Studies On Antifungal Mechanism, Residue Determination Method Of Kresoxim-methyl And Trifloxystrobin Belonging To Strobilurin Fungicides And Its Degradation Pattern In Citrus Fruits

Citrus fruits are vulnerable to fungal infection during postharvest period,which could lead to significant economic losses. The application of fungicide oncitrus fruit after harvest could be the efficient means for controlling the fungaldiseases. Kresoxim-methyl and trifloxystrobin, two strobilurin fungicides, have sethigh standards for controlling fungal diseases due to their broad-spectrum activityand outstanding environmental tolerability. But there were less study about actionmechanism and residue detection of kresoxim-methyl and trifloxystrobin in citrusfruits. In this paper, the antifungal mechanism, determining methods and residuedegradation about kresoxim-methyl and trifloxystrobin in citrus were studied throughseveral experiments. The results were followed:1. Antifungal activities and mechanism of kresoxim-methyl and trifloxystrobinThree pathogen separated from citrus, Penicillium italicum, Penicilliumdigitatum, Colletotrichum gloeosporioides, were cultured in the mediumsrespectively including kresoxim-methyl and trifloxystrobin.The result showedthat the EC50of kresoxim-methyl and trifloxystrobin inhibiting P. italicum，P. digitatum, C. gloeosporioides mycelia growth were13.22,66.92,273.74μg/mL, and0.18,0.32,1.37μg/mL, respectively. The soluble proteincontents of three fungis were examined as the evaluation indices when culturemedium contained the fungicides for24h. For P.italicum and P.digitatum, theprotein content began to descend after3h and1h, but protein content hadbeen declining during the early3h and ascended after3h forC.gloeosporioides. The EC50of kresoxim-methyl and trifloxystrobininhibiting three fungis were significantly declined along with the sol ubleprotein content reduction when100μg/mL of Salicylhydroxamic acid (SHAM)was added into the mediums. There was the synergistic interaction betweenSHAM with kresoxim-methyl and trifloxystrobin against three fungis.The fungal mitochondria were obtained through differential centrifugation to detect the activities of complexI-IV of mitochondria in thepresence of0.2-200μg/mL of kresoxim-methyl and trifloxystrobin in themedium, respectively. The result showed there was little difference ofkresoxim-methyl and trifloxystrobin in inhibiting complex activity, and theinhibition ratio was positively correlated to the concentration of fungicides.The activity of complexⅢ declined more significantly than complex I, II andIV for three fungis, and that of P.italicum and Penicillium digitatum werevulnerable than C. gloeosporioides in the same fungicide concentration.Kresoxim-methyl and trifloxystrobin may play important roles in regulatingcomplex activity of mitochondria to control fungal growth.2. QuEchERS-HPLC method for monitoring kresoxim-methyl and trifloxystrobinin citrus.Modified QuEchERS based on the former recognized by AOAC wasintroduced to samples preparation. The extract was purified by PSA and C18(4/1, m/m). The detected conditions: UV detection at210nm, the mobilephase was methanol: water(80:20). The retention time of kresoxim-methyland trifloxystrobin were about9.3min and12.3min, respectively. Therecoveries of kresoxim-methyl in peel and pulp ranged from80.08%~93.77%and83.93%~95.50%, RSD%were of1.80%~4.24%and1.55%~4.50%; Therecoveries of trifloxystrobin in peel and pulp ranged from80.00%~91.00%and84%~93.47%, RSD%were of1.40%~5.20%and3.00%~4.20%. The lowestdetectable concerntration of kresoxim-methyl and trifloxystrobin were0.005and0.008mg/kg. The developed method is in line with demands for theinternational residue analysis standard.3. ELISA method for analyzing kresoxim-methyl and trifloxystrobin1) The carboxyl was synthesized through reaction of mercaptopropionic acid andOEBr, containing the active group of kresoxim-methyl and trifloxystrobin, and wasas the conection arm to prepare the immunogen and coating antigen throughNHS-DCC method and mixed-anhydried method, respectively. The products wereidentified by ultraviolet and infrared absorption spectrum, MS, NMR andSDS-PAGE. The hapten-to-protein molar ratio in the mixture was approximately12.8:1, and the product was appropriate as the antigen for the preparation of vaccine. 2) Vaccine was prepared through emulsification of antigen and adjuvants.Three NewZealand white rabbits, which had been immunized through subcutaneous and legmuscle injection, were fed to get antiserum for3months. The titers of antiserumwere detected by ELISA and could reached64000,32000and32000, respectively.The highest titer antiserum was chosen for the followed test.3) The IC-ELISA was conducted with the coating antigen(Hapten-OVA) and the bestantiserum. The IC50of universal hapten was9.5μg/mL and IC10was0.005μg/mL.The antiserum showed high affinities and sensitivity to kresoxim-methyl andtrifloxystrobin, and its IC50and IC10to fungicide were15.3and0.0074μg/mL forkresoxim-methyl, and19.6μg/mL and0.011μg/mL for trifloxystrobin. The crossreaction rate was below0.1%for pyraclostrobin. The results showed that theantibody had higer specificity and sensitivity for the detection of kresoxim-methyland trifloxystrobin.4) Determining kresoxim-methyl and trifloxystrobin in citrus by IC-ELISA. Withthree spiking levels,100,10and1μg/mL,the recovery of kresoxim-methyl were104.35%，95.57%，82.73%and RSD were11.1%，10.8%,8.8%, respectively;therecovery of trifloxystrobin were92.43%，91.75%，91.10%, and RSD were7.4%，10.2%，10.9%, respectively.4. Residues Degradation of kresoxim-methyl and trifloxystrobin in citrus.The degradation pattern of fungicide residues were studied byQuEchERS-HPLC method. More than97%of the kresoxim-methyl andtrifloxystrobin deposits gradually dissipated from the citrus peels within12days. Theresidue in edible part was lower than the MRLs of kresoxim-methyl andtrifloxystrobin (0.5-2ppm).The half-lives of kresoxim-methyl and trifloxystrobin inthe peels were in the ranges of2.63–2.66d and3.12–3.15d, respectively, and thepattern of decline in the peels followed first-order kinetics.