The EU requires Member and Acceding States to be competent
in diagnosing almost 300 plant pathogens listed under plant
health legislation. Causal agents are diverse, from nematodes
to viruses, so a range of expensive equipment and expertise
is required. A group of European researchers are assessing
the possibility of using gene chip technology to combine diagnosis
of all these pathogens in a single test. As a case study,
the project is producing a microarray that tests for notifiable
pathogens of the potato. A full protocol for its use will
be tested by diagnostic laboratories.
|The Diag Chip consortium.
range of organisms can attack plants, from viruses to insects.
Those that are serious pests and diseases are quarantined
under the EU Plant Health Directive which lists almost 300
pathogens. This legislation controls the passage of plant
material between countries, and is designed to prevent introduction
of new pests or infectious diseases that threaten agriculture
and the wider environment. To implement the legislation,
Member States and Acceding countries must be competent in
diagnosing all the pests and pathogens, some of which are
obscure. Currently, diagnosis requires a wide range of expertise,
from traditional taxonomy to classic microbiology and ultra-modern
molecular biology. In some cases, expensive specialised
equipment is required.
The Diag Chip project, with a consortium of plant disease
scientists from Germany, France, Spain and the UK, recognised
the potential of new genetic techniques to combine diagnoses
in a single test. The partners are involved in a three-year
project to study the feasibility of creating a ‘diagnostic
chip’ that covers all types of notifiable plant pathogen.
“DNA and RNA, the nucleic acids that hold genetic
information, unify all life,” explains project coordinator
Ian Barker of the UK’s Department for Environment,
Food and Rural Affairs. “So they can form the basis
of a method to deal with all the different pathogenic organisms
A new potato chip
The technology exploited by the project is the DNA microarray.
This is a series of unique nucleic acid sequences, or probes,
usually held as spots on a glass microscope slide or chip.
Because strands of nucleic acid will hybridise into double
strands with corresponding sequences, single-stranded pieces
can be used to detect the presence of particular sequences
from any sample of biological material. Label the nucleic
acid in your sample with fluorescent tags and wash it over
the chip. Any spot on the chip containing a genetic sequence
that is present in the sample will fluoresce.
As a test case, the project is creating a microarray for
potato pathogens. “We want to assess the applicability
of highly parallel testing,” says Barker. That means
using a common method to test for many different organisms.
“We chose the potato, because it is an important European
crop, with a manageable list of pests and diseases that
includes most types of organism.” The chip will cover
24 potato pests and diseases, including 15 viruses and virus-like
organisms, six nematode worms, one fungus and two bacterial
Many dangerous potato pathogens still reside in South America,
from where the potato itself came. Historically, there has
been little or no trade in potatoes from the New World to
the Old World, so we have escaped many of its diseases.
But breeding material is often imported from South America,
and has to be carefully monitored in potato quarantine stations.
To create a microarray for potato pathogens, a unique piece
of genetic sequence must be selected for each organism.
Some of the organisms, like Potato virus Y, are well studied,
and their sequences are available on public access databases.
“We are using the bioinformatics capability at the
University of York to do the number-crunching involved in
identifying a distinctive sequence for each species,”
says Barker. “Other organisms, like the obscure South
American viruses, have never been extensively characterised,
so we are sequencing their genomes ourselves.” The
partners in the project have different areas of expertise
– the French scientists are working on nematodes,
the Spanish group on bacteria and the Germans are focusing
The tiniest trace
The main technical challenge is making the test sensitive
enough. “The problem with highly parallel testing
is that it is not as sensitive as ‘high-throughput’
tests, in which you test a lot of samples but only for one
thing,” Barker explains. In high-throughput testing,
known nucleic acid sequences are amplified many times, so
the test can detect tiny quantities from the original sample.
The conventional method of amplification, PCR, has to be
done separately for each different sequence. “We need
a generic way of amplifying all the nucleic acid in the
sample in a single process. Otherwise, our test will only
work if large amounts of pathogen are present, which will
severely limit its application.” The team are currently
testing a novel strategy to solve this problem.
Six routine testing laboratories around Europe have agreed
to test the chip. “These are the people who hope to
take up the technology,” says Barker. “To ensure
that they feel comfortable with it, and that the methods
we devise are practical, we have involved them right from
“Microarrays have the potential to hold tens of thousands
of sequences,” he continues. “If we can produce
a chip for 23 potato pathogens, then it can certainly be
done for the 300 pathogens in the EU Plant Health Directive.”
The Diag Chip consortium includes a small Bavarian company,
MWG, which brings a commercial perspective. “This
may not be the final format,” Barker predicts. “Other
microarray formats such as three-dimensional liquid arrays
are emerging, and a huge investment is being made in miniaturisation
and cost reduction. We are just proving the feasibility
of highly parallel genomic testing for plant health.”