Fingerprinting plant killers

David Cooke

David Cooke, a researcher at the Scottish Crop Research Institute (SCRI) explains how DNA-based molecular diagnostics is helping researchers to fight plant diseases caused by a notorious family of fungi.

Ranking high on the list of the world’s worst invasive species is Phytophthora infestans, the fungus responsible for potato blight, a disease that devastated potato crops throughout Europe in the 1840s. The species was identified in 1876 by Anton de Bary, a German botanist, who aptly named the genus Phytophthora – Greek for ‘plant destroyer’.

Since the discovery of potato blight fungus, over 64 species of Phytophthora have been identified. It is likely that many more varieties occur in natural communities as only those that cause serious plant disease have been recorded. It is this specialised ability to infect plants that has given Phytophthora its reputation as one of the world’s most devastating family of plant pathogens. Transported around the world in shipments of agricultural produce, the fungus causes root, crown and stem rots and also infects the leaves and fruits of its many host species.

Phytophthora species are often named after their most common host. Thus, Phytophthora citrophthora is a notorious ‘citrus fruit killer’, while Phytophthora sojae typically causes soybean root and stem rot. However, the most widely distributed species is Phytophthora cinnamomi. Although named after the cinnamon tree, which originates from Sri Lanka, it is now better known in South Africa for the damage it does to avocados (root rot) and in the Caribbean, for spoiling pineapples (due to root and heart rot).

Controlling Phytophthora species has proved challenging. To begin with, they are difficult to detect in the field. Although there are some distinctive leaf blights, in most cases a ‘trained eye’ is needed to spot secondary symptoms such as wilting, since the species tend to attack stem bases and roots. Once isolated from field material, identification is the next problem. In the absence of distinct and stable species-specific morphological characteristics, even specialists can have difficulty in identifying Phytophthora beyond the genus level.
Many isolates are therefore misidentified or remain unclassified, resulting in delays in identifying new threats, and in failure to rapidly evaluate current problems. Accurate identification is also crucial for compliance with international plant health legislation and agricultural export quarantine regulations.

DNA-based molecular diagnostics now offers a valuable tool to assist in the identification of Phytophthora species, and to trace genetic linkages between species and isolates from different geographical origins. Researchers at the Scottish Crop Research Institute (SCRI) have developed a diagnostic method that uses a fragment of ribosomal DNA, termed the ‘internal transcribed spacer’ (ITS) region. When these fragments are digested with a range of suitable enzymes, researchers can generate a unique ITS digest profile or ‘fingerprint’ that can be visualized on an agarose gel.
Subsequently, these gel images are digitized and analyzed with bioinformatics software. Researchers at SCRI use GelCompar, a sophisticated software package that allows them to analyze and compare ITS digest profiles of each Phytophthora isolate against their reference database of profiles from over 400 isolates. Increasingly, they are directly sequencing the DNA of the ITS regions and comparing against their reference sequence database in a similar manner to the ITS digest patterns.

Molecular diagnostics of Phytophthora is specialized, time-consuming work and the laboratory equipment and software needed is often not available in developing countries. SCRI is therefore cooperating with CAB International (CABI), a UK-based organisation specializing in bioscience for development that offers a Phytophthora identification service. Through CABI’s regional centres in Africa, the Caribbean, and East and Southeast Asia, researchers in developing countries can send Phytophthora isolates to SCRI in Scotland for analysis when accurate identification is essential.

The case of cocoa pod rot in Ghana illustrates the importance of accurate species identification for the development of effective control methods. A number of different Phytophthora species are known to cause the disease. In Ghana, cocoa producers had learned to cope with pod rot due to P. palmivora until, in 1985, a severe outbreak of the disease caused serious crop losses. Eventually, the perpetrator was isolated and identified as P. megakarya, a much more aggressive pathogen than P. palmivora that had spread from Ivory Coast. Currently, molecular diagnostic methods are helping researchers from CABI Africa and the Cocoa Research Institute of Ghana to assess the variation between and within isolates of Phytophthora species found on cocoa pods as part of a project to develop environmentally friendly control measures.

To extend the benefits of DNA-based Phytophthora diagnostics to scientists in developing countries, SCRI and CABI have developed a website ( www.phytid.org) that provides detailed protocols and access to a database containing the ITS digest profiles of 46 Phytophthora species. At this website, researchers anywhere in the world can enter the profile of an unidentified Phytophthora species for comparison with the database. In this way, unknown isolates can be identified in a matter of hours rather than the several days required for traditional morphological analysis.

Dr David E.L. Cooke (email: dcooke@scri.sari.ac.uk ) is a researcher with the Host-Parasite Co-evolution (HPC) programme of the Scottish Crop Research Institute (SCRI).

For further information, visit the SCRI website and the CABI Identification and Diagnostics Service.

25 February 2005

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