A Portable Real-Time Quantitative Colorimetric LAMP Workflow for Early In-Situ Detection of Botrytis cinerea in Tomato Plants

In the present study, a novel method was developed and validated for the early, rapid and on-site detection of Botrytis cinerea in crude tomato samples using a newly designed loop-mediated isothermal amplification (LAMP) assay and the portable device PEBBLE. LAMP primers (targeting the IGS region of rDNA gene) were designed, and the sensitivity, specificity and accuracy of the developed method were determined (97.96%, 100% and 98.70%, respectively). The LAMP assay, combined with the portable device, enabled real-time quantitative colorimetric (qcLAMP) detection of B. cinerea in less than 30 min. The limit of detection (LoD) of the qcLAMP assay was 1 pg genomic DNA (≈20 genome copies) per reaction, whereas conventional PCR (utilized as the gold standard method) detected no less than 100 pg of genomic DNA per reaction. In terms of B. cinerea infective propagules, qcLAMP could detect as few as 10² spores in both pure and crude DNA samples extracted from artificially inoculated tomato leaves, whereas PCR detected no fewer than 10⁴ spores in pure DNA samples, failing to achieve detection in crude extracts. In in-vivo bioassays, qcLAMP successfully detected B. cinerea immediately following spore deposition on leaf tissues (0 hours post inoculation) in both pure and crude DNA extracts. In contrast, conventional PCR was successful only in pure DNA samples from leaves collected 2 days post inoculation (dpi) and in crude extracts at 3 dpi, when symptoms were already evident. Evaluations in field samples demonstrated that qcLAMP detected B. cinerea in 100% of crude extracts from symptomatic tissues and in 45% of asymptomatic (latently infected) samples. Conversely, conventional PCR failed to detect the pathogen in crude extracts, regardless of the disease status (symptomatic or asymptomatic). The developed method can serve as a rapid, reliable, and user-friendly tool for the early detection of B. cinerea in the field, enabling prompt assessment of inoculum pressure, thereby facilitating timely management interventions.