Antimicrobial Activity Of Botanical Compounds Cinnamaldehyde On Phytophthora Capsici

The oomycete Phytophthora capsici (Leonian) is a destructive fungus-like plant pathogen, which infects solanaceous and cucurbitaceous hosts. Cinnamaldehyde (CA), which is a botanical compound, can efficiently inhibit virus, bacteria and fungi. CA is widely used in medicine, food, cosmetics and other fields. According to early research, we found that CA could effectively inhibit growth of Phytophthora capsici (P. capsici). This thesis mainly focused on P. capsici and CA, which were mainly studied from three aspects.Firstly, this section includes the inhibitory effects of CA on mycelial radical elongation and zoospore germination of P. capsici and field efficacy. According to the dose-dependent inhibition of CA on mycelial growth of P.capsici, the EC50 value was calculated as approximately 0.75 mM. Under a low concentration, CA could efficiently inhibit respiration of zoospores, which led to the inhibitory effects on zoospores. The MIC value of CA against zoospore germination and growth was 0.4 mM. In addition, the effect of CA (81.73%) treatment group is obviously better than the control group of metalaxyl (50.63%). The field efficacy of CA increased 31.1% compared with metalaxyl.Secondly, CA induced immediate Ca2+ efflux from P. capsici zoospores. It was surprising to find that exposure to CA resulted in an immediate decrease in Fluo-3 fluorescence intensity (RFU), which indicated a decline of free Ca2+ level in zoospores. The content of extracellular Ca2+ was determined using ICP-AES. Extracellular Ca2+ increased in the zoospore suspension under stimulation with CA. The addition of ruthenium red and verapamil, two voltage-dependent calcium channel blockers, led to rapid decreases in intracellular Ca+ level in zoospores indicated with Fluo-3 fluorescence. This result suggested that Ca+ influx occurred simultaneously in response to the loss of Ca+ due to Ca2+ efflux in the action of CA. Free intracellular Ca2+ level was decreased by the addition of EGTA, which could inhibit the growth of P. capsici zoospores. Treatment with PA (without α,β-unsaturated bond) led to a Ca2+ influx but not efflux in zoospores with a dose-dependent manner and PA did not show any inhibitory effects on the growth of zoospores even up to the concentration of 2 mM. The nucleophilic mercapto group cysteines of TRP can attack the α,β-unsaturated bond of (CA) via a Michael addition leading to a Ca2+ influx, which suggests that the α,β-unsaturated bond is essential for CA targeting to TRP. The parallel antimicrobial assay indicated that the addition of cysteine significantly antagonized CA-induced growth inhibition of P. capsici zoospores. These results indicated that Michael addition to a,p-unsaturated bond of CA was very important for stimulating Ca2+ efflux and the subsequent growth inhibition of P. capsici. Our results showed that CA-disturbed Ca2+ homeostasis was involved in CA-induced growth inhibition of P. capsic.Thirdly, CA is marginally soluble in water and can be easily oxygenated, which makes its challenging to the practical applications and results in low antimicrobial efficacy. In the present study, Nano-CA complexes were prepared with a modified kinetic and thermodynamic stability system, and had the properties of transparency and low viscosity. Free CA and Nano-CA were compared in terms of their antimicrobial efficacies against P. capsici. The results indicated that Nano-CA had well water-solubility and dispersion stability. The average grain diameter was 38.9 nm by nanocrystallization. According to TEM, the particle size distribution is uniform. In addition, Nano-CA demonstrated enhanced antimicrobial activity with the increase comparing to the free CA. Zoospore germination could be significantly inhibited in the presence of 0.2 mM of Nano-CA. Few zoospores could germinate normally from an exposure to more than 0.3 mM Nano-CA. Compared with free CA, Nano-CA had better efficiency on P. capsici zoospores. P. capsici zoospores treat with Nano-CA, Ca2+ level (RFU) in intracellular, decreased obviously much more than free CA treatment. Nano-CA enhanced the stability and inhibitory efficiency on P. capsici.Our results showed that CA could significantly inhibit P. capisici in vivo or vitro. CA with a α,β-unsaturated bond, as an activator of TRP channel, which may be attacked by nucleophilic mercapto group cysteines (Cys) of TRP via a Michael addition leading to a Ca2+ efflux. CA-disturbed Ca2+homeostasis was involved in CA-induced growth inhibition of P. capsic. Protein of TRP channel could be used as a target on screening new drugs. Besides, Nano-CA enhanced dispersibility and increased antimicrobial efficiency against P. capsic. These results were the basis of practical application of CA and provided new clues for nanominiaturization.