Detection and diagnosis of plant disease

J.A. Lucas

Plant diseases caused by fungi cause significant damage

and economic losses in crops every year. It is difficult to
predict, however, whether disease is likely to occur in a
particular crop, and how severe it will become. As a consequence fungicides are often applied inappropriately, at the
wrong time, or when they are not required. Early detection
and diagnosis of plant pathogens can provide more accurate forecasts of disease, and improve the precision of
fungicide application and other control measures.

Current research in the Plant-

ronment, and within infected tis-

Pathogen Interactions Division in

sues. These methods can also pro-

IACR is developing novel molecu-

vide important information on the

lar methods for identifying fungal

properties of fungal pathogens,

pathogens in crops. Using these

such as the presence of different

techniques it is now possible to

genes determining the ability to

detect and quantify the presence of

reproduce sexually, and mutations

such pathogens in the crop envi-

conferring resistance to fungicides.

Detecting airborne inoculum

Many plant pathogenic fungi are
spread by microscopic airborne
spores (inoculum). Detection of
such inoculum is potentially useful in disease forecasting and
management, as it gives an early
warning of the risk of infection.
Conventional methods for measuring spore concentrations in the
air rely on trapping followed by
microscopy to identify and enumerate fungal spores (Fig. 9). This
is time consuming, and often it is
difficult to identify specific fungal
plant pathogen spores on morphological grounds alone. Thus,
routine inoculum detection, using
conventional methods, is usually
impractical for disease management purposes. Alternative methods based on DNA analysis are
attractive as they are potentially
both highly specific and highly
sensitive. Such methods have
been used to develop diagnostic

Fig. 9. A Burkard spore trap, based on a design developed at Rothamsted during the 1950s,
operating in a linseed crop. Samples are collected on an adhesive tape mounted on a rotating drum (top right). Spore concentrations are estimated by counting the deposit on the
tape using conventional microscopy (bottom right). Molecular methods are now being
developed to analyse such samples

20

tests for plant pathogenic and

other fungi. While the potential of
these methods for detecting air-

Institute of Arable Crops

Research Report 2000-2001

detected, indicating that PCRbased assays are potentially very

sensitive.
The spore disruption and DNA
extraction protocol was tested on
spore samples placed on Burkard
spore trap tapes.

The results

showed that the protocol could

successfully extract and purify
DNA from spore trap tapes with little affect on the sensitivity of the
PCR assay.
the

Experiments done in

wind

tunnel

at

IACR-

Rothamsted, using P. roqueforti

spores, showed that the methods
could detect target spores in air
samples even in the presence of
Fig. 10. Light leaf spot (Pyrenopeziza brassicae) symptoms on oilseed rape
leaves. Airborne ascospores infect the crop in autumn. These spores can be
detected in DNA samples purified from spore trap tapes (below). The numbers
of spores estimated to be present in each PCR sample are shown beneath each
lane. P indicates purified P. brassicae DNA and N is a no DNA control

large

numbers

of

non-target

spores, with a sensitivity of less

than ten spores (DNA equivalent) in
the PCR. Burkard spore traps were
operated in oilseed rape crops, and
the tapes analysed conventionally

borne fungal spores has been

recognised, little progress in their
use for this purpose has been
made to date.
Research to assess the potential of
polymerase chain reaction (PCR)
based assays for detecting airborne fungal inoculum is now in
progress at IACR-Rothamsted.
Experiments have been done with
four different fungal species,
Sclerotinia
sclerotiorum,
Leptosphaeria maculans and
Pyrenopeziza brassicae, all important airborne pathogens of oilseed
rape, and Penicillium roqueforti, a
typically air dispersed fungus.
Pairs of PCR primers specific for P.
roqueforti, P. brassicae, and L.
maculans were already available.
Two sequences of the internally
transcribed spacer region of ribo-

somal DNA were identified as

potential specific primers for S.
sclerotiorum. Tests showed that
these primers could differentiate
between S. sclerotiorum and a
range of fungi commonly found in
the air in oilseed rape crops,
including the closely related
species Botrytis cinerea.

and by PCR-assay. To do this the

Three methods of processing

spore samples were tested: adding
the sample direct to the PCR;
breaking open the spores to
release DNA before adding it to the
PCR; and purifying the DNA from a
disrupted spore sample and adding
the purified DNA to the PCR. For all
four fungi the best results were
obtained when purified DNA was
added to the PCR. In some cases
the DNA equivalent of less than
one spore added to the PCR was

detected DNA purified from spore

tapes were cut into sections representing 12 h exposure periods and

one half of each section was
processed for PCR analysis, and
the

number

of

target

spores

deposited on the other half counted. PCR assays using primers specific for the three target fungi
trap tapes, even when the tapes
contained large numbers of nontarget spores. In some cases as few
as 1 or 2 spores in the PCR reaction
were detected. PCR-assays also
detected fungal DNA in DNA
extracted from Burkard spore trap
tapes exposed in Mexico City.
These samples contained large
numbers of inorganic and non-fungal organic particles as well as fungal spores, showing that DNA can
21

Institute of Arable Crops

Research Report 2000-2001

be extracted from spore trap

samples taken from highly polluted
atmospheres.
The results of
these experiments demonstrate
the potential of DNA analysis for
detecting airborne inoculum of
plant pathogenic fungi. The methods should be equally applicable
for detecting other airborne biological particles such as pollen
grains and animal and human
pathogens.

Discriminating between mating

types
Epidemics of light leaf spot
(Pyrenopeziza brassicae) on winter

oilseed rape crops are initiated by

ascospores of the fungus, which
infect newly emerging crops in the
autumn (Fig. 10). The ascospores
are released from apothecia that
develop on debris left on the ground
following harvest of the previous
crop. As the available fungicide
treatments are most effective at the
start of epidemics, the main fungicide application to control light leaf
spot is timed to coincide with the
arrival of ascospores. However, previous studies have shown the severity of disease to be very variable
between seasons and hence fungicides are often applied unnecessarily. The ability to predict the availabil-

ity of light leaf spot ascospores prior

to the emergence of the crop may
enable more rational decisions
about fungicide application to be
taken at an appropriate time.
The formation of P. brassicae
apothecia is dependent upon the
availability of both mating types
(MAT-1 and MAT-2) to interact and
undergo sexual reproduction within
the fungal population. The mating
type genes of P. brassicae have now
been cloned. In common with other
ascomycete fungi these genes contain regions with sequences which
differ between the two mating
types, flanked by sequences which

Fig. 11. Powdery mildew (Erysiphe (Blumeria) graminis) symptoms on cereal leaves. Using
a DNA diagnostic specific for a fungicide resistance gene it is possible to monitor the
incidence of resistance in mildew populations exposed to different treatment regimes

22

Institute of Arable Crops

Research Report 2000-2001

are conserved between the two

mating types. This feature has
enabled the design of a PCR-based
diagnostic test that can differentiate
between the two mating types. The
test uses a three-primer approach in
which primers specific to each mating type are combined with a common primer, which anneals to a
sequence located within the fully
homologous flanking regions of
both mating types.
This PCR-based diagnostic test will
enable surveys to be conducted on
P. brassicae populations, to determine whether both mating types
are present in a crop and therefore
predict whether sexual fruiting
bodies and ascospores are likely to
occur before they are even formed.

Detecting fungicide resistance

Fig. 12. Detection of strobilurin resistance in populations of wheat powdery mildew: real-time
allele-specific PCR using SYBR Green I

For Erysiphe graminis f. sp. tritici

gies in order to prolong the cost-

Fungicides are widely used for the

(wheat powdery mildew, see Fig

effectiveness and lifetime of fungi-

maintenance of healthy crops and

11) we have identified a mutation in

cides. This will lead to more prof-

reliable yields of high-quality pro-

the

itable disease control with less

duce. However, their effectiveness

cytochrome b gene that confers

has been seriously affected in some

resistance to strobilurin fungicides.

situations by the development of

resistance

in

target

fungi.

Understanding how such resistance

develops and spreads is vital to
safeguard the efficacy of fungicides.
We have developed methods to
detect the occurrence, mechanism
and spread of resistance to some of
the main fungicide groups (e.g.,
benzimidazoles, 14-demethylase
inhibitors and strobilurins) in important cereal pathogens. This will help

mitochondrially-encoded

With real-time PCR based assays

impact on the environment.

For further information contact
john.lucas@bbsrc.ac.uk

using fluorescent DNA-intercalating dyes, such as SYBR Green I, or

DNA sequence-specific TaqMan
probes and Molecular Beacons, we
can now detect and monitor the
spread of strobilurin-resistance
genes in populations of the wheat
powdery mildew fungus from field
crops grown under different fungicide regimes (Fig. 12).

to devise strategies to prevent or

Early detection of fungicide resis-

delay the development of resistance

tance genes at low frequencies in

and to support the introduction of

plant pathogenic fungi, coupled

new fungicide products to the mar-

with risk evaluation, will aid imple-

ketplace.

mentation of anti-resistance strate23