Development of NIS1-PCR-RFLP marker system to effectors of Colletotrichum spp. from infected pepper plants

【Objective】 Based on the complex population of pepper anthracnose and unclear composition of compound infection population in the fields,it was relatively difficult to control capsicum anthracis. Therefore,a rapid and intuitive marking system for pepper anthracnose was established to provide scientific basis for field control of the disease.【Method】 The rDNA-ITS primers were used to clone 22 isolates of pepper Colletotrichum spp. isolated from field. The phylogenetic tree was constructed by sequence alignment analysis,and the species and taxonomic status of 22 isolates were preliminarily determined. An effector NIS1 gene as target,its alignment was performed between Colletotrichum gloeosporioides and Colletotrichum orbiculare to design degenerate primers of NIS1 by genome database. NIS1 gene was isolated and cloned of 22 different strains of pepper Colletotrichum spp.,and phylogenetic tree of NIS1 gene was constructed with their NIS1 sequences alignment. Analyzed and compared the genes of rDNA-ITS and NIS1 gene to distinguish the difference of the tested strains,and then analyzed the NIS1 gene of 22 different strains of pepper Colletotrichum spp.,selected restriction endonuclease,analyzed the difference of PCR product restriction fragment length polymorphism(RFLP)of NIS1 gene,and established the NIS1-PCR-RFLP labeling system.【Result】 Phylogenetic analysis of rDNA-ITS sequences showed that 22 strains of Colletotrichum spp. belonged to 5 species of C. gloeosporioides,C. brevisporum,C. acutatum, C. truncatum and C. capsici,and their sequences homology was 85.6%-99.8%,but C. capsici and C. truncatum were in the same mixed group. The NIS1 gene sequences of 22 strains of Colletotrichum spp. were conserved,and there was variable region in the middle,which was 34.6%-78.9% homologous with the NIS1 gene of C. orbiculare. The NIS1 gene phylogenetic tree could divide 22 strains of Colletotrichum spp. into 5 groups,and C. capsici and C. truncatum were in different groups,indicating that their differentiation was better than rDNA-ITS. NIS1-PCR-RFLP showed that the maximum fragments of C. gloeosporioides and C. brevisporum were both 210 bp,but the number of fragments was different,while those of C. acutatum,C. capsici and C. truncatum were 292,685,and 345 bp,respectively. The reference strain C. orbiculare was 497 bp,which can be visually distinguished.【Conclusion】 The NIS1-PCR-RFLP marking system was established in this study,it can easily and directly distinguish the differences of different Colletotrichum spp. infected pepper plants,Therefore,the NIS1-PCR-RFLP marking system is expected to be developed into a new fungal molecular marker for rapid identification of Colletotrichum spp. from infected pepper plants in field.