Molecular Identification And Detection Of Apple Anthracnose Pathogen And Molecular Variation Of Its Pathogenicity

The similarity of rDNA nucleotide sequence is most extensively used in the classification and identification of fungi.The ITS sequence of rDNA has been used for the classification and identification of many Colletotrichum pathogens for its relative big variation.To determine the classification status of pathogen causing apple anthracnose more clearly,and to improve its detection efficiency and enable the early diagnosis of apple anthracnose,the research work on the molecular identification of apple anthracnose pathogens was carried out.The specific primers were also designed to be used for the rapid diagnosis of apple anthracnose.It was confirmed that apple anthracnose pathogen should belong to C.gloeosporioides through molecular identification.RAPD and ISSR molecular markers methods were utilized to analysis molecular diversity of low toxic strains of apple anthracnose pathogen on the basis of physical mutation screening low toxic strains of C.gloeosporioides.The rDNA sequence of apple anthracnose pathogen was determined.Sequence comparing of ITS of Colletotrichum was conducted and a phylogenetic tree based on alignment of their ITS nucleotide sequences was constructed.The results showed that Cg-1 and Cg-2 shared the highest sequence similarity(99.8%) with C.gloeosporioides and they could be grouped into one branch with C.gloeosporioides.It was confirmed that apple anthracnose pathogen should belong to C.gloeosporioides.We found that there is a 379 bp sequences residue in 3' ends of 18S rDNA of apple anthracnose pathogen comparing with the other C.gloeosporioides.Specific primer CgF1 was designed according to the 379 bp sequence,and only a 1232 bp specific band could be amplified using primer pair CgF1 and ITS4.Apple fruits were inoculated with apple anthracnose pathogen.Total DNA was extracted from the diseased tissue,and an identical 1232 bp band was amplified.It was suggested that the method could be applied for the detection and rapid identification of apple anthracnose pathogen.Primers CgF/CgR were utilized to clone the rDNA sequence of low toxic strain of apple anthracnose pathogen which was screened by the ion implantation and magnetic field exposure methods.The rDNA sequence of the low toxic strain was cloned and sequenced.The full-length sequence of the rDNA is 3156 bp.Comparison with the original pathogen strain,the mutation strain has only 1-2 nucleotides different.They shared the sequence similarity as high as 99.8%,and the mutation strain had hardly any molecular variation. RAPD and ISSR molecular markers methods were utilized to amplify DNA polymorphism of the normal strain of apple anthracnose pathogen and its low toxic strains.Thirty RAPD random primers were screened,and most random primers can not amplify abundant patterns except for the primer C-9.But C-9 can only amplify the identical polymorphism patterns between the normal strain and its low toxic strains. Almost all the 20 ISSR primers can amplified multiple polymorphism patterns,but the polymorphism patterns amplified from the normal strain and its low toxic strains were identical completely.The differences of the molecular markers between the strains still could not be found.The molecular diversity also could not be found in the low toxic strains during the preliminary research work.