A DNA extraction method that allows reliable PCR amplification of MLO DNA from "difficult" plant host species.

Abstract

Faculty of Science, Northern Territory University, Casuarina, Northern Territory, Australia 0811 Molecular techniques have facilitated the study of plant pathogenic mycoplasma-like organisms (MLOs) in the last few years, and PCR has been used to detect MLOs in infected plant and insect tissue. (1-3~ Where very little is known about MLOs and wide-scale screening of plant host species is required, or quarantine restrictions prevent the propagation of infected plant material, it is essential that a universal DNA extraction be developed that can be applied to a range of plant host species to give good quality DNA suitable for molecular applications such as PCR. Some of these species may contain high levels of polyphenolics, making it difficult to obtain good quality DNA. (4) To study MLOs in Australia, a screening program was initiated in which a range of plant host species with suspected MLO-associated diseases were collected. A DNA extraction method was needed to obtain DNA suitable for use in PCR. A number of techniques have been published for extraction of MLO DNA. (1-3s'6) One of the problems we encountered was that DNA extracted by some of these methods deteriorated with t ime when stored at -20~ Furthermore, DNA extracted from some plants with classical MLO-associated disease symptoms did not give an amplified product using PCR. This posed significant problems for longterm genetic studies using stockpiled DNA as well as severe l imitations for a screening program in which a wide range of plant host species have to be accommodated. The method described here gave DNA that produced consistent results in PCR after storage at -20~ It also allowed the detection of MLO DNA by PCR from plant host species that had symptoms but were consistently negative using DNA extracted by other methods. Our method is based on modified protocols and combines MLO enrichment and maintenance in an osmotically supplemented buffer, with polyvinylpyrrolidone (PVP) to complex polyphenolic compounds (7~ followed by selective precipitation of nucleic acids using ce ty l t r i -methylammonium bromide (CTAB) and sodium chloride. (3~