Development of rapid DNA extraction and PCR amplification methods for fungi and parasitic plants

Abstract

The main culprits of catastrophic agricultural losses or crop mortality are phytopathogenic fungi and parasitic plants. To manage the plant pathogens, a simple and rapid disease diagnosis is needed. The aim of the experiment was to develop simple and rapid genomic DNA extraction and PCR amplification methods for fungi and parasitic plants. For the DNA extraction, mycelia from 16 fungi species and stems from two parasitic plants species were incubated in a lysis buffer and were homogenised using a sterilised wooden stick. The homogenates were incubated at 95°C temperature for 10 min. Crude extracts served as a template for the PCR amplification containing UKOD polymerase. The application of lysis buffer, mechanical and heat disruption resulted in a fast DNA extraction from fungi and parasitic plants. DNA amplification time is reduced when using Master Mix containing UKOD polymerase. The presented results confirm that these simple and rapid DNA extraction and PCR amplification methods are applicable to diverse fungi species and parasitic plants.