Studies On Antimicrobial Action And Mechanism Of HSAF To Alternaria Alternata

Heat stable antifungal factor（HSAF）is a kind of macrolide antibiotics produced by the metabolism of Lysobacter enzymogenes C3 and OH11.It has inhibitory activity to a variety of filamentous fungi and has good development and application.Pear black spot is one of major diseases in pear production.It is caused by Alternaria alternata,which damages the leaves and fruits of pears and forms the symptoms of black spots on leaves and fruits,which seriously affect the yield and quality of pears in China.In the earlier period,pear black spot samples were collected from the main pear producing areas in the country,and Alternaria alternata were isolated and identified.In order to clarify the inhibitory effect of HSAF on the population of Alternaria alternata and its mechanism of pear black spot,the following researches have been carried out:1.Isolation and identification of A.alternata and screening of tested strainsFifty-seven pear black spot pathogens were isolated from 66 samples of pear black spot disease which collested from 18 pear producing areas across the country.These 57 black spot disease were identified with the nucleotide sequences of rDNA-ITS,Gpd,RPB2,OPA2,and EF1α.For the three major categories and their representative strains were selected,based on pathogenicity and sensitivity to HSAF,a representative strain of HN-5（Henan-5）was selected as the test strain.2.Determination of HN-5 inhibitory activity against A.alternata by HSAFThe growth rate method was used to determine the virulence level of HSAF against A.alternata HN-5,and the virulence regression equation of HSAF against A.alternata HN-5 was obtained.It was found that the concentration of HSAF against A.alternata was determined by the analysis.50%inhibition EC₅₀=1.54μg/mL,95%inhibition EC₉₅=6.75μg/mL.HSAF treatment of HN-5 conidia found that HSAF can inhibit conidial germination,the 50%inhibition of the HSAF concentration about 1μg/mL.Further study found that elder mycelium became significantly thicker and swollen under the HSAF-treated,with severe mycelial distortion and increased mycelial branching.It was found through cell wall staining that HSAF could inhibit mycelial cell wall synthesis,and nuclear staining revealed:mycelium nuclei disappear,cells apoptosis.3.Field efficacy experiment of HSAF against A.alternataThe field efficacy experiment of HSAF against A.alternata were conducted.The results of Wuhan showed that:compared with control without spraying,the disease index on huali-1 pear dropped from 19.13 to 1.33,and the disease index on Cuiguan pear dropped from 13.89 to 2.06,And compared to conventional pesticides 10%difenoconazole,HSAF control effection is also better.The control efficacy of HSAF in Chongqing was also better than the control of mancozeb.4.Preliminary study on the Bacteriostatic Mechanism of HSAF to A.alternataTo clarify the molecular mechanism of HSAF to A.alternata,conducted transcriptome sequencing analysis of the HSAF-treated A.alternata（treatment group）and wild-type（control group）and found that there were 9854 and 10561 gene expressions in the treatment group and control group,respectively.152 genes were specifically expressed in the HSAF treatment group.Differentially expressed genes were analyzed and 7369 differentially expressed genes were obtained,which 3729were up-regulated and 3640 were down-regulated.The GO and KEGG enrichment analysis of these differentially expressed genes revealed that after HSAF treatment,metabolic processes,catalytic activity and membrane-associated genes,amino acid metabolism,drug metabolism,and tricarboxylic acid cycle and carbon metabolism were down-regulated,whereas proteolysis,RNA Degraded endoplasmic reticulum protein processing,cytochrome P450 and AMPK signaling pathways were significantly enriched in up-regulated expression genes.Further analysis of genes related to metabolic pathways and signal transduction pathways revealed that down-regulated expression of key genes in carbon metabolism pathways suggests that HSAF may block the glycolytic pathway and the tricarboxylic acid cycle,resulting in aerobic respiration of mycelium being inhibited and affecting cells Internal sugar metabolism and energy gain.Corresponding to carbon metabolism,most of the nitrogen metabolism-related genes also show down-regulation expression patterns.Further analysis revealed that the metabolic pathways associated with nitrogen metabolism and carbon metabolism,such as the sphingolipid metabolic pathway and the AMPK signaling pathway,respond to the toxicity of HSAF against A.alternata.5.Establishment of genetic transformation system of A.alternataThrough the optimization of the preparation conditions and transformation methods of the protoplasts,the genetic transformation system of A.alternata was established.The A.alternata HN-5 was cultured in MR medium for 36 h,and the hyphae were collected in the hydrolysate 1%driselase+1%lysing+1%Cellulase+1%snailase was digested for 3 h.The protoplasts were collected by centrifugation and the protoplasts were washed with an infiltration solution（0.7 M NaCl+0.8 M Mannitol）and resuspended in STC Buffer.Suspension protoplasts were used for transformation;2μg of plasmid was transformed into A.alternata,12 transformants were obtained,and the PEG-mediated protoplast transformation system of A.alternata was successfully established.