Preparation And Antifungal Activity Of Pectinase-responsive Prochloraz Controlled Release Formulation Based On Mesoporous Silica Nanoparticles

Agricultural production benefits from pesticide application to protect the crops from harmful pests.Decrease the utilization efficiency of these chemicals led to their enormous accumulation in the environment,which caused many problems to humans and non-target organisms.Stimuli-responsive carriers as delivery systems of agrochemicals into plants can increase the utilization efficiency of pesticides against target pests and reduce hazards on non-target living organisms and the environment.In this study,the mesoporous silica nanoparticles（MSNs）as a nanocarrier were synthesized.Moreover,prochloraz as a non-systemic fungicide model was loaded into MSNs and coated by pectin on the particle surface as a gatekeeper（Pro@MSN-Pec）to study the translocation and fungicidal activity of prochloraz in rice plants.The main results were as follows:1.The synthesis and characterization of MSNs and Pro@MSN-Pec were conducted.The structure and morphology of the MSNs and Pro@MSN-Pec samples were characterized by scanning electron microscopy（SEM）and transmission electron microscopy（TEM）,respectively.The variation in the functional groups on the samples was recorded on a Fourier transform infrared（FTIR）spectrophotometer.The modification and pesticide loading of the particles were determined by thermogravimetric analysis（TGA）.The size distribution was measured by dynamic light scattering（DLS）.The prochloraz levels were determined by high-performance liquid chromatography（HPLC）.The MSNs were labeled using fluorescein isothiocyanate（FITC）via amino groups（MSN-FITC）to track the distribution of the carriers in the rice plant.The results of SEM and TEM showed that MSNs were observed to have a uniform structure and spherical shapes with a visible mesoporous structure in a size range of 20 to 50 nm as a nanocarrier to transfer and distribute the pesticide in the plants,and Pro@MSN-Pec coating with pectin completely presents good dispersibility and a uniform shape.The result of FTIR spectrophotometer showed successful grafting for FITC onto MSN-NH₂Pro@MSN-Pec was successfully fabricated with a good loading efficiency（30%w/w）.2.The prochloraz release behaviors at different p H values as well as enzyme-responsive release were investigated.The results showed that the release of prochloraz from Pro@MSN-Pec reached 30.85%,59.62%,and 77.36%at p H 7.0,5.0,and 9.0,respectively after 30 days of treatment.Also,a high cumulative release of prochloraz was found after adding pectinase enzyme to the samples（74.55%）compared with absence of enzyme（27.78%）after 21^st day of treatment.3.The cross-sections of roots,stems,and leaves were examined by the green channel of the confocal laser scanning microscopy in order to monitor the MSN-FITC in the different parts of the rice plant.The results showed that MSN-FITC could transfer through the treated leaves to all plant parts,such as the stems and roots.Moreover,MSN-FITC could penetrate the rice roots and translocate to all plant tissues,such as the stem and leaves.Moreover,the translocation and distribution of prochloraz in rice plants were determined using high-performance liquid chromatography.The results demonstrated that Pro@MSN-Pec could transfer through the rice plant parts,such as the roots,stems,and leaves.Compared to the conventional prochloraz EC,Pro@MSN-Pec had a better uptake and translocation performance in the rice leaves,stems,and roots.4.The final concentrations of prochloraz were determined in different parts of the rice plant,namely,the leaves,stems,roots,and seeds,in addition to the soil using ultrahigh-performance liquid chromatography/mass spectrometry（UPLC/MS）under field conditions before harvest.The residue concentrations in the rice treated with Pro@MSN-Pec were slightly higher than those of the rice treated with the prochloraz EC in the leaves,stems and roots,while the residue levels in the seeds and soil were the same for both treatments.A comparison of the residue values with the maximum residue limit（MRL）shows that the final concentrations of prochloraz in the rice were lower than the maximum allowable levels,which indicated a low hazard with Pro@MSN-Pec application on rice plants.5.Biological activity of prochloraz fungicide in nanocapsule formula against Magnaporthe oryzae was assayed in the laboratory.The fungicidal activity results indicated that the efficiency of Pro@MSN-Pec after 7 days was better than that of prochloraz EC and technical prochloraz.Over time,the fungicidal activity of Pro@MSN-Pec after the 14^th day was higher than that of prochloraz EC and technical prochloraz under the same treated concentrations.6.Efficacy of prochloraz fungicide in nanocapsule formula against rice blast disease was conducted in greenhouse.The different treatments were sprayed before inoculation（SBI）and after inoculation（SAI）by fungus.The result showed that Pro@MSN-Pec had better antifungal activity against rice blast disease for SBI treatment,while there are no significant differences between Pro@MSN-Pec and prochloraz EC for SAI treatment.Also,the results showed significant differences between the recommended dose（RD）and0.5-fold RD,while there are no significant differences between RD and 2-fold RD for Pro@MSN-Pec.MSN-Pec（without fungicide）showed antifungal activity on rice blast disease.There are no obvious differences in chlorophyll content,which indicating the absence of phytotoxicity in all treatments.Furthermore,the enzyme activity levels for peroxidase（POX）,ascorbate peroxidase（APX）,chitinase,and phenylalanine ammonia-lyase（PAL）were determined in the rice plant for the different treatments.The rice plant leaves had higher concentrations for enzyme activity in all treatments compared with untreated ones.Moreover,the leaves for infected treatment（without fungicide）recorded a gradual increase in enzyme activity compared with healthy treatment.Also,the treatment of SAI had the highest concentrations compared with SBI.7.The efficacy of Pro@MSN-Pec,prochloraz EC 45%,and MSN-Pec was evaluated under field conditions during the 2021 season in two different locations in Egypt（Sakha and Qoutor city,Egypt）.The result showed that the control efficacy of the Pro@MSN-Pec formula was better than the prochloraz EC 45%.Also,MSN-Pec（without fungicide）showed the antifungal activity against rice blast disease.Moreover,Pro@MSN-Pec treatment had a high yield of rice compared with other different treatments,where the yield of rice plants at Sakha and Qoutor locations was 9.90 and10.17 tons/hectare.These results suggest that the use of stimuli-responsive carriers as pesticide-delivery systems in plants can be potentially implemented in integrated pest management.