitivity and specificity of the LAMP inner primers were determined by optimised PCR. The end-point detection involved gel electrophoresis, turbidity and eriochrome black T (EBT) colour reaction. The sensitivity, specificity and limit of detection (LOD) of the developed LAMP assays were then characterised using L. monocytogenes, non-monocytogenes Listeria and non-Listeria bacterial strains. Results and conclusions. Both the alkaline cell lysis-based sodium hydroxide and Tris-HCl (HotSHOT)+Tween buffer and the Triton X-100 and sodium azide-based TZ buffer generated amplifiable DNA templates under isothermal conditions for LAMP. However, the TZ buffer produced a significantly higher DNA yield than the HotSHOT+Tween buffer. LAMP primers were designed to target the hlyA and inlA virulence genes of L. monocytogenes. The sensitivity and specificity of the LAMP inner primers were 100 % for both genes; however, the PCR reaction targeting the inlA gene generated fewer non-specific PCR products than the hlyA-targeting PCR. The sensitivity of the InlA LAMP assay was 100 %, while its specificity was 96 %. The LOD was 500 fg per reaction, which corresponds to 157 genome copy numbers. The combination of DNA extraction, LAMP amplification, and colorimetric endpoint detection in a single tube resulted in a LAMP assay suitable for the detection of L. monocytogenes in food under laboratory conditions, with potential for further development for on-site detection with microfluidic platforms. Novelty and scientific contribution. To the best of our knowledge, this is the first report of a LAMP assay targeting the inlA gene of L. monocytogenes. The developed single-tube LAMP assay is well-suited for integration with microfluidic systems.