Chitosan nanoparticles as a tool for sustainable crop protection: synthesis, characterization and evaluation of their antifungal activity

Chitosan (CS) is a biopolymer resulting from the deacetylation of chitin, which is the most representative molecule of the fungal cell wall, the exoskeleton of crustaceans and insects. Its success in nanotechnology is due to its unconstrained biological properties like the antimicrobial and plant growth regulatory activity, biocompatibility and non-toxicity to humans. To date, many studies about the exploitation of chitosan in agriculture have been carried on, although the applications of chitosan-based nanoparticles (CS NPs) needs to be deeply explored to understand their true potential. Moreover, the advantages of CS NPs over bulk materials relies in their small size and large surface area. In the present study, CS NPs were synthesized via ionotropic gelation method using sodium tripolyphosphate (TPP) as cross-linking agent. Two protocols were carried out, one of which foresaw the sonication. The resulting NPs were characterized by DLS and Zeta potential analysis. Commercial CS (ChitoPlant®) and synthesized CS NPs were tested in vitro using a microdilution assay (serial two fold dilutions from 10000 to 0 ppm) in 96-well microtiter plates (reads at 595 nm) against 11 fungi: Botrytis cinerea, Colletotrichum lupini, C. nimphaeae, C. acutatum sensu stricto, Fusarium oxysporum species complex, F. oxysporum f.sp. basilici, F. graminearum, Gibberella fujikuroi species complex, Cylindrocarpon macrodidymum, Trichoderma capillare and Clonostachys byssicola. The nucleic acids leakage after a 72-h treatment with CS and CS NPs along with negative control (untreated) was assessed by measuring 260 nm absorbance with a spectrophotometer. In vivo assays with CS NPs on lupin seeds inoculated with C. lupini, and on raspberries inoculated with B. cinerea were performed. As regards in vitro assays, fungi showed very different results at the 5000-ppm concentration. CS and CS NPs (not sonicated) have proven a high efficacy against Fusarium species, G. fujikuroi, C. macrodidymum and T. capillare with a growth inhibition percentage ranging from 93.7% to 100%. C. byssicola was strongly inhibited by CS NPs (98.9%) and less by CS (65.2%). Colletotrichum species were highly inhibited (90-100%) by CS NPs, and much less by CS (5.5-28.6%). B. cinerea showed percentage of growth inhibition of 22.8 and 99.7 when treated with CS NPs and with CS, respectively. These results were in agreement with the findings from the nucleic acids leakage assay. The 260 nm absorbing cellular material increased significantly when compared to untreated control samples indicating a membrane damage mediated by CS or CS NPs depending on the fungal species. CS and CS NPs (size 98 nm and Z potential +12mV), applied as a seed dressing, were tested against C. lupini on artificially inoculated lupin seeds (L. albus cv. Multitalia, L. angustifolius cv. Tango and L. luteus cv. Mister). They showed a good efficacy compared to the untreated control when applied on L. angustifolius cv. Tango seeds. Regarding the raspberry fruits assay, treatments with CS seems to reduce the decay caused by B. cinerea, however CS NPs showed the best performance. The analysis on fatty acids composition of fungal cell was carried out to understand whether it could explain the sensitivity or the tolerance of fungi towards CS. Preliminary results highlighted a good correlation between tolerance/sensitivity of fungi and their fatty acids composition. CS NPs exhibited a more outstanding antifungal activity compared to bulk materials. The promising results obtained open up opportunities for further research on the development of CS NPs based nano fungicides for a sustainable crop protection