Abstract:

Fungal pathogens causing poplar canker diseases are cosmopolitan in species, and have a wide range of hosts. These pathogens have diverse anamorphs and their morphology overlaps with each other; Their teleomorphs are hardly discovered in nature. Furthermore, the identification of these pathogens is usually limited by the geography, host and taxonomic knowledge. Therefore, a culture-independent method used to identify determine pathogens is expected to be developed for field diagnostics. Through amplifying, sequencing and analyzing multiplex nucleic acid templates and genetic marked target sequences, a multiplex PCR technique has been established and used to directly detect various environmental samples. In this study, four pathogen strains and environmental samples were amplified using fungal universal primers ITS1/ITS4 and EF1-728F/EF1-986R by general PCR and multiplex PCR. The amplicons were sequenced, and then aligned in GenBank. Our result showed that the multiplex PCR was able to successfully amplify the target gene and identify the pathogens from environmental samples in the condition of an annealing temperature of 55.6 ℃ and primers final concentration of 0.2 μmol·L^-1. The multiplex PCR could amplify the target at the concentration level of approximately 1ng of genomic DNA. This method would provide a useful tool for the identification of canker pathogens by the multiplex PCR and the high-throughput DNA microarray detection of environmental samples.

Key words: poplar canker, multiplex PCR, pathogen identification, environmental samples