Abstract

Many fungal plant pathogens can be spread over long distances by airborne or rain splash spore dispersal. These spores can infect susceptible hosts causing disease and in some cases mortality and for this reason spore monitoring followed by control is needed. Air sampling can be combined with modern sensitive molecular methods such as quantitative polymerase chain reaction (qPCR), real-time PCR (West et al., 2008, 2009). A method for absolute quantification of Dothistroma septosporum (teleomorph Mycosphaerella pini) spores by real-time PCR is being developed. We report here the optimisation of plasmid standards and standard curves that are required for quantification using qPCR. Preparation of a DNA standard is necessary to make a robust and reliable estimation of the number of spores. The most consistent results have been obtained by using plasmid DNA with insert containing target sequence (Dhanasekaran et al., 2010). The standard can be used to determine the number of DNA copies in a spore trap sample, and thus the number of spores. Two plasmids, with different lengths of the target gene inserted (single copy gene), were prepared and both linear and circular forms of these were used for standard curve generation. Linear plasmid amplified in average of 2 to 3.5 cycles (C_q - quantification cycle) earlier than circular plasmid when using a SYBR Green detection system. However, no differences in C_q value were recorded when using a hydrolysis detection system. Addition of carrier tRNA to plasmid solution caused inhibition of PCR at high plasmid concentration. Plasmid with the short insert, the same length as the amplified product, generated standard curves with efficiencies closer to 2.0 than plasmid with long insert. The final standard curve was prepared with circular plasmid containing short insert without adding the tRNA. DNA extraction from D. septosporum spores will be optimised and then tested with spore trap tape samples. Standard curves developed from DNA extracted from spores will be correlated with those issued from plasmid as a DNA template. The method for quantification ofD. septosporum spores will increase accuracy of epidemiological studies. This type of spore quantification method will be suitable to form part of the complex method for management of disease caused by Dothistroma septosporum.

Key words: Dothistroma septosporum, quantification, real-time PCR, spores.