Control of Bacterial Wilt in Tomato using Bio-Antagonists Adsorbed on Chitosan-Silica Nanocomposites

Abstract

Ralstonia solanacearum L, the causal agent of bacterial wilt, is a devastating and persistent pathogen in solanaceous crops. In severe infections, up to 100 % crop loss may occur. Chemical control is difficult due to the pathogen’s variability and persistence in the soil. Biological control holds promise in managing the disease. However, the efficacy of biological control agents (BCAs) is limited by inability to adapt immediately to the environment and their ineffectiveness after in situ application. Hence, the need for suitable carrier materials to enhance their adaptation and efficacy. In addition, for effective management of diseases, precision in disease diagnosis is paramount. The study therefore, entailed synthesis of a suitable nanocarrier material for the delivery of BCAs and development of a diagnostic nanoprobe for R. solanacearum. The materials used to synthesize the carriers of the BCAs and diagnostic nanoprobe were selected based on their good sorption, ease of functionability, anti-bacterial properties, colorimetric effect and non-polluting properties. Chitin was deacetylated and functionalized to form chitosan nanoparticles with a crystallite size of 3.2 nm. It was then immobilized on mesoporous silica nanoparticles (4.0nm) to form chitosan immobilized silica nanocomposites (CISNC) gel with a crystallite size of 5.8 nm. Biological antagonists (Bacillus subtilis, Trichoderma viride, Glomus mosseae, effective micro-organisms, and bacteriophage) were adsorbed on CISNC gel and their effect on R. solanacearum tested. Green synthesized iron oxide nanoparticles were added to the CISNC gel and the mixture adsorbed on nylon nanofibres to form a R. solanacearum diagnostic nanoprobe. Characterization of the CISNC carrier showed physisorption properties, making it ideal for adsorption and desorption of the BCAs. BCAs adsorbed on CISNC carrier were more efficacious than those applied without nanocomposite. Successful adsorption of BCAs on CISNC gel significantly (P<0.05) increased the inhibition of the pathogen, enhanced germination rate and growth, increased chlorophyll in treated plants, reduced wilt incidences, increased yield and prolonged shelf life of tomato fruits. Molecular analysis of treated tomato plants showed increased resistance related biochemicals; chitinase and glucanase. Microscopic observation of the treated root hairs confirmed successful colonization by the BCAs. The enhanced activity of the BCA-CISNC complex (bionanocomposite) was attributed to effective delivery, release and synergy of BCAs and the nanocomposite. The mode of action of the diagnostic nanoprobe for R. solanacearum was based on; chitosan molecules rupturing the bacterial cell wall, complexation of iron oxide with the bacteria cell constituents and the resulting colour change. The grey-like nanoprobe turned brown in the presence of the pathogen due to reduction of iron oxide through the process of mineralization. There was also an effect on the nanoprobe crystallinity and peak reduction of the x-ray powder diffractograms for nylon-ferrous oxide chitosan-silicacomposite (nanoprobe). The crystallite size of nanoprobe changed from 20.4 nm to 21.8 nm after complexation with the R. solanacearum bacteria. The diffractogram peaks of the nanoprobe shifted from 29, 35 and 38 to 32, 38 and 45 nmafter complexation with the pathogenic bacteria. The change indicated a reaction between the nanoprobe and the pathogen. Adsorption of microbial antagonists on CISNC gel enhanced efficacy of the beneficial microbes against R. solanacearum in vitro and in the greenhouse. Finally, the developed nanoprobe heralds precision of bacterial wilt diagnosis. Utilization of the developed nanocomposites enhances tomato production by accurate detection of the pathogen, effective wilt management, increased yield and shelf life of the harvested tomato fruits.