This was written by by Sarah Green, Forest Research. This is the report from a BSPP Senior Fellowship. Click here to read more/apply for one yourself.
In January/February 2008 I spent three weeks at the Norwegian Forest and Landscape Institute, located in Ǻs, Norway.
The purpose of this fellowship was to focus on learning the theory and techniques associated with real-time PCR for detection of diseases in tree hosts. I work as a forest pathologist based at Forest Research’s Northern Research Station and carry out research into tree diseases as well as managing a tree disease diagnostic service for northern Britain. I have a strong interest in increasing our utilisation of recently developed molecular tools for the detection and study of tree diseases, and with the recent acquisition of a real-time PCR machine at our research station, I set off to Norway for a three week real-time PCR ‘immersion’ to learn how to develop and use our own detection assays with this tool.
My hosts at the Norwegian Forest and Landscape Institute were the staff of the molecular biology lab, in particular Senior Research Scientist Carl Gunnar Fossdal (molecular lab supervisor) who works mainly with topics related to forest health. The section for forest health is lead by Professor Halvor Solheim and this includes, among others, the researcher in molecular pathology Ari Hietala and the biotechnology technician Inger Heldal. The group studies disease resistance in Norway spruce (Picea abies), the pathogen Heterobasidion annosum s.l. and other serious pathogens of forest trees using molecular tools. The group have considerable experience in developing real-time PCR assays and methods for the detection and study of a number of tree diseases important to Norwegian forestry.
Real-time PCR differs from classical PCR in that successful amplification of target DNA is recorded and monitored as the PCR reaction progresses (i.e. in real-time). This is achieved by either combining a primer pair with a fluorescently labelled internal probe which only attaches to the correct sequence of target DNA, or by the use of a highly specific primer pair together with SYBR Green dye which binds to double stranded DNA. Both methods enable detection of the PCR product by measurement of fluorescence as it accumulates during PCR cycles. Since the amount of PCR product generated depends on the amount of DNA added to the reaction, this method can accurately quantify the level of infection in the host tissue. Therefore, due to greater rapidity, sensitivity, specificity and capacity for DNA quantification, real-time PCR has a number of advantages over classical PCR for detection of disease in host tissues.
The Norwegian group had developed a real-time PCR assay of particular interest to me, for the specific detection of an unnamed Phomopsis sp. occurring on spruce (Picea spp.) and possibly other hosts. This Phomopsis sp. was isolated frequently from damaged Norway spruce seedlings in Norway and is identical to a Phomopsis sp. isolated consistently from lesions on drought damaged Sitka spruce (Picea sitchensis) in Scotland. I brought samples of Sitka spruce to Norway which I’d inoculated with the Phomopsis sp. during pathogenicity tests18 last summer, as well as non-inoculated, healthy Sitka spruce trees. I used realtime PCR to detect and quantify the level of Phomopsis DNA in phloem and wood tissues of Sitka spruce at different locations from the point of inoculation, in relation to the amount of host DNA present. I intend to use this method to determine the frequency of occurrence and quantity of this Phomopsis sp. in drought damaged and non-drought damaged Sitka spruce in Scotland during 2008 to help determine the pathogenic status of this fungus in relation to drought stress. My second area of work during my visit was to design primers, develop and test a real-time PCR assay for the detection of an important bacterial disease of horse chestnut in the UK, Pseudomonas syringae pv. aesculi. This work, which is still ongoing, was carried out based on DNA sequences for a number of isolates of target and closely related bacterial species isolated from horse chestnut in Scotland prior to my visit to Norway.
This visit has provided me with the skills necessary to take forward my own work incorporating real-time PCR as a tool to study and detect tree diseases in the UK, and has also resulted in a new collaboration between Forest Research and the Norwegian Forest and Landscape Institute. I wish to thank the BSPP for awarding me this Senior Fellowship without which I could not have undertaken this study visit. I also wish to thank Carl Gunnar Fossdal for facilitating my visit, and Ari Hietala for help and advice during the stay. Finally (but not least!) I am extremely grateful to Inger Heldal for her time spent assisting and guiding me with my laboratory work during my visit.