Control Of Peach Brown Rot With Berberine-Preparation Of Dosage Forms And Assay For Drug Resistance

Compared with traditional chemical pesticides, botanical pesticides have advantages of low toxicity, limited residue, high efficiency, safety to mankind and environment, and thereby satisfy the requirement of sustainable development. In recent years, it has become a research hot spot to seek antimicrobial active ingredients from botanical sources. This study focused on the inhibitory activities of berberine and salicylic acid on Monilinia fructicola and other plant pathogenic fungi. The EC50 of berberine against M. fructicola was found to be merely 4.55μg/mL, and berberine showed dose-dependent inhibitory activity. At a concentration of 1.47-23.44μg/mL, the higher concentration was, the higher inhibitory activity was. The minimum inhibitory concentration (MIC) and EC50 of salicylic acid to M. fructicola was 0.75mg/mL,0.435mg/mL, respectively. Similarly, at a concentration of 0.125-0.5mg/mL, the higher concentration was, the higher inhibitory activity was. Besides, the inhibition of salicylic acid against spore germination of M. fructicola was also investigated. It was found that salicylic acid of high concentration could obviously inhibit spore germination, but low concentration had little effect.Based on these results, compound micro-emulsion consisting of berberine and salicylic acid was prepared using phase transfer method. According to single-factor test, orthogonal test was designed to determine the optimum microemulsion preparation conditions, which are consequently obtained as follows:solvent of 80mL, emulsifier Tween-80 of 8%, stirring at 60℃, mixing time of 25min. Under these optimum conditions, the average particle size, emulsion stability and the emulsifying of the microemulsion were all qualified.Besides, the in vitro inhibitory activity and fresh-keeping effect of micro-emulsion were also studied, and the MIC and EC50 on Monilinia fructicola was 1/100C, 1/1250C(C=10mg/mL, the concentration of compound microemulsion), respectively. Moreover, the in vivo fresh-keeping effect of compound microemulsion on peaches at room temperature and low temperature were also investigated, the compound microemulsion showed evident inhibition and fresh-keeping effect.To explore the inhibition mechanism of berberine against M. fructicola along with the drug resistance, the likelihood of berberine penetrating cell was tentatively studied by HPLC. The result demonstrated that berberine could enter into cell. Meanwhile, upon berberine stress, the growth rates of M. fructicola of six consecutive generations were measured. It was found that the mycelial growth of each generation varied little in diameter, and the EC50 of sixth generation strain was up to 14.43μg/mL, which showed little difference with untreated strains EC50 (4.55μg/mL). To clarify the mechanism, the genes encodingβ-microscopic protein(β-tub2) and cutinase(cutl) were chosen to investigate the differential expression between untreated strain and berberin-treated strain by reverse transcription PCR. The two genes in berberine-treated strain displayed higher reverse transcriptase level than that of the untreated.