Sensitivity Differentiation Of Colletotrichum Gloeosporioides Causing Pepper Anthracnose To Chemical Imazalil And Its Derivations

Pepper anthracnose is a serious and important fungi disease in pepper production in the worldwide,which was cuased by different colletotrichum spp.Strains.The pathogens are mainly making damage to stem,leaves and fruits on host plants peppers.Yield is loss up to 40%-60%,when anthracnose was broken with good conditions.Totally five different strains,colletotrichum gloeosporioides?C.g?,colletotrichum acutatum?C.a?,colletotrichum truncatum?C.t?,colletotrichum brevisporum?C.b?and colletotrichum capsici?C.c?,were documented in China.Rich genetic diversity and obvious pathogencity differentiation were proved within different species by numerous researches.According to our detection results,co-infection with different strains is very popular in the fields of China,C.gloeosporioides as a dominant strain was found in the different pepper production regions.Interestingly,up to now it is unkown that how to vary on the genetic diversities and pathogencity differentitation within C.gloeosporioides groups,as well as the relationship of host preference.At present chemical control as main methods to pepper anthracnose is popularly accepted for all pepper-growth farmers,however,environmental pollution was brought about by low levels with unscientifically and improperly of employing fungicides,as well as the development of the risk resistance to target fungicides with accerlation speed.Therefore,it is pressing need that how to enhance of sensitive to fungicides and improve of control effects to the pathogens of pepper anthracnose,which is help us to make better strategy to copy with this disease.In this study,to combine with C.gloeosporioides-CSLL11 strain genome,the SSR-PCR detection systems based on microsatellite DNA with 6 nucleotides repeats was developed successfully,the distinct genetic diversity as well as pathogencity within C.gloeosporioides groups was elucidated by SSR-PCR;to seek for reforming common fungicide to pepper anthracnose,we hypothesized to alternate imazalil chemicals by ionic liquid preparation,under keeping skeleton structure of imazalil,which to obtain different derivatives compounds by adding six different selected natural anionic ligand.Naturally,the chemical characterization biological activity as well as broad-spectrum on those ionic liquids are determined.at the same time,the sensitive of the pathogens C.gloeosporioides-CSLL11 to imazalil and six derivatives was detected by bioassay methods,which is benefit for extending to explore and delaying to development of target fungi resistance to fungicides.To further clarify interaction of target gene and different derivatives,the system of genetic transformation of C.gloeosporioides-CSLL11 was successfully developed.Taking all above-mentioned,the corresponding results as following:1.The system of SSR-PCR was successfully developed to detect the pathogens within C.gloeosporioides groups;seven very specific primers of 213 SSR primers by microsatellite DNA of C.gloeosporioides-CSLL11 genome were screened and determined.The screened 7 pairs primers was very specific and easily distinguished from different colletotrichum species infecting pepper plants as well as C.gloeosporioides strains infected different host.To combine the results of SSR-PCR detection of 55 tested C.gloeosporioides strains in the study were divided into 5groups through cluster analysis,there are three groups includ ing group2,group3 and group4 with rich genetic diversity,partly geographical distribution characteristics were observed in the three groups.To compare with CSLL11,ten of 55 tested C.gloeosporioides strains with significantly differentation strains were further screened and carried out biological characteristics with growth rate,colony morphology,conidia,conidia-producing as well as pathogenicity,the results showed that there are significant differences in growth rate,colony morphology and pathogen icity.2.Six different imazalil ionic liquids?derivatives?were prepared by acid-base neutralization reaction with different chemical ligands including acetic acid,nonanoic acid,oleic acid,benzoic acid,salicylic acid and nicotinic acid,the relevant yield of those derivatives was about 89%-98%,their yields are rank of imazalil-nonanoic acid ionic liquid>imazalil-benzoic acid ionic liquid>imazalil-oleic acid ionic liquid>imazalil-acetate ionic liquid>imazalil-salicylic acid ionic liquid>imazalil-nicotinic acid ionic liquid.Through nuclear magnetic resonance spectroscopy,those derivatives structures were characterized with 0.1 ppm-0.4 ppm N-CH-N hydrogen spectrum shift on the imidazolium ring from the synthesized product to the high field and the active hydrogen of the carboxyl group in all ligands without its peak as well.The results showed that the imazalil ionic liquid derivatives were made preparation in the experiments.3.All 5 different strains including HHBY48?C.acutatum?,YYGXZ07?C.brevisporum?,HBLF01?C.capsici?,CZHP03?C.truncatum?and CSLL11?C.gloeosporioides?isolated from chili plants as tested strains,and imazalil as tested chemicals,the sensitive tested experiments were performed by mycelia growth rate methods.In the experiments with designed concentration of tests chemicals of 0,0.5,1.0,2.0,4.0,8.0 and 16.0mg/L,the results showed that the sensitivity of different strains to imazalil was varied with the EC₅₀0 value from 2.85 mg/L and 176.34 mg/L,we found 62 fold in the EC₅₀ value between C.truncatum?176.34mg/L?and C.acutatum?2.85mg/L?.The dominant strain C.gloeosporioides was further used as test strains,the sensitive to imazalil and its 6 different derivatives were also determined,the results showed that the EC₅₀ value of sensitivity to imazalil and 6 different derivatives is 6.23mg/L,4.31mg/L,4.37mg/L,3.88mg/L,3.29 mg/L,8.49 mg/L and7.19 mg/L,respectively.To compare with the EC₅₀ value of imazalil,the results showed that those derivatives with acetate,nicotinic acid,oleic acid and salicylic acid4 ligands is help imazalil to enhance tested strain CSLL11 sensitive to tested chemicals,vary from 29%to 47%,while others ligands reduce its sensitivity.Therefore,those 4 enhance-sensitivity derivations would be as candidates further to define and evaluate their potents.To detect antifungi broad-spectrum,4 different strains Phytophthora capsici,Magnaporthe oryzaeand,Fusarium,were used as tested strains,the results showed that imazalil and 4 its derivaties had no effect on Phytophthora capsici,however,the EC₅₀ value of Magnaporthe oryzae and Fusarium to 4 derivaties is lower or the same level than that of im azalil,Magnaporthe oryzae was sensitivite to imazalil-oleic acid ionic liquid and imazalil-acetate ionic liquid with enhancing effect up to 3%-5%;while Fusarium was sensitivite to imazalil-salicylic acid ionic liquid with enhancing effect up to12.91%.However,all tested derivations had no any effect on Phytophthora capsici,it could be confirmed that ionic liquid derivatives with different active ligands could not expand their antifungi spectrum.4.Using PEG-CaCl₂-mediated protoplast transformation,we used the C.gloeosporioides-CSLL11 as the tested strain,and the DNA fragment containi ng hygromycin B resistance gene and eGFP expression gene was successfully transferred into CSLL11 strian.eGFP was stably expressed with green fluorescence in the obtained transformants.The positive transformants were screened with the concentration of hygromycin B 500 mg/L,PCR and Southern blot showed that the eGFP expression gene was integrated into the genome of the transformant.The hyphae and the conidia of the transformants after the first generation and subculture showed strong green fluorescence signals,indicating that GFP can be stably inherited in the transformants.Moreover,there was no significant difference in colony morphology,growth rate and pathogenicity between the transformants and the wild-type strains.