Development and Validation of a Colorimetric Probe for Detection of Bacterial Wilt of Tomato in Kenya

Abstract

Kenya is ranked 6th among the African countries leading in tomato production, with approximately 14% of total vegetable production and 6.72% of horticultural crops. Despite its importance, tomato production is threatened by a wide range of diseases and pests, which result in up to 42% yield losses. Bacterial wilt disease caused by Ralstonia solanacearum. a soil-borne pathogen is a severe threat to the production of over 400 plant species, particularly crops in the Solanaceae family, including tomato and potato. Crop losses can be reduced, and specific treatments developed to control the pathogen if the disease is accurately diagnosed and identified prior to spreading. Existing bacterial wilt identification and detection methods present a wide range of limitations in their use. These conventional methods employ many techniques that involve extensive sample preparation steps before analysis, making them a potential source of error. They are also expensive, time-consuming, and require skilled labour to utilize, thus limiting the effectiveness of any remedial strategy. A rapid, efficient, and user-friendly detection method for most host crops is still lacking. This necessitates the development of methods that are; simple, selective and capable of being used by non-skilled persons in the field. Colorimetric probes can overcome the challenges presented by most conventional detection methods since signaling of the targeted event can be visualized in real-time by the “naked eye”. This study presents the development of a gold-nanoparticle-based colorimetric probe to detect Ralstonia solanacearum through gibberellic acid exudation signalling event. The objectives of this study were to develop a diagnostic probe using gold nanoparticles, quantify the gibberellins produced during Ralstonia-tomato interaction and validate the efficacy of the designed probe under in-vitro conditions. The gold nanoparticles (AuNPs) were chemically synthesized through reduction with citrate ion and characterized using Transmission electron microscopy (TEM) and Dynamic light scattering (DLS) to determine their size. The optical properties of AuNPs solution and its interaction with Gibberellic acid (GA) secreted by Ralstonia Solanacearum was evaluated using Uv-vis spectrophotometry. The formation of gold nanoparticles was visually observed through a change in the colour of the solution confirmed by a broad absorbance peak centered at 520 nm in the UV-vis spectrum. Upon interaction with synthetic gibberellic acid (GA), there was a bathochromic shift in the Surface Plasmon peak of AuNPs to higher wavelengths. A change accompanied this shift in the colour of the reaction media from wine-red to purple. To determine the efficacy of the developed probe, pure cultures of R. Solanacearum were isolated from soils sampled from bacterial wilt infected farms in Kajiado, Kirinyaga, Kiambu and Bomet counties of Kenya, using Casamino acid Peptone-Glucose (CPG) medium with tetrazolium chloride (TZC) at 28°C for 48h. Gibberellins were extracted from the R. Solanacearum cultures and characterized using High-Performance Liquid Chromatography (HPLC) and Fourier transform infrared spectroscopy (FT-IR). Significantly (p ≤ 0.05) varying concentrations of GA3 were obtained from 108, 106, and 104 CFU/ml of R. solanacearum inoculum. The colorimetric probe elicited colour changes from wine-red to purple upon binding selectively with GA-mediated R. solanacearum. The results illustrated the use of Surface Plasmon Resonance (SPR) wavelength-shift sensing and visual colour change of wine-red nanoparticles to purple nanoparticles to detect gibberellic acid mediated R. solanacearum. The distinguishable colour change facilitated the development of a simple sensor for bacterial wilt detection. The high sensitivity of gold nanoparticles played a crucial role in designing the simple colorimetric pathogen probe. This study reports for the first time the use of a colorimetric Nano sensor for bacterial wilt pathogen detection in Kenya. These findings are important for early and pre-detection of the pathogen by small scale farmers all over the country and contribute to management strategies for bacterial wilt.