- 1 - Accelerated Detection and Diagnosis of Invasive Alien Pests and Pathogens in Imported Plants Steve Woodward 1 , Eleni Siasou 1 , Anna Brown 2 , Benjamin Drake 3 , Rainer Ebel 4 , Paul Gascoine 5 , Jean-Claude Grégoire 6 , Gail Preston 7 , Michael Shaw 8 & Stewart Snape 9 1: University of Aberdeen, Institute of Biological and Environmental Sciences, Department of Plant and Soil Science, Cruickshank Building, St. Machar Drive, Aberdeen AB24 3UU, UK. 2: Forest Research, Alice Holt Lodge, Farnham, Surrey GU10 4LH. 3: ADAS, Pendeford House, Wobaston Road, Wolverhampton WV9 5AP 4: University of Aberdeen, School of Natural and Computing Sciences, Department of Chemistry, Meston Building, Aberdeen AB24 3UE. 5: Smiths Detection plc, Bushey, Watford WD23 2BW. 6: Lutte Biologique et Ecologie Spatiale , CP160/12, av. F.D. Roosevelt 50, 1050 Bruxelles, Belgium. 7: Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, OX1 3RB. 8: University of Reading, School of Agriculture, Policy and Development, Whiteknights, Reading RG6 6AR. 9: Forestry Commission, Silvan House, Corstorphine Road, Edinburgh EH12 7AT. Contacts: [email protected]; [email protected]
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Accelerated Detection and Diagnosis of Invasive Alien Pests and Pathogens in Imported Plants
Steve Woodward1, Eleni Siasou1, Anna Brown2, Benjamin Drake3, Rainer Ebel4, Paul Gascoine5, Jean-Claude Grégoire6, Gail Preston7, Michael Shaw8 & Stewart Snape9
1: University of Aberdeen, Institute of Biological and Environmental Sciences, Department of Plant and Soil Science, Cruickshank Building, St. Machar Drive, Aberdeen AB24 3UU, UK.
2: Forest Research, Alice Holt Lodge, Farnham, Surrey GU10 4LH.
Volatile Organic Compounds Produced by Plants .................................................................................................. - 6 -
Changes in VOC Signatures in Stressed Plants........................................................................................................ - 7 -
Volatile Organic Compounds from Microorganisms ............................................................................................ - 15 -
The application of VOC profiling in disease detection.......................................................................................... - 20 -
Methods for detecting volatile chemicals ............................................................................................................ - 22 -
Statistical and economic aspects of improved biosecurity .................................................................................. - 26 -
Economic justifications for additional detection technologies ............................................................................ - 30 -
How Can These Methods be Applied in Practice? ................................................................................................ - 32 -
The possibilities of utilizing state-of-the art chemical detection instrumentation and image analyses to diagnose the presence of alien invasive pests and pathogens in imported plants and plant products are reviewed.
All organisms produce volatile organic compounds (VOCs) which can be detected by a range of instruments already widely in use at ports.
Over 100,000 VOCs produced by plants are known.
VOCs produced by some of the organisms that interact with plants are known. For example:
o Bark beetles are known to both alter organic molecules already present in host tissues, and to produce others themselves. These VOCs have roles in communicating with other individuals of the same species.
o Bacteria and fungi produce a range of relatively simple and easily detected VOCs during different phases of growth.
The suite (profile) of VOCs produced by an organism following stress differs from those produced by the healthy organism.
VOCs produced by healthy versus damaged plants differ significantly, both quantitatively and qualitatively. Moreover, VOCs produced by the same plant may differ depending on the precise cause of the damage/stress.
There is increasing research on the production of VOCs by microorganisms and by many insects, presenting a wide range of specific VOCs which can be used in developing this science for the detection of pests and pathogens.
Given the packing and transport systems used to export plants and plant products, it is likely that VOCs analyses could be utilized in the detection and diagnosis of the presence of alien invasive pests and pathogens in consignments.
Moreover, the widespread presence and use of highly sensitive and discriminatory instrumentation at ports throughout the world means that, with further calibration, existing equipment could be used in this work.
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Introduction During importation, the detection and diagnosis of potentially invasive pests and pathogens
on plants at air- and sea-ports of entry into the UK is a major focus of phytosanitary
inspections. The authorities intercept many such organisms in consignments of plants and
plant-based products during the routine surveys undertaken, but when the numbers of units
inspected, approximately 2%, is set against the discovery rate, it is clear that the number of
invasive pests and pathogens of plants coming into the UK may be very high. The number of
recent pest and disease epidemics that have arisen over the last 20-30 years is also clear
evidence of a failure in phytosanitary regulation on an international scale (Brasier 2008;
Santini et al. 2013).
Many of the invasive organisms not detected at ports of entry have or are
establishing in our forests and woodlands, resulting in high rates of damage and threatening
entire ecosystems. The public outcry in late October 2012, following the confirmation that
the ash dieback pathogen Hymenoscyphus pseudoalbidus was already present in the UK ash
woodlands, illustrated how seriously the general public now takes these problems. Yet the
ash dieback outbreak was only one in a series of serious influxes of alien pests and
pathogens that have become established in our forests and woodlands in the last 20 – 30
years (Brasier 2008; Santini et al. 2013). Clearly, if global trade in plants and plant products,
which is worth billions of Euros world-wide, is to continue, our ability to detect potential
problematic organisms in trade must be very much improved.
The problems posed by alien invasive pests and pathogens and the lack of ability to
make wide-ranging inspections of incoming plants and plant materials at ports, illustrate the
urgent need for more stringent techniques for detection and diagnosis of these damaging
organisms. Current methods of detection, relying on visual and/or molecular (DNA)-based
detection techniques, are extremely useful and powerful, but have serious constraints in
terms of their overall effectiveness:
1. Both methods must be highly targeted to be successful
a. Inspectors cannot examine (visually) more than a very small proportion of
imported plants;
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b. The molecular approach has similar constraints, as the detection of pests and
pathogen depends on sampling tissues that contain DNA of the pest or
pathogen.
2. Both visual and molecular methods are labour intensive and require highly trained
personnel.
3. Both methods are expensive to implement.
The purpose of this review, initiated under DEFRA contract TH0122, is to examine the
potential for alternative methods for detecting potentially damaging invasive pests and
pathogens in imports of plants and products. It is not the intention to suggest that the
current methods should be replaced with this technology, but to add another level of
detection capacity to the system, enabling a more targeted approach for subsequent visual
and molecular detection and diagnosis techniques.
All organisms produce an array of volatile organic compounds (VOCs), which are in
themselves, or as a profile, apparently characteristic of the emitting organism. Moreover,
the profile of VOCs for a given organism changes when that organism is under stress,
whether from a disease-causing agent, or an abiotic agent. It is not the intention in this
review to list all VOCs produced by plants under non-stressing and stressing conditions, but
it is clear that a huge number of plant VOCs can be used to distinguish between species and
within even cultivars and varieties to determine health status, possibly even the cause of
the stress, whether abiotic or biotic. These quite simple organic molecules can be rather
easily detected by the instrumentation currently available at ports (see below).
A first challenge facing researchers aiming to use plant VOCs to detect attack by
pests or disease-causing organisms, however, is selecting which VOCs to study. VOCs used
for the presence of pests or disease can originate from three main sources: the plant, the
pest/pathogen or the action of the pest/pathogen on plant tissues. VOCs are produced by
both aerial and root tissues, although most disease detection focused studies published to
date have focused on foliar VOCs, which are typically released to the atmosphere in higher
concentrations than are root-associated VOCs.
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Volatile Organic Compounds Produced by Plants The production of a wide variety of VOCs by plants is well known and was the subject of a
recent review (Dudareva et al. 2013). Humans have used many such products from species
such as basil, garlic, lavender, rosemary, camphor, damask and other roses, pine (and other
gymnosperms), and many spice plants, in perfumes, food and drink flavourings,
preservatives and solvents for centuries. Many other fragrant plants feature strongly in
human history and are well documented in ancient texts. For example, the aromatic
properties of food plants are of immense importance to feeding choices of animals,
including humans (Goff & Klee 2006).
More than 100,000 metabolites produced by plants are known, many of which are
classified as VOCs (Dicke & Loreto, 2010). For example, over 1700 volatile compounds floral
scent compounds have been identified (Knudsen et al. 2006); a high proportion of the 7000
flavour VOCs known from food and beverages are of plant origin (Nijssen et al. 1997). VOCs
can be regarded as ‘infochemicals’, which are released, and which act in plant-plant, plant-
animal and plant-pathogen interactions (Maffei, 2010).
Plant VOCs have a highly significant contribution to VOC emissions overall, with over
50% of VOC emissions per annum in the USA, and over 67% of emissions world-wide coming
from plants (Guenther 1997). It has been estimated that monoterpene emissions from
plants alone are responsible for the release of between 127 and 480 megatonnes of carbon
into the atmosphere globally per annum (Kesselmeier & Staudt 1999).
Plant VOCs fall into two main categories:
constitutive VOCs, which are frequently stored in plant tissues and volatilised
according to their concentration and physicochemical properties, and
inducible VOCs, which are synthesised in response to environmental stimuli.
The distinction between constitutive and inducible VOCs is somewhat ambiguous, however,
as many of the constitutive VOCs normally released from healthy intact plants become
inducible volatiles (IVOCs) after foliar damage, when larger quantities or different ratios are
produced (Holopainen, 2004). Both have uses in the detection of stressed plants, as novel
IVOCs may be produced in response to infection, while changes in the profile and quantity
of constitutive VOCs can also be used to provide information on plant health and disease.
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Typical examples of fragrant VOCs produced by plants are the monoterpenes
produced as significant components in the essential oils of the Pinaceae and Citrus spp.,
Hanover, 1992; Zhang et al. 1999; Staudt, Lhoutellier 2007; Foster et al. 2013
Benzyl acetate Shikimate pathway
Plant Yes (laurel) Piesik et al. 2011; Politeo et al. 2007
*Please note that this column lists examples of tree species in which specific volatile chemicals have been detected. This list is not exhaustive: the absence of a species does not necessarily indicate that a specific chemical is not produced by this species.
monoterpenes to attract further beetles to the tree, most commonly through oxygenation
of specific monoterpenes produced by the host, although in certain cases the beetles may
produce the oxygenated monoterpenes by de novo synthesis (Seybold & Tittiger 2003). This
kairomone (attractant) effect is well known in several species of Dendroctonus and Ips, for
example (Wood 1970; Silverstein 1977; Seybold et al. 2006). The fact that the oxygenated
monoterpenes are produced specifically by the insect pests gives a ready target for
detection using high resolution chemical techniques.
Changes in plant host VOCs following attack by herbivorous insects are known in
several other host-insect systems (Pare & Tumlinson 1999; Kessler & Baldwin 2001;
Gouinguene et al. 2003; Zhang & Wang 2007; Staudt & Lhoutellier 2007). In an experimental
system examining responses of silver birch (Betula pendula) to infestation with larvae of
Epirrita autmunata and the leaf spot fungal pathogen Marssonina betulae, it was shown
that changes in host-produced VOCs differed substantially between plants damaged by the
insect and by the fungus (Vuorinen et al. 2007). Production of both methyl salicylate and (E)-
4,8-dimethyl-1,3,7-nonatriene was stimulated by insect attack, but not by the pathogen,
whereas infection by M. betulae caused significantly increased concentrations of (Z)-
ocimene and (E)-β-ocimene. Clearly, the system demonstrates differential responses in birch
which could be used to determine the possible cause(s) of alterations in VOC production. In
holm oak (Quercus ilex), attack by gypsy moth (Lymantria dispar) caterpillars also lead to a
significant increase in VOC release from the foliage of attacked trees (Staudt & Lhoutellier
2007).
Experimental systems have already been developed to enable detection of certain
insect pests in glasshouse crops. For example, Miresmailli et al. (2010) showed that
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caterpillars of the cabbage looper moth, Trichoplusia ni, could be detected in a tomato crop
using GC-MS analyses of VOCs produced from affected plants. A zNose™ system was also
calibrate to detect (Z)-3-hexenyl acetate, (E)-β-ocimene and ϐ-caryophyllene, three VOCs
associated with the insect, and not with tomato plants. These methods were useable in the
glasshouse, demonstrating that, with further development, such systems may be utilized to
detect infestations early in the development of the pest. Different levels of infestation by
one of the most damaging rice pests, the brown plant hopper (Nilaparvata lugens), could be
detected on rice plants grown in a glasshouse using an E-nose system calibrated to sense 10
compounds in the VOCs from the affected plants (Zou & Wang 2011), although the system
relied on glasshouse grown plants enclosed in chambers in order to sample the headspace
volatiles.
Changes in VOC production following damage by phytophagous insects is a well-
known phenomenon, occurring in many, if not all, insect herbivore-plant host interactions
(Pare & Tumlinson 1999; Arimura et al. 2005), and presents an excellent opportunity for the
detection of pest problems inside plants and plant products during shipment. A further
intriguing possibility here is the use of excised insect sensor organs (usually antennae) to
detect tiny quantities of kairomones/pheromones released by insects attacking plants or
plant products (Sevonkaev & Katz 2008). Antennae are attached to electronic amplifiers to
determine changes in signal potentials (Cooper & Cass 2004). As insect antennae are
naturally calibrated to detect highly specific chemical cues in tiny quantities, the method
could be used in the detection of known problematic insect pests.
Volatile Organic Compounds from Microorganisms Bacteria: The production of VOCs by prokaryotes is a well-known phenomenon, which may
be involved naturally in cell-to-cell signaling and in the inhibition of potential competitive
microorganisms (Schultz et al. 2010). The range of VOCs known from bacteria is extensive,
and includes many small molecular weight compounds, some of which are identical to those
produced by plants. Soil-borne Pseudomonas spp., for example, produce compounds
including benzothiazole, cyclohexanol, nonanal, decanal, and dimethyl trisulphide (Fernando
et al. 2005; Sauer & Kliem 2010), each of which is relatively easily detected using routinely
available chemical methods (see below). Dimethyl sulphide and other related compounds
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are further easily detected compounds, produced by bacteria in many substrates, including
cheese (Seefeldt & Weimer 2000).
Although many bacterial VOCs appear to be produced by a wide range of
actinobacteria, myxobacteria and cyanobacteria, other compounds are strain specific
(Schultz & Dickschat 2007) and could prove useful in diagnosing the presence of bacterial
pathogens in plants.
Fungi: The fungi also produce many volatile compounds (Splivallo et al., 2011; Kramer and
Abraham, 2012) in a number of chemical classes including monoterpenes, sesquiterpenes,
alcohols, aldehydes, aromatic compounds, esters, furans, hydrocarbons, ketones, and
nitrogen- and sulphur-containing compounds (Splivallo et al., 2007a; Campos et al., 2010;
Kramer and Abraham, 2012). As with bacteria, it is obvious in mycology laboratories or in
damp buildings that fungi produce characteristic odours. The odour of the common
mushroom, Agaricus bisporus, results partly from the production of 1-octen-3ol, for
example (Wąsowicz 1974). Recently, it was proposed that a combination of morphological,
molecular and metabolite data be used in the identification and classification of fungi
(Frisvad 2011), and some published work confirms the utility of this approach (Zhang et al.
2010).
A great deal of research carried out to date has focused on the volatiles produced by the
genera:
Penicillium, which includes many spoilage organisms, plant pathogens, and species
such as P. roqueforti, P. camemberti and P. chrysogenum, of use in the food and
pharmaceutical industries (Whilborg et al. 2008) and P. expansum, P. italicum and
P. digitatum attacking fruits (Chitzandis 1988a,b; Archer, 1988). It is pertinent to
note here that VOCs produced by some strains of P. expansum may have
exploitable repellent activity against the pine weevil, Hylobius abietis (Azeem et al.
2013).
Aspergillus: again, spoilage organisms that, in this genus, produce aflatoxins which,
being carcinogenic and mutagenic, are regulated in human foodstuffs. Aspergillus
spp., particularly A. flavus and A. parasticus, commonly contaminate food crops,
such as maize, peanuts, true nuts, dried fruits and vegetables. A range of VOCs is
produced by Aspergillus spp. (Kaminski et al. 1972; Zeringue et al. 1993; Roze et al.
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2007). Recent work suggests that VOCs from Aspergillus play a major role in the
production of the toxins (Roze et al. 2007). Hence, the production of the toxins can
be accurately estimated through analyses of VOCs.
Fusarium: targeted to enable rapid determination of mycotoxins or mycotoxigenic
fungi in stored food, particularly cereals (Magan & Evans, 2000; Demyttenaere et
al. 2004; Zhang et al. 2007). The availability of electronic detection methods
(‘electronic noses’; ‘e-noses’) enables rapid detection of toxins from fungi such as
Fusarium in grain stores, reducing public health risks (Schnürer et al. 1999; Magan
& Evans, 2000). There are many Fusarium species that are pathogenic on growing
plants, however, including the highly damaging alien invasive species, F. circinatum,
cause of pitch canker on pines.
Trichoderma, a genus of very common fungi which appear to be ubiquitous. Several
species in this genus are exploited for the control of a range of plant diseases,
particularly those caused by soil-borne fungi (Elad et al. 1982; Papavizas 1985).
There are several mechanisms proposed for the inhibition of other fungi by
Trichoderma spp., including production of lytic enzymes, competition for infection
sites on roots or for nutrients and induction of host resistance (Benitez et al. 2004).
VOCs, however, are also long-known components in the arsenal used by
Trichoderma to protect the occupied niche (Rathore et al. 1992; Pandey &
Uapadhay 1997; De Respinis et al. 2010; Patil et al. 2012).
Tuber: the truffles. The drive in this research has been the development of
portable, field-useable instrumentation to detect truffles in forests and to
determine the presence of mixed truffle species in goods sold as a single species
(Bellesia et al. 1996; 2001; Diaz et al. 2002). Considerable success has been
achieved in this application, suggesting that the approach could be modified for use
with other fungi.
There is, however, an increasing body of work published on VOCs from other fungal genera.
Interesting recent work of direct relevance to the present review is the development
of VOC methods to determine fungi growing in water-damaged buildings (Polizzi et al.
2012). The method could be used to rapidly discriminate between strains of Aspergillus,
Chaetomium and Epicoccum isolated from inside damp buildings. The fungi were first
cultured in the laboratory, before analysis. Discrimination based on VOCs profiles was
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obtained through Although it was recognised by the authors that low concentrations of the
VOCs would occur in the buildings (cf. Pasenen et al. 1998; Kim, Elfman et al. 2007;
Schleibinger et al. 2002, 2008), and that different VOCs may be produced by the same
fungus at different points in the life cycle (Matysik et al. 2008), it was concluded that VOCs
analyses could be used to complement classical and molecular identification techniques.
Discrimination between various fungal groups was also achieved in recent work
using mycorrhizal, pathogenic and saprotrophic species (Műller et al. 2013); VOCs produced
by the three groups were analyzed using GC-MS and differences determined using principal
component analysis (PCA). In this type of work, PCA uses data on the occurrence of all
compounds detected to discriminate species or strains, and is an essential tool when single
species-specific compounds are not present.
Further work on three species of Penicillium, along with other true fungi and a
species of the Oomycota genus Saprolegnia, demonstrated that different groups of fungi
and fungus-like organisms could be distinguished based on VOCs analyses (Wihlborg et al.
2008). Distinguishing between different fungi using these methods strengthens the case for
extending research into the volatile compounds produced by plant pathogenic fungi, to
determine if the method discriminates between these organisms. VOCs can be highly
discriminatory in Penicillium: P. roqueforti is a species complex, yet the different biological
species within the complex can be distinguished by VOCs analysis (Karlshøj & Larsen 2005).
Aromas of wine may sometimes be affected by the presence of fungi such as Botrytis
cinerea or Penicillium expansum in the must obtained from pressed grapes. This problem
lead to the development of GC-MS methods to detect VOCs produced by the two fungi in
must (Morales-Valle et al. 2011). Geosmin, a VOC produced by P. expansum when
competing with B. cinerea (La Guerche et al. 2007), was easily detected in contaminated
must, giving a marker metabolite for the presence of both fungi in this particular substrate
(Morales-Valle et al. 2011).
It is known from work in various laboratories, that many species of fungi have
characteristic smells when in culture. Species in the genus Ceratocystis, for example,
produce fruity odours in culture (Christen et al. 1997); as C. platani and C. fagacearum, both
formerly placed in the ‘C. fimbriata group’, cause highly damaging tree diseases, the
discrimination of these species based on human sensing of volatiles suggests that the highly
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sensitive instrumentation available for VOCs analysis may be able to detect the presence of
these pathogens in plants. Some unsubstantiated reports also suggest that it is possible to
distinguish between different species of Phytophthora based on the human sense of smell
too; unfortunately, there is little information in the literature on production of VOCs by
Phytophthora spp., with reports confined to aromas produced following infection of potato
by P. infestans (de Lacey Costello et al. 2001; Laothawornkitkul et al. 2010).
There is growing evidence that interactions between microorganisms can lead to
dramatic changes in the metabolite profiles of the interacting partners, and these changes
can thus be of enormous diagnostic relevance. A recent example (Rateb et al. 2013)
demonstrated this phenomenon in the interaction of the fungus Aspergillus fumigatus MBC-
F1-10 and an actinobacterium, Streptomyces bullii, isolated from hyper-aridic Atacama
desert soil. In competing co-cultures of these two microorganisms (Figure 2) induction of a
plethora of secondary metabolites produced by the fungus was observed (Figure 3), i.e.
ergosterol 1, seven metabolites belonging to the diketopiperazine alkaloids; brevianamide F
2, spirotryprostatin A 3, 6-methoxy spirotryprostatin B 4, fumitremorgin C and its 12,13-
dihydroxy derivative (5–6), fumitremorgin B 7, and verruculogen 8, in addition to 11-O-
methylpseurotin A 9 and its new isomer 11-O-methylpseurotin A2 10. Neither the fungus
(chart 1a) nor the actinobacterium (chart 1b) produced these compounds when cultured
alone.
Figure 2. 1 HPLC-MS profiles of different bacterial/fungal co-culture approaches. (a) bacteria (Streptomyces bullii) alone; (b) fungus (Aspergillus fumigatus) alone; (c) fungal-bacterial co-cultivation (Rateb et al. 2013).
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The application of VOC profiling in disease detection Despite the possible negative factors associated with diagnosing problems in plants
discussed above, there are multiple reasons to believe that VOCs provide a valuable tool in
the development of advanced technologies for disease detection. One particularly strong
argument supporting this approach for distinguishing plant-pest/pathogen interactions from
other stressors comes from molecular studies of plant signal transduction during disease.
Three major defense signaling pathways have been recognized as important for plant biotic
interactions and for the production of defence associated VOCs: the jasmonic acid, salicylic
HO
HN HN
N
O
O
H
H
N
N
HN
O
O
H
H
O
MeO
N
N
HN
O
O H
O
MeO
NH
N
N
MeO
O
O
H
RR
5 R = H6 R = OH
NN
N
OHMeO
O
O H
OH
NN
N
O O
O
O H
OHOH
NHO
O
OHO
OMe R
OMe
O
172
3
5
6
8
9
21
11 10
15
16
1
2
3
4
7
8
9 R = -10R =-
MeO
Figure 3. Structure of fungal metabolites induced by competing co-culture between Aspergillus fumigatus MBC-F1-10 and Streptomyces bullii (Rateb et al. 2013).
In evaluating any novel detection method, this table is key. In any plausible case h >> i. The
large costs of an incursion or eradication are associated with Pr{-|+}. Because h >> i, the
false positive proportion of intercepts is likely to be high. This is minimised by instituting a
series of further checks, including visiting the importer after internal transit. But minimising
Pr{+|-} will increase Pr{-|+}. Reducing Pr{+|-} + Pr{-|+} as far as possible will increase
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complexity and time, and therefore reduce θi . Any new technology improving detection
therefore needs Table 1 to be evaluated in the context not just of the immediate
equipment, but also the effect on follow-up and trace-forward operations.
Sampling strategy at the level of individual consignments: This is a question about how to
best use established (primarily visual and olefactory) and novel (auditory, expanded
spectrum visual and volatile detection) methods to detect diseased plants in a large
consignment in limited time. Methods depending on altered volatile emissions (whether
detected by animals or machinery) will have the best chance of positive detection if the
volatiles have been emitted into a closed space and thereby concentrated. Containers with
fan recirculation systems are ideal in this way. There will be conventional statistical issues
to do with the variation in concentration within a consignment and the variation in
concentration in similarly infected consignments. These need solution, but cannot be
further specified without the detail of the technology to be deployed. Similarly, there is
both an optimisation and a pathology question about how to use a positive result which
cannot immediately be linked to visible symptoms.
Measuring the effectiveness of interceptions: The most robust way to measure the risks run
would be to “spike” trades at their origin with differing proportions of plants inoculated
with known pathogens already established in the UK and therefore not posing additional
risk. This would allow formal comparisons of novel detection techniques with visual
inspections, and provide a means of evaluating problems caused by the need to move
packaging in order to see the hosts, latency, and other factors. Again the cost of this
approach would make a formal statistical approach important.
Economic justifications for additional detection technologies IAS are estimated to cost the EU at least €12 billion per annum, based on data from the past
20 years; as increasing numbers of IAS enter and establish in Europe, the costs are
increasing (Kettunen et al. 2009). Impacts in the UK of all invasive species were estimated by
Williams et al. (2010) to cost approximately £1.7 billion per annum. The financial impact of
invasive alien pests and pathogens to forestry are difficult to quantify in absolute terms
(Price 2010), but it was predicted that the annual costs to the forest environment of a
restricted number of tree diseases alone (Dothistroma septosporum on Pinus nigra var.
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laricio, Phytophthora ramorum, acute oak decline and a ‘nasty surprise on Sitka spruce’ [sic])
could be in the region of £ 172 million (Price 2010). Despite the apparently dramatic figures
for losses and other costs following establishment of an invasive pest or pathogen, it is
essential that any additional biosecurity systems put in place at borders or elsewhere are
adequately predicted, to be certain that there is an economic benefit in their
implementation.
There are a number of publications in which the costs and benefits of biosecurity protocols
at national and state borders are modelled (e.g. Hinchey and Fisher 1991; Cook et al. 2007;
Cook and Matheson 2008; Sonigo et al. 2011).
A model for the development of a system to determine the reductions in economic losses
resulting from preventing the entry of a given invasive and damaging pest or pathogens is
given in Figure 4, using the agent causing pine pitch canker, Fusarium circinatum, as an
example, and based on Cook and Matheson (2008). The model could be adapted for a range
of pests and pathogens, using appropriate data.
In order to develop an understanding of the economic viability of any proposed changes to
phytosanitary procedures, the model below (Figure 4), prepared based on the threat posed
by Fusarium circinatum, cause of pitch canker of pines, could be followed. The sequence of
events to be followed is:
1. Run the model (Figure 4) to obtain probability weighted losses from damage (pest or
infection) under the status-quo scenario (current standard government response to
spread, existing probability of entry).
2. Modify the probability of entry to reflect the changes from the introduction and
adoption of the new detection technologies.
3. Re-run the model. This repetition should show a reduction in the probability
weighted losses incurred by the forestry or horticulture sector.
4. The reduction in probability weighted losses will also entail development and
adoption costs associated with the new detection technologies. Clearly, for an
efficiency gain, the reduction in losses must be greater than the costs of
development and adoption of the novel technology.
A sensitivity analysis using different discount rates is also required, as the choice of discount
rate may heavily influence the probability weighted losses: higher discount rates will
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diminish the benefits of reducing future spread much more rapidly compared to lower
discount rates. It is also pertinent to consider the non-market benefits of forests and
landscapes, particularly when the affected plant species at risk are highly visible to the
general public, i.e. outside of commercial timber plantations.
A highly relevant recent publication in which the economic impacts of an invasive tree
pathogen are considered in detail is that of Cook and Matheson (2008), where the economic
implications of the establishment of F. circinatum in the pine plantations of Australia are
modelled. It was suggested that large economic benefits would accrue from specifically
targeting the likelihood of pathogen arrival and the speed of spread:
“Using a hypothetical example to illustrate this point, let us assume the probability
of arrival can be lowered by 25% as a result of $0.25 million per year being
invested in pre-border and border security measures to avoid F. circinatum
incursions over 30 y. If we consider our distribution of entry and establishment
probabilities, Uniform (0.035, 0.3), and think in terms of the median (0.13), this
change would be equivalent to decreasing the likelihood of an arrival event
occurring once every 7.5 y to once every 10 y. If this were achieved we predict a
benefit/cost ratio of 1.7:1 would result with about $13 million prevented damages
over time” (Cook and Matheson 2008).
Clearly, preventing the ingress of alien pests and pathogens into a given state (or even at
the continental level) will have immense economic advantages, supporting the introduction
of further, highly sensitive and accurate detection methods into the plant and plant
products trade routes.
How Can These Methods be Applied in Practice? Two approaches can be used during the technological development phase:
1. Detection of volatile metabolites of bacterial and fungal pathogens, and of pest
invertebrates characteristic of the organisms (to family/genus level, or species where
possible);
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Figure 4. Model for a system to determine reductions in economic losses resulting from reducing the
probability of entry of a given invasive and damaging pest or pathogens. The example used here is Fusarium
circinatum.
2. Detection of characteristic changes in the volatile components produced by affected
plants following attack by pests and/or pathogens, including transformation of plant
metabolites by pest and pathogen activities.
It may also be possible to utilize a combination of these two approaches to detect the
presence of threatening organisms. As plants are shipped both inter and intra-continentally
in containers, sometimes heavily wrapped and/or sealed, concentrations of volatiles will be
higher in transit compared with ambient conditions.
Calibration of the currently available instruments enables the detectors to respond
to the target molecules amongst a very diverse background of confounding material,
including common chemicals such as cleaning materials, pollen, benign organisms and other
particulate matter such as diesel elements and dust. The literature, coupled with the
- 34 -
experience of the chemists and engineers at Smiths Detection, strongly suggest that the
approach is feasible for the detection of organisms producing defined VOCs.
The data already published strongly suggest that a range of indicative and potentially
diagnostic molecules are produced by different micro-organisms and insect pests. The
information generated in the development phase, however, will require careful handling
and analysis. It may be most effective to target a matrix of molecules from some species
along with Bayesian analyses to determine what potential problems are being transported
in the shipment examined. These initial screening tests could then indicate which
consignments need further, possibly visual inspection and molecular analysis, as suggested
above. Analysis of the volatile components using GC-MS, IMS or other approaches would
serve to identify ‘healthy’ or acceptable shipments, freeing up the time of the phytosanitary
inspectors to examine the more questionable consignments.
Conclusions The further development of chemical analytical instrumentation to detect pests and
pathogens in plant consignments requires an innovative, forward-thinking and
interdisciplinary approach, utilizing the expertise of pathologists, entomologist, chemists,
engineers, IT specialists and end-users. In addition, economic aspects of taking this
approach rather than one focused solely on molecular methods must be considered.
It is evident that the currently available detection instrumentation, following
appropriate parameterization, could be used in the detection of VOCs produced by
microorganisms or by infected plants. The feasibility for this approach requires in depth
investigation, including determination of the costs and benefits of applying these
technologies over the current systems based on visual assessments and, when indicated,
molecular techniques. It is not envisaged that VOC detection techniques would replace the
current methods, but they will add a further technique to the arsenal utilized by
phytosanitary officials when dealing with large numbers of plants and plant product
consignments at ports of entry into the EU and the UK. Moreover, a positive result in
chemical detection will indicate to the current inspectors where more detailed
investigations could be made for example, taking tissues samples for molecular analyses.
- 35 -
The instrumentation to detect VOCs produced by pests and pathogens, or by
attacked plants, is already available. Moreover, the machinery is highly sensitive to a wide
range of small molecular weight compounds; currently, the instrumentation is calibrated for
the detection of several compounds that are derived from plants, and are chemically related
(through metabolic pathways) to known VOCs released by both plants and microorganisms.
Hence, with suitable parameterization based on further knowledge of VOCs produced in
plant-pest and plant-pathogen interactions, it will be possible within a small time scale, to
use these machines in the detection of potentially invasive pests and pathogens of plants.
It could be argued that systems relying on the detection of VOCs in plant and plant
product consignments would not be of use in diagnosing the presence of soil-borne pests
and pathogens, but emerging research indicates that soil sampling for VOCs is also highly
likely to be feasible in the near future (Insam & Seewald 2010). Such adaptations of the VOC
detection technologies could prove invaluable in detecting the presence of pathogens such
as the Oomycota (Phytophthora, Pythium) and true fungi, including many species of
Fusarium or Rhizoctonia, or root-feeding insects, such as vine weevils, cut worms and wire
worms.
As there are likely to be large numbers of currently unknown invasive agents
threatening forest ecosystems, it is important that detection methods recognize signature
metabolites of groups of pests or pathogens (e.g. by genus or family), rather than
discriminating to the species level. This requirement does not preclude using the technique
to detect to the specific level, of course. The IMS and/or GC/MS or GasID instruments will
provide, initially, a technique to focus sampling for other diagnostic methods, such as highly
discriminatory molecular tools.
With such large numbers of plants imported into European countries, it will remain
true that sampling will be imperfect; the potential for increased detection rates using state-
of-the-art chemical methods, however, will greatly enhance the efficacy of phytosanitary
control at borders.
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