Preliminary Study On Anti-fungal And Insecticidal Activities And Anti-fungal Mechanism Of Isodon Rubescens Extract

Background:Isodon rubescens is a herbal drug from dry ground part of Isodon rubescens(Hemsl.)Hara,which is bitter in taste,slightly cold in nature.It has the functions of clearing away heat,detoxifying,antiphlogistic,analgesic and anti-tumor.The herb is commonly used for diseases such as stomach aches,pharyngitis,cough,sore throat,wrestling injuries as well as tumors.At present,there are 1 000-1 200 kinds of medicinal materials of the Chinese pharmacy in the national 3A hospitals,of which about 300 medicinal materials are frequently used and mainly from artificial cultivation.During large-scale cultivation of Chinese medicinal herbs,pesticide and heavy metal might be introduced to Chinese medicinal herbs.It is said that detection of pesticide and heavy metal would be required in the new Pharmacopoeia published in 2020.For the previous edition of Pharmacopoeia published in 2015,detection of pesticide and heavy metal was only required in a few medicinal materials such as liquorice,astragalus membranaceus,ginseng and American ginseng.Moreover,a large gap may exist between the legal requirements and practice.Current pest control mainly relies on chemical pesticides which display obvious and enduring residual,on the contrary,biopesticide shows less residual.Objective:In this study,anti-fungal and insecticidal activities of I.rubescens extract,and anti-fungal mechanism were investigated,which might lay the foundation for further research.Methods:(1)I.rubescens was first extracted in ethanol using reflux gradient extraction as total extract,and further extraction using organic solvents was preformed to obtain separate parts.The growth rate method was used to determine effect of the extract on Ascochyta moelleriana G.Winter and plant-derived fungi,and control effect on A.moelleriana was evaluated by leaf method;effect of extracts on honeysuckle powdery mildew by spray method.(2)Toxicity of the extract to honeysuckle aphids by leaf soaking method and the research on honeysuckle aphids was conducted in the field;role of the extracts on cotton bollworm and white grub by the soaking method.(3)Anti-fungal mechanism was explored by the determination of mycelial morphology,protein content,total polysaccharide content,DNA content and so on.Results:(1)I.rubescens extract showed somewhat anti-fungal effect on A.molleriana by in vitro experiments,of which n-butanol extract part showed the best,with a inhibition rate at 94.61%and an EC50 value at 0.67 mg/mL,which was the research focus of this study on bacterial activity.It also demonstrated anti-fungal effect on Curvuaria lunata,Fusarium graminearum,Alternaria alternata,Botryosphaeria dothidea,with EC50 values at 0.261 mg/mL,0.689 mg/mL,0.487 mg/mL,0.419 mg/mL.n-butanol extraction part demonstrated best control effect on A.molleriana,and the control effect was 75.52%.The ethyl acetate extraction part showed good control effect on honeysuckle powdery mildew,and a long action time.(2)Insecticidal experiments indicated that the extracts from separate parts had toxic effects to the Honeysuckle aphids,of which ethyl acetate extraction part showed the best.The control effect was 72.73%and 97.67%,respectively when plants grown in indoor chamber or field at a concentration of 20 mg/mL.It's natural that ethyl acetate extraction part was chosen for further insecticidal activity tracking.In addition,after five days treatment to cotton bollworm,the death rate reached to 83.33%when using total extract.However,extracts show almost no resistant to white grub.Therefore it is not recommended to use Donglingcao extract for insecticidal pesticide against white grub.(3)To illustrate the anti-fungal mechanism of Dongligncao,liquid fungi culture was treated with n-butanol extract part or not.Results showed that the conductivity,total polysaccharide content,protein content and PI dyeing rate were significantly increased by treatment,in addition,the total polysaccharide content,protein content,DNA content and ergosterol content were obviously decreased by treatment,indicating that the extract could damage the permeability of cell membrane,and cell metabolic disorders of occurred after I.rubescens extract treatment and then inhibition of fungi was observed.Conclusions:In this paper,the anti-fungal effects of extracts from different polar parts of Donglingcao against A.moelleriana,honeysuckle powdery mildew and other 7 plant-derived fungi,and their insecticidal effects on honeysuckle aphids,cotton bollworm,and white grub were first studied.In addition,its anti-fungal mechanism was explained briefly.The research results above suggests that Donglingcao extract has great potential to control agricultural diseases and pests,which provides a research basis and theoretical support for the development of I.rubescens.