Simple direct DNA extraction protocols for efficient routine detection of plant pathogenic bacteria via conventional PCR

Abstract

ABSTRACT Rapid, easy and direct DNA extraction protocols are required for use in routine analyses to detect plant pathogenic bacteria. For this purpose, five protocols for extracting bacterial DNA, which do not use toxic chemicals such as phenol and chloroform, were optimized for the detection of plant pathogenic bacteria quickly and directly from plant material using conventional polymerase chain reaction (PCR). The protocols are based on the use of different maceration and extraction buffers to ensure quality bacterial DNA extraction and to remove the PCR inhibitors. Results showed that the protocol #1 based on the use of Tris and TNPEE buffers and protocol #3 based on the use of 0.4% bovine serum albumin and 0.05% Tween 20 were able to detect Erwinia amylovora from apple and pear samples. Protocol #2 that used 2% polyvinyl pyrrolidone in maceration and extraction buffers was found to be efficient for detecting Erwinia amylovora, Agrobacterium tumefaciens, and Allorhizobium vitis directly from plant tissues. However, protocols #4 and #5 based on the use of 1% Triton X-100 and 0.1% Tween 20, respectively, were unable to detect the studied bacteria. Accordingly, the protocol #2 is proposed for efficient, direct and rapid detection of plant pathogenic bacteria in plant materials via PCR.