The Action Characteristics And Mechanism Of ?-poly-L-lysine Against Plant Viruses And Plant Pathogenic Fungi

?-poly-L-lysine(?-PL)is a water-soluble,heat-resistant,biodegradable,and broad-spectrum antimicrobial activity of peptides produced by microbial metabolism,which is non-toxic to human health and the environment.The existing reports mainly focused on its application in food preservatives,but its mechanism of action as a biocontrol agent against plant diseases has not been elucidated.?-PL used in this paper was obtained by separation and purification from Streptomyces in this laboratory,and has independent intellectual property rights(invention patent acceptance number: 201910330737.2).Therefore,inhibition and mechanism of ?-PL on tobacco mosaic virus(TMV)was studied by pot culture,fluorescence detection,Northern Blot analysis and transmission electron microscopy.The anti-bacterial effect and mechanism of ?-PL on plant fungal diseases were studied by plate inhibition test,in vitro leaf inoculation test,spore germination inhibition test and quantitative real-time PCR(q PCR)test.In addition,the induced resistance of ?-PL on tobacco plants was studied by transcriptomic analysis of BY-2 tobacco cells cells treated with ?-PL.The important research results are as follows:1.In greenhouse pot experiment,?-PL with different concentrations were used to treat Nicotiana glutinosa(N.glutinosa)and Nicotiana tabacum L.cv.NC89 inoculated with TMV.When the N.glutinosa was treated with 2000 ?g / ml ?-PL,the passivation effect and preventive effect on TMV was 90.6% and 79.3%,respectively.The same concentration of ?-PL treated N.tabacum L.cv.NC89,the passivation and prevention effects of TMV were 72.35% and 56.7%,respectively.Fluorescence detection of Nicotiana benthamiana(N.benthamiana)seedlings inoculated with GFP-TMV showed that the fluorescent spots in the inoculated leaves,upper leaves or parietal leaves were significantly reduced compared with the control group.The top leaves of N.benthamiana treated with 100 ?g/ml ?-PL showed no fluorescent spots,while the green fluorescent areas of the control leaves were obvious.RNA accumulation of TMV-infected tobacco leaves and tobacco protoplasts treated with different concentrations of ?-PL were analyzed by Northern blot.The results showed that the TMV-RNA accumulation in the sample treated by ?-PL was significantly reduced,indicating that ?-PL affects the synthesis and accumulation of viral nucleic acid.Observation by transmission electron microscopy revealed that ?-PL can induce cleavage of virions.2.Different concentrations of ?-PL were used to carry out the inhibition zone experiments on three pathogenic fungi on three plants including tobacco brown spot disease,cucumber brown spot disease and rice bakanae disease.The results indicated that the plates of tobacco brown spot and cucumber brown spot produced clear and distinct inhibition zones under ?-PL treatment,and the minimum inhibitory concentration of ?-PL on tobacco brown spot disease was 5 ?g/ml.In contrast,rice seedling disease is not sensitive to ?-PL treatment.The results showed that ?-PL also inhibited the spread of lesions on tobacco and cucumber leaves,and compared with the control,?-PL inhibited tobacco brown spot lesions more significantly.Using different concentrations of ?-PL to test the tobacco inhibition of mycelial growth of Alternaria alternata showed that the inhibition rate of hyphae was 92% after treatment with 200 ?g/ml ?-PL for 3 days,and the inhibition rate was also as high as 87% after 7 days.The results indicated that ?-PL has good stability and strong inhibition,and pocess sustainable control effects on mycelial growth.?-PL also has a good effect on the spore germination,germ tube morphology and elongation of tobacco brown spot disease.Result of microscopic observation showed that 25?g/ml ?-PL treatment induced spores wrinkle and malformation at 24 hpi;when the concentration of ?-PL reached 200 ?g/ml,the inhibition rate of spore germination was close to 98%.Furthermore,the q PCR detection of the genes expression of protein kinase PKA,AAPK1 and glyceraldehyde-3-phosphate dehydrogenase gene GAPDH involving in mycelial germination and growth of A.alternata were down-regulated by ?-PL treatment and ribosomal r RNA was used as a control.3.To investigate whether ?-PL treatment induce resistance in the host plant.Transcriptome analysis was performed and revealed various differentially expressed genes induced by ?-PL in tobacco BY-2 cells.Through differential gene cluster analysis,it was found that the expression level of tobacco heat shock homologous protein 70 k Da gene and 1350 genes related to aging-related proteins was significantly up-regulated compared with the control.While the expression levels of more than 1,700 genes such as tobacco anthocyanin 3-O-glucosyltransferase gene and tobacco leaf epidermis-specific secreted glycoprotein were down-regulated compared with the control.The differential gene Gene Ontology(GO)enrichment analysis indicated that the significantly differential expressed genes of tobacco cells after ?-PL treatment were mainly enriched in the transcriptional regulation of biological functions.The differential gene Gene Ontology(GO)enrichment analysis found that the significantly different genes of tobacco cells after ?-PL treatment were mainly enriched in the transcriptional regulation of biological functions,and the cell groups related to the composition of nucleus,cytoplasm,plasma membrane and membrane.It is also enriched in molecular functions related to protein binding and DNA binding functions that affect transcription and specific sequences.The above functions indirectly regulate plant growth,development,evolution and resistance to stress,therefore,the results suggested that ?-PL caninduce resistance in host plant tobacco.