Forensic plant pathology

C H A P T E R

5 Forensic plant pathology Jacqueline Fletcher1, Neel G. Barnaby2, James Burans3, Ulrich Melcher4, Douglas G. Luster5, Forrest W. Nutter, Jr. 6, Harald Scherm7, David G. Schmale, III 8, Carla S. Thomas9, Francisco M. Ochoa Corona10 1

Department of Entomology & Plant Pathology, National Institute for Microbial Forensics & Food and Agricultural Biosecurity, Oklahoma State University, Stillwater, OK, United States; 2FBI Laboratory, Quantico, VA, United States; 3National Bioforensic and Analysis Center, Frederick, MD, United States; 4 Oklahoma State University, Department of Biochemistry & Molecular Biology, Stillwater, OK, United States; 5USDA ARS, Foreign Disease - Weed Science Research Unit, Fort Detrick, MD, United States; 6 Iowa State University, Department of Plant Pathology and Microbiology, Ames, IA, United States; 7 University of Georgia, Department of Plant Pathology, Athens, GA, United States; 8Virginia Tech University, School of Plant and Environmental Sciences, Blacksburg, VA, United States; 9University of California, Plant Pathology Department, Davis, CA, United States; 10Department of Entomology & Plant Pathology, National Institute for Microbial Forensics & Food and Agricultural Biosecurity, Oklahoma State University, Stillwater, OK, United States

Acronyms used in this chapter

NIMFFAB National Institute for Microbial Forensics & Food and Agricultural Biosecurity NPDN National Plant Diagnostic Network NPDRS National Plant Disease Recovery System OIG Office of the Inspector General PCR Polymerase Chain Reaction PPQ Plant Protection and Quarantine SDA State Department of Agriculture SNP Single-Nucleotide Polymorphism SOP Standard Operating Procedure SPHD State Plant Health Director SPRO State Plant Regulatory Official UAS Unmanned Aircraft System USDA United States Department of Agriculture

APHIS Animal and Plant Health Inspection Service ARS Agricultural Research Service CBP Customs and Border Protection CRISPR Clustered Regularly Interspersed Short Palindromic Repeat DHS Department of Homeland Security EAN Emergency Action Notice ELISA Enzyme-Linked Immunosorbent Assay FBI Federal Bureau of Investigation NBFAC National Bioforensic Analysis Center NIFA National Institutes for Food and Agriculture

Microbial Forensics, Third Edition https://doi.org/10.1016/B978-0-12-815379-6.00005-2

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5. Forensic plant pathology

Introduction Plant resources in the United States (US), which include crops, forests, range, nurseries, and orchards, as well as natural and landscaped spaces, are essential for human and animal life. In addition to providing food, feed, fiber, and recreational opportunities, they harness sunlight energy, utilize carbon dioxide, and recycle oxygen. Plants are affected naturally by a wide range of microbial pathogens that colonize their surfaces, invade their interior spaces, compete with them or metabolize their tissues for nutrients, upset the balance of their growth hormones, contaminate their tissues with toxins, and trigger or suppress their gene activity. The science and practice of plant pathology are targeted to the prevention, detection, diagnosis, response, and recovery from such naturally induced disease outbreaks. Heightened biosecurity concerns in the early 2000s brought focus to the possibility that crops and other plant resources could be targeted directly by individuals or groups motivated to cause harm. Intentional targeting of plants by release of pathogens could reduce crop yield and quality, as well as erode consumer confidence, affect economic health and the environment, negatively affect human nutrition and international relations, and undermine the public’s confidence in the government’s ability to provide adequate security (Casagrande, 2000; Madden and Wheelis, 2003; Whitby, 2002). Since that time, a number of countries have implemented steps to enhance agricultural biosecurity. New programs in microbial forensics and criminal attribution have strengthened national security capabilities in the US (American Phytopathological Society Public Policy Board, 2002).

Naturally caused versus intentional introduction The vast majority of plant disease outbreaks occur as a result of natural events. In most cases,

a familiar set of diseases for any given crop will appear repeatedly in a given location, depending on weather and cropping conditions. An unfamiliar disease could prompt a call to a County Extension agent or crop consultant or the submission of a plant sample to a state’s plant disease diagnostic clinic. However, new symptoms alone would be unlikely to raise alarm about the possibility of human intentda phenomenon that could be termed “suspicion inertia.” What features of a plant disease outbreak might trigger concern, on the part of a first detector, that a crime had occurred? What would prompt a call to law enforcement, and when would that call be made? Certain indicators, alone ordmore likelyd in combination, are more likely to trigger a consideration that a disease should be examined more closely, and that a criminal investigation is needed (FDA et al., ND; Rogers et al., 2009). Factors such as a new geographical location (disease not seen in this area before), absence of an insect vector required for natural introduction, unusual pattern of disease in the field, unusual pathogen attributes (such as abnormally high virulence or aggressiveness, or evidence of novel nucleic acid sequences including signatures associated with genetic engineering), weather history nonconducive to pathogen survival or disease development, disease occurring at an unusual time of year, disease present in one field but not in surrounding ones, physical evidence of inoculation (spray equipment, inoculum containers, gloves or masks, etc.) or of unauthorized human visitors (tire tracks, footprints, gates left open, etc.), or recognized motivation (recent argument, firing of an employee, money owed, etc.), all are potential indicators of human involvement in a pathogen release (Fig. 5.1). To assist law enforcement personnel in determining if an agricultural crime has occurred, a decision tool, in which criteria were assigned weights and values to assess the probability of intent, was developed (Rogers et al., 2009). An accompanying worksheet and fact sheet aid inexperienced users in the application of the

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History of agricultural bioweapons

Inputs *Symptoms *Host & pathogen diversity *Environment & season *Soil, water & microclimate *Epidemiological data *Reference material & literature

SAMPLING TECHNIQUES & CHAIN OF CUSTODY

Assay Technologies

PATHOGEN DETECTION

DIAGNOSIS

(+/-)

Decision Making *Initial investigation *Investigation *Response *Delimiting surveys *Eradication or management *Remediation *Attribution

*Microscopy *Biological assays *Mass spectrophotometry *Serological assays *Molecular assays *Next-generation sequencing & bioinformatics

PRESUMPTIVE DIAGNOSIS

FIGURE 5.1 Flow of activity and information for decision-making based on pathogen detection and disease diagnosis. Field data, including symptoms and epidemiological information, are compared with reference material and databases to determine appropriate sampling and analysis techniques. Samples are subjected to laboratory assays that detect and identify microbes present; further testing may be used to discriminate among strains or isolates of a pathogen. The compiled field information and test results inform a final diagnosis. A “presumptive diagnosis” is sometimes made when circumstances require a rapid response (before or in the absence of conclusive diagnosis), allowing responders to act based on the evidence at hand until a definitive diagnosis is completed. In microbial forensics applications, sample management must follow appropriate chain of custody and standard operational procedures.

tool. The tool also was adapted to a web-based survey format to aid law enforcement professionals in assessing the likelihood that a disease outbreak was the result of criminal activity (Rogers et al., 2009).

History of agricultural bioweapons Motives for intentional plant pathogen introduction could include economic gain (within a farm community, between residents of different states, perhaps between nations) due to effects on marketing and trade; revenge (the disgruntled neighbor or employee); or publicity (making a statement about an ideological position such as

genetic engineering, stem cell research, or animal rights). It is also possible to deploy plant pathogens criminally, yet unknowingly. Some introductions of the citrus canker bacterium Xanthomonas axonopodis pv. citri into Florida were likely to have occurred due to the illegal importation of citrus planting stock from canker-affected countries; those responsible may have known that bringing exotic plant stock into the US was illegal, but it is unlikely that they knew that the plant pieces carried X. axonopodis pv. citri. The history of state-sponsored programs to develop and weaponize biological agents for use against agricultural or other nonhuman targets is well documented (Fletcher et al., 2006;

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Harris and Paxman, 2002; Huber, 2006; Whitby, 2002). Germany is believed to have used biological weapons in World War I against the US, inoculating horses with Burkholderia mallei, which causes the disease glanders (Harris and Paxman, 2002). During and after World War II, research was conducted on the efficacy of various bioagents, optimal dissemination methods, and defensive countermeasures. The US, Russia, and other countries are known to have developed weapons against numerous crop species including maize, rice, wheat, potato, soybean, sugar beet, and cotton (Harris and Paxman, 2002; Huber, 2006). Most antiplant biological weapons are not harmful to humans and animals, and therefore are safer than zoonotic pathogens to handle, develop, and deploy. In most state-sponsored programs in which biological weapons were developed, they were considered to serve more as deterrents than as actual offensive weapons (Huber, 2006). However, the 2001 anthrax mailings raised awareness of the potential for the use of biological weapons for nonstate-sponsored terrorism, including the possibility of agroterrorism, an attack on a nation’s agricultural systems in the furtherance of political or social objectives. The simple introduction of a foreign disease agent to a nation’s agricultural enterprise could produce economic devastation or public alarm as confidence in the food supply is impacted (Fletcher et al., 2006; Madden and Wheelis, 2003; Wheelis et al., 2002).

The need for forensic plant pathology If plant pathogens or their products are used deliberately to cause social or economic damage, or are introduced inadvertently by illegal actions, forensic and law enforcement officials are responsible for determining the source, method, and time of the introduction, and identifying those responsible (Budowle and Chakraborty, 2004; Budowle et al., 2005a, 2005b; Cooper, 2003; Fletcher, 2008; Jones and Gladkov, 1999;

Kaffarnik et al., 2003). Forensic science provides scientific analytical support for the ultimate goal of attribution of a criminal act (Budowle and Chakraborty, 2004; Budowle et al., 2005a, 2005b, 2005c; Fletcher et al., 2008). The significant legal ramifications resulting from criminal attribution and prosecution necessitate higher degrees of scientific validation and stringency than those normally used in disease diagnosis and plant pathogen identification (Budowle, 2003; Fletcher et al., 2008). The ideal bioforensic investigation will be supported by scientific data that will assist investigators in linking a crime to its perpetrator(s), including the identification and characterization of a specific crime-associated microbe and determination of how the microbe was prepared and introduced. The bioforensic investigation should prioritize the use of defined and validated techniques, such as those developed by the International Organization for Standardization, for the collection and preservation of samples and other evidence, and of robust and reliable assays for pathogen identification and characterization. Maintaining rigorous chain-of-custody records is essential. While it is desirable to minimize the time between on-site sample collection and arrival at a forensics laboratory, and the time required for completion of controlled laboratory analysis, the fact that some case investigations continue for months and even years highlights the importance of rigor in early-stage collection and preservation activities. Microbial forensic capabilities targeted specifically toward pathogens and toxins of humans and animals have increased over the past several years; however, fewer specific methods or standard operating procedures (SOPs) have been developed and rigorously validated for application to plant pathogens. The emerging science of forensic plant pathology requires the adaptation and validation of protocols for crime scene sampling, evidence handling, laboratory testing, and analysis. As the discipline of plant pathogen forensics continues to evolve, existing methods,

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Pathogen detection and diagnostics

SOPs, and protocols continue to be assessed, standardized, and validated so that their use will be meaningful in a criminal investigation. Plant pathologists and forensic scientists must work together closely in groups (such as the American Phytopathological Society’s Microbial Forensics Interest Group) and in collaborative teams.

Pathogen detection and diagnostics The flow of activity and information for decision-making with respect to reaction and response to a plant disease outbreak is shown in Fig. 5.1. A plant disease outbreak is often recognized initially by the presence of symptoms. Depending on the plant host species and the nature of the pathogen, these may include stunting, wilting, chlorosis, necrosis, soft rotting, malformations, tissue proliferations, prevention or reduction in flowering, seed, or fruit production, and other phenomena. The nature of the symptoms on a particular host plant can provide clues todbut not proof ofdthe identity of the pathogen and the disease. Challenges arise when several pathogens induce similar symptoms or when multiple pathogens occur in the same plant. One pathogen can mask symptoms of another, infect several hosts, cause different symptoms in each, act synergistically with another pathogen, or produce a distinctive and sometimes more severe disease in combination with another pathogen than either does alone (Nyvad, 2004; Ochoa-Corona et al., 2007, 2010). Detection of a microbe or associated secondary metabolite in a plant sample, whether by observation of pathogen signs (fungal mycelia or spore-producing structures; bacterial exopolysaccharide slime, nematode galls, etc.) or by a specific assay, establishes that an organism is present, but does not prove a causative role for that microbe. Plant disease diagnosis is the establishment of the cause of observed damage, generally accomplished by a combination of careful observations of plant and microbial

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growth, signs, and symptoms, soil, water, and environmental conditions, seasonality, host and pathogen diversity, epidemiological data, and serological or nucleic acid-based assays. For new diseases, an additional requirement is the fulfillment of Koch’s postulates (Grogan, 1981). In the last several decades, serological and nucleic acidebased assays have allowed precise but inconclusive presumptive diagnosis of a plant disease (associating the presence of a pathogen with a disease but falling short of proof-of-cause) (Nyvad, 2004), a service frequently offered by plant diagnostic clinics and used at the farm level for making crop management decisions. Presumptive diagnosis is normally insufficiently rigorous for applications in agricultural biosecurity and forensic plant pathology, but in the absence of any other evidence could be used in an investigation or legal proceeding. Diagnostic and detection procedures for agricultural biosecurity and forensics should include multiple methods: light and/or electron microscopy, biological assays (culturing, indexing, and mechanical transmission), chemical assays for secondary metabolites such as mycotoxins (HPLC, GC/MS, LC/MS), and serological and molecular tests (Lebas and Ochoa-Corona, 2007). The number of methods applied in a given case will depend on the pathogen type, the availability of validated methodologies, and the pathogen’s genomic stability (Martin et al., 2000). Diseases most often reported are those occurring in plant populations that have monetary value: crops, orchards, and vineyards, nurseries, forests, rangelands, or ornamental landscapes. For example, significant recent plant disease outbreaks in the US include a damaging 2009 epidemic in the northeastern US of tomato late blight, caused by the Irish potato famine pathogen, Phytophthora infestans, and the still-spreading outbreak of sudden oak death, a disease caused by Phytophthora ramorum, which affects many tree and shrub species in both natural and landscaped settings. In addition, of concern are pathogens that slip across a nation’s

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borders (ports of entry) during international trade of produce, bulbs, ornamentals, seeds, and wood or other biological products, those that are detected at quarantine transitional facilities or mail centers, or even those that can move over long distances through the atmosphere. The sudden oak death pathogen, for example, is believed to have entered the US inadvertently in a shipment of nursery plants to the West Coast but has since been widely disseminated throughout the TABLE 5.1

continental US; and the bacterium Ralstonia solanacearum race 3 biovar 2, a Select Agent (Table 5.1) causing a damaging disease in potatoes, was introduced into the US on shipments of infected ornamental geranium cuttings from production sites outside our borders (Kim et al., 2002). Soybean rust, caused by the fungus Phakopsora pachyrhizi, entered the US in 2004 (Schneider et al., 2005) via Hurricane Ivan from the tip of South America (Isard et al., 2005). Finally, a

USDA APHIS-PPQ plant pathogen select agent list, 2018 (7 CFR part 331). Subspecies and disease name

Taxon

Established in the United States

Ralstonia solanacearum

Race 3, biovar 2; bacterial wilt of potato, tomato, etc.

Bacteria

No

Rathayibacter toxicus

Gumming disease of grasses, Bacteria causal agent of annual ryegrass toxicity

No

Xanthomonas oryzae

pv. oryzae; bacterial leaf blight Bacteria of rice pv. oryzicola; bacterial leaf streak of rice

No

Listed at species level

Peronosclerospora philippinensis (peronosclerospora sacchari)

Philippine downy mildews of corn and sugarcane

Oomycete

No

Species status not established

Phoma glycinicola (formerly pyrenochaeta glycines)

Red leaf blotch of soybean

Fungus

No

Renamed Coniothyrium glycines

Sclerophthora rayssiae var. zeae

Brown stripe downy mildew of corn

Oomycete

No

Synchytrium endobioticum

Potato wart, black scab of potato

Fungus

No

Phakopsora pachyrhizi

Soybean rust

Fungus

Yes

Removed

Plum pox virus

Pox of stone fruits

Virus

Yes

Removed

Ca. Liberibacter africanus

Citrus greening (African)

Bacteria

No

Removed

Ca. Liberibacter asiaticus

Citrus greening (Asian)

Bacteria

Yes

Removed

Xylella fastidiosa (citrus variegated chlorosis strain)

Citrus variegated chlorosis

Bacteria

No

Removed

Scientific name

Comment

Currently listed Listed at species level

Formerly listed

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Pathogen detection and diagnostics

diversity of waterborne plant pathogenic microbes and viruses can be found naturally or could be unlawfully introduced, in aquatic environments (Ravnikar and Mehle, 2012). Rapid detection is critical to effective and timely disease management, response, and timely mitigation (Miller and Martin, 1988; Schaad et al., 2003), but in cases of biocrime or agroterrorism additional forensic analyses are necessary. Symptomatology alone is too variable for a reliable diagnosis. Data from biological assays such as plant host range evaluation, metabolic usage profiling via commercial tests (such as Biolog, Vitek, and API), and chemical analyses such as fatty acid methylester composition can not only be highly accurate but also are costly, time consuming, and unsuitable for high throughput (Schaad et al., 2003). In contrast, enzyme-linked immunosorbent assays (ELISA) and polymerase chain reaction (PCR) assays allow rapid and sensitive detection and timely decision-making (Miller and Martin, 1988; Boonham et al., 2007; Clark, 1981; Halk and De Boer, 1985; Henson and French, 1993). ELISA and PCR are both relatively economical, and ELISA allows high numbers of predetermined tests to be processed. PCR, real-time PCR, and their variants provide high sensitivity with more limited capability for multiplex applications (Miller and Martin, 1988; Schaad et al., 2003; Clark, 1981; Halk and De Boer, 1985; Henson and French, 1993). High sensitivity is desirable for samples collected out of season or those with low numbers of pathogens. Isothermal nucleic acid amplification technologies (Zhao et al., 2015; Donoso and Valenzuela, 2018) such as recombinase-polymerase amplification (Babu et al., 2018) and loop-mediated isothermal amplification (Crandall et al., 2018) offer high sensitivity and field-site application using field-portable devices and have been rapidly adopted by agricultural diagnostic companies offering pathogen-specific assay kits, assay services, and instrumentation for viral and bacterial plant pathogens. Several generations of biosensor instrumentation have been developed

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and deployed based on pathogen-specific nucleic acid sequences (Khater et al., 2017; Neethirajan et al., 2018) and antibody affinity to specific target antigens on the pathogen surface (Rider et al., 2003). Although not widely deployed in the agricultural field, biosensors designed for sampling sensitive and secure areas may be coupled with air sampling and filtration devices for continuous monitoring of the environment over extended periods and automated reporting to local, regional, or state agencies. Advances in the speed and cost of DNA and RNA sequencing, which makes possible very deep sequencing (multiple read coverage), have obvious utility in forensic investigation, allowing high confidence levels in matching nucleic acid sequences from crime scene and suspect samples. Coupled with the exponential increase in plant pathogen genomic sequence data available in open-source public databases, these technologies provide an increasingly cost-effective method to identify pathogens. Where resources are sufficient to process and sequence large numbers of pathogens, it is possible to submit raw sequence reads or partially assembled genomic data into databases for comparison purposes to identify an unknown. Computational biologists link bioinformatics tools into scripted “pipelines” to perform database matches and identify unknowns from raw read genomic sequence data (Feau et al., 2018). Alternatively, virtual “e-probes” of pathogen-specific nucleic acid sequences that have been mined from genomic data may be applied to query custom databases composed of raw sequence reads gathered from environmental, diagnostic, or other unknown samples to identify the presence of any microbe (Stobbe et al., 2013; Melcher et al., 2014; Pena Zuniga et al., 2017; Pena et al., 2017). Methods developed for environmental metagenomic analysis, employing computational analyses to identify multiple sources of DNA in environmental samples, offer a further opportunity to identify multiple microbial pathogens in an agricultural or horticultural commodity, soil or plant sample,

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once challenges in sample preparation, extraction, and deep sequencing have been addressed. Furthermore, recent sequencing breakthroughs have enabled the characterization of the microbiome (the entire collection of microorganisms in an environment) (Vonaesch et al., 2018) and the phytobiome (the entire living and nonliving elements impacting a plant (Beattie et al., 2016)), which could be used to assist in source attribution efforts. Sequencing advances have impacted microbial forensics in other ways. Sequencing has become high throughput, enabling computational screening of samples for the presence of suspect pathogenic species or strains. As a result, farmers will be able to assay their crops for the presence of microbes including human foodborne pathogens such as Escherichia coli, Salmonella species, or Listeria monocytogenes, or plant pathogens that produce toxins affecting humans and could lead to outbreaks of human disease. The absence/presence of such certification also may play a role in forensic investigation. Because of the availability of multiple sequences for a wide variety of strains, it may be possible to associate a particular geographic location as a probable (or improbable) source of the outbreak pathogen. Methods in phylogenetic analysis are becoming phylogeographic tools, steadily being honed by the determination of more and more isolate genomes. HPLC, GC/MS, and LC/MS are routinely used to determine the types and concentrations of mycotoxins in grain samples, but these methods rely on expensive equipment, internal and external controls for the toxins of interest, and well-trained operators at private and government-funded laboratories, for safety certification of grain for feed and food processing. Recent technologies with unmanned aircraft systems (UASs, or drones) have created new capabilities for detecting high threat plant pathogens in the atmosphere (Schmale and Ross, 2015). Small, hand-launched, fixed-wing UASs have been equipped with remote-operated sampling devices to collect atmospheric samples at

target altitudes over regions of interest (JimenezSanchez et al., 2018). Rotary-wing UASs have been equipped with wind and particle monitors in an attempt to characterize and predict the long-distance transport of plant pathogens in the atmosphere (Fig. 5.2). Near real-time detection methods based on surface plasmon resonance have been used onboard a large fixedwing UAS for sampling and detection during flight (Palframan et al., 2014). Coordinated unmanned systems in the air, land, and water (Powers et al., 2018) are likely to play an increasing role in the detection, tracking, and source localization of high threat plant pathogens in the future. A relatively recent development in plant pathogen detection has been the use of trained dogs to detect volatiles from host plants infected with specific pathogens and/or the pathogen itself (Simon et al., 2017). While detector dogs have been widely deployed to detect illicit plants and plant-derived narcotics at ports of entry for many years, applications for phytosanitary purposes have only recently been developed. Dogs are capable of detecting volatile compounds at levels of parts per trillion level. Once trained, they can rapidly identify any object associated with the specific compound(s) (Angle et al., 2016). Current efforts supported by the United States Department of Agriculture Animal and Plant Health Inspection Service (APHIS) focus on training detector dogs to rapidly identify Candidatus Liberibacter asiaticus-infected citrus trees before symptoms of citrus greening (huanglongbing) appear in orchards having scattered pathogen infections as well as at ports of entry for surveillance of imported plants (https://www. aphis.usda.gov/aphis/ourfocus/planthealth/ppqprogram-overview/plant-protection-today/articles/detector-dogs). Because a forensic investigation, unlike the work of traditional plant disease diagnosticians, is designed to lead to criminal attribution, it is often necessary to identify and characterize associated pathogens to a degree well beyond what is

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Epidemiology in forensic investigation

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FIGURE 5.2 Hand-launched unmanned aircraft systems (UASs) can be used to collect and monitor plant pathogens in the atmosphere (left). Rotary-wing UASs equipped with weather and particle monitors can be used to collect weather and pathogen data (right). Images courtesy of D. Schmale.

required for disease management. The critical step in criminal attribution is to discern a reliable match between an outbreak pathogen and one associated with a suspect, a process that often depends on the ability to discriminate with confidence among pathogen strains or isolates. However, although the ability to discriminate confidently among similar microbial species, strains, or even isolates may be an essential element of the attribution process, it likely will be easier to generate data that can be used to establish exclusion (that a particular pathogen or person is not involved in the incident) than absolute attribution (evidence that uniquely associates a particular pathogen or person to the incident). Despite recent impressive advances in diagnostic technologies, accurate and timely plant disease diagnosisdin the enddis a human interpretation of a preponderance of evidence. Technology supports (or is an adjunct to) hands-on experience of a diagnostician, information available from databases and journals, and

consultation and validation with external laboratories (Ochoa-Corona et al., 2010).

Epidemiology in forensic investigation Plant disease epidemiology, the study of populations of plant pathogens in populations of plants, is a key element of forensic investigation in that it provides data, analyses, and quantitative interpretation supporting the attribution of biocrimes involving plant pathogens. Epidemiological analyses can help assess the localities and/or plant systems at greatest biological risk for a biocrime, pinpoint the location where an outbreak originated, support the determination of whether the outbreak was natural or deliberate, predict whether and where the outbreak will spread, and develop appropriate sampling schemes. In the context of forensic investigation, epidemiology is most powerful when combined with remote sensing

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(Nutter et al., 2009) and population genetics (Milgroom and Peever, 2003) for near real-time attribution.

Vulnerability assessment Plant disease epidemics are strongly influenced by meteorological and edaphic factors, and it is therefore possible, over larger geographical and temporal scales, to predict the relative likelihood of successful disease establishment based on the pathogen’s environmental requirements. Climate-matching using CLIMEX software (Sutherst et al., 2007), for example, an approach used commonly in this context, has been applied to identify geographical regions at greatest risk of sudden oak death caused by P. ramorum (Ireland et al., 2013), of citrus black spot caused by Guignardia citricarpa (Yonow et al., 2013), and many others. In addition to environmental conditions, the likelihood of disease establishment and spread also depends on the density and/or connectedness of susceptible crops (Margosian et al., 2009) or horticultural operations (Dehnen-Schmutz et al., 2010), which can be quantified using epidemiological approaches such as network analysis (MoslonkaLefebvre et al., 2011). Using this methodology, Margosian et al. (2009) determined that maize and soybean in the continental US are highly connected at the national scale (and therefore at greater risk of disease establishment and spread), compared with the more discrete regions of wheat and cotton production.

Source attribution Pathogen dispersal and associated disease spread from the site of the initial infection results in characteristic disease gradients that can be analyzed to pinpoint the source of the pathogen introduction. This information, in turn, can be used by forensic investigators to search for physical evidence of a deliberate pathogen introduction and to sample pathogen isolates for strain analyses. Remote sensing imagery derived from satellites or UAS can be analyzed to

quantify healthy green leaf area (an inverse proxy for disease intensity) at field or regional scales, and the resulting gradients can be used to locate the primary disease focus. Nutter and coworkers (2009, 2010) studied focus expansion of Asian soybean rust using IKONOS satellite images at the field scale (1-m2 resolution). Image intensity in the near-infrared (NIR) was used to quantify healthy green leaf area and to identify the primary focus from which disease expanded (Fig. 5.3). In a case study using data from nine soybean research plots in which the disease had been introduced, this approach allowed determination of the primary disease foci to within 1.8
1.3 m (Nutter et al., 2009, 2010). Concentrations of plant pathogen propagules at different altitudes in the atmosphere (e.g., measured with an UAS) can similarly assist in source localization efforts. For example, Aylor et al. (2011) collected sporangia of P. infestans using UAS at different distances above and downwind of large area sources of inoculum. Prussin et al. (2014) conducted releaserecapture experiments near ground level for the small grains pathogen Fusarium graminearum and demonstrated (via microsatellite markers) the collection of a released clone of the fungus up to 750 m away from the source. Such aerobiological sampling methods could be used to localize potential sources of inoculum and thus assist in source attribution efforts.

Natural versus deliberate introduction Once the primary disease focus or foci have been located using the aforementioned methodology, they can be georeferenced and analyzed to determine if their spatial pattern is indicative of a natural or deliberate introduction. For example, a regular (as opposed to random) shape of the disease focus, such as a line pattern within an individual field or regular foci along a major highway across multiple fields, may indicate a deliberate pathogen introduction (Nutter and Madden, 2008). Given this information, ground crews could be directed to the geospatial

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Mutation, evolution, and forensic plant pathology

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FIGURE 5.3 Contour map for a primary focus of Asian soybean rust based on 2-unit interval pixel intensity values extracted from an IKONOS satellite image obtained on 27 August 2006 over Quincy, FL (Nutter et al., 2009). Image consists of 22  22 pixels, each providing 1-m2 resolution.

coordinates of the primary disease foci to search for chemical (e.g., surfactants) or physical (e.g., inoculation tools) evidence of artificial introduction and to sample pathogen isolates to assess whether their genetic structure is typical or atypical of the natural population.

Sampling considerations To be successful, forensic investigations are critically dependent on early detection of a deliberate plant disease introduction, yet sampling theory for early detection is largely underdeveloped. Given the vast (e.g., 102 million hectares of cropland in the US) and heterogeneous environment in which plant disease outbreaks can be initiated and develop, surveillance for early detection is extremely challenging. During the past decade, Parnell et al. (Chavez et al., 2016; Mastin et al., 2017; Parnell et al., 2012, 2015, 2017) have developed new tools to improve the efficiency and cost-effectiveness of surveillance for early plant disease detection by linking

statistical theory with epidemiological information about disease spread. Among other things, these approaches help to determine where to deploy sampling resources to detect the disease as early as possible; ascertain the likelihood of disease absence if no symptomatic plants have been found; calculate disease incidence at the time it is first detected; and incorporate “passive detections” by growers, land managers, or the general public into disease surveillance schemes (Parnell et al., 2017). Although these approaches were designed primarily to support quarantine and regulatory surveillance efforts, they have considerable appeal for use in forensic investigations as well.

Mutation, evolution, and forensic plant pathology Mutation and evolution play a role in forensic plant pathology, as they impact the linkage between the organism(s) that caused the disease

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and those associated with suspects. Comparison of pathogen genomes is central to the process of deciding how likely it is that microbes from two sources came from a recent common source (attribution) or, conversely, that they did not have a common recent ancestor (exclusion). Like other microbes, plant pathogens undergo mutations that, if not repaired, become variations on which selection acts, leading to evolution (Fletcher et al., 2006). Such mutations are both a boon to and a problem for the microbial forensic investigator. On one hand, evolution shows that differences among strains of an organism are plentiful enough that many sources of a phytopathogen can be excluded from consideration simply on the basis of their genetic distance from the crime scene organism. On the other hand, depending on the length of the investigation, evolution may cause the genomes of the crime scene and suspect source organisms having a common ancestry to not be identical due to changes occurring since their derivation or to selection of different individuals from the source strain pool. Such differences, which make reliable attribution or exclusion more difficult, necessitate carefully controlled statistical analyses of likelihoods of changes and the use of methods beyond those based on DNA. The above factors should be considered when selecting a method by which to compare a crime scene organism with a suspect pathogen. A variety of methods can be used to assess genetic differences between organisms, including allelespecific PCR, single-strand conformation polymorphism analysis, multi-locus variable-number tandem repeat analysis, amplified fragment length polymorphisms, and restriction fragment length polymorphisms of PCR products, as well as nucleic acid sequencing. The methods used produce different kinds of data and differ in resolution, reliability of discrimination, and sensitivity to mutation and selection. Sequencing of the genome of the crime scene isolate and control organisms is regarded as the ultimate analysis, from which data on all the other kinds of DNA

tests can be derived, and is recommended for producing a report and/or supporting a courtroom presentation. The improved speed and low cost of next-generation sequencing provides a greater capability to discriminate between closely related plant pathogens. Open-source comparative genomics data analysis toolkits can speed the identification of nucleotide differences once genomic data have been collected from sample and reference strains. Genome sequencing coupled with comparative genomics also has led to the widespread use of very large numbers of single-nucleotide polymorphisms to aid in more rapid discrimination of plant pathogens at the subspecies level (Rep and Kistler, 2010; Olivera et al., 2015). The discovery that bacterial cells harbor clustered regularly interspersed short palindromic repeat-Cas (CRISPR-Cas) genetic elements, comprising a system capable of “memory” in the form of spacer regions with ordered sequences representing genetic invasions from bacteriophages and plasmids (Vergnaud et al., 2007; Horvath et al., 2008), provides a new discriminatory tool to trace the evolution of individual plant pathogen species and strains (Jeong et al., 2013; Davis et al., 2018; Getaz et al., 2018). However, in initial investigations in which exclusion is the principal objective, less sensitive (and often less expensive) methods of surveying phytopathogen genomes are useful as long as there are sufficient data on expectations of mutations in natural populations. Mutation and evolution do not stop after a crime is committed or discovered but continue as long as organisms continue to live and replicate their genomes, possibly for many years in agricultural settings. In that time, the crime scene pathogen and a sample associated with a suspect can diverge from their most recent common ancestor and accumulate multiple mutations (i.e., base substitutions). Even in the absence of replication, spontaneous mutations occur through deamination and other base changes. It is often necessary to propagate suspect organisms in plants before

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genomic analyses, but the selective environment of laboratory hosts may differ from the original such that multiple adaptive mutations occur. Such genome changes are particularly well documented for many phytopathogens, for example, in the adaptation of Plum pox virus, once limited to Prunus species as hosts, to growth in peas (Wallis et al., 2007). Many plants, particularly perennials, can harbor multiple microbes and multiple strains of individual phytopathogens. For example, grapevines carry multiple strains of the phytopathogenic bacterium Xylella fastidiosa, distinguishable by restriction patterns (Naraghi-Arani et al., 2001). The population composition of such mixtures changes significantly during as few as three propagation cycles. Even triply cloned isolates of bacterial phytopathogens can undergo significant genome alterations during prolonged passage (Ye et al., 1996).

Investigation Forensic investigation of a plant disease outbreak requires careful assessment of disease characteristics, sample collection, pathogen identification, identification of likely pathogen sources, and attribution or exclusion of pathogen(s) as the causal agent (Nutter, 2004a). While the most intuitive setting for a deliberate introduction may be the release of a live pathogen into a production field setting, other potential pathways of deliberate introduction include imported plants, seeds, and plant products moving through US ports of entry, by air, sea, or land. While agricultural inspectors of the Department of Homeland Security (DHS) Customs and Border Protection (CBP) are stationed at some major ports of entry, the large volume of plants and agricultural products and the often-cryptic nature of microbial plant pathogens moving through these facilities, typically in large shipping containers with mixed cargo, hinder effective screening for pathogens. USDA APHIS is investigating the use of canine detection

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technologies for large-scale screening of cargo at ports of entry; however, there are limitations in the numbers of volatiles dogs can be trained of which to respond. Therefore, the number of pathogens that individual animals can be trained to detect is limited. “First detectors” on the scene of a deliberate plant pathogen introduction are likely to be growers, crop consultants, Master Gardeners, extension agents, or other local, nongovernmental personnel. “First responders,” authorized to take action after a potential deliberate introduction, generally arrive later, after notification by first detectors. Timely and effective management of a crime scene requires that both of these groups be able to recognize that a crime has occurred and to react appropriately. A National Plant Diagnostic Network laboratory (NPDN; https:// www.npdn.org/home) may become involved if tissue samples are submitted for diagnosis (Stack et al., 2006, 2014). Initial disease assessment should be done before any field disturbance and should include the pattern of disease occurrence and any relevant or unusual field characteristics. Sample collection and handling should follow a chain of custody, and each sample is assigned an identifying designation (Miller and Martin, 1988; Schaad et al., 2003). Forensically relevant samples may include whole plants, plant parts, plant swabs, soil, insects, water, air, and/or biological samples such as alternate hosts. Documentation should include an administrative log, a sample log, complete chain of custody, collection site map(s), and detailed information on the crop, field history, and environment. Photographs, GPS coordinates, and other aids are useful supplements to this documentation. What constitutes a “good” sample depends on the disease incidence, the pathogen, and the host. Samples should be collected from a representative number of disease foci (see Section 6), from outside the focal areas, and from different plants and plant parts (Nutter, 2004b). Pooling samples from several sources allows a larger proportion

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of the plant population to be tested and improves the detection limit (Hughes and Gottwald, 1998); positives can then be tested individually if appropriate. Sampling of necrotic lesions should be from their edges, as the centers may be invaded by saprophytic microbes. Seeds are a good source for seed-borne pathogens, while underground stems and tubers are suitable for other pathogens. Specialized pathogen structures, such as galls or tumors, may also be collected. Epidemiological considerations for sampling for early disease detection are discussed in the section on Epidemiology in forensic investigations. Sampling for pathogen detection (i.e., presence or absence) requires different sampling patterns and sample sizes than those used to determine disease incidence or severity. Sampling the atmosphere for plant pathogens requires some knowledge about the potential scales of atmospheric transport for the agent in question (Schmale and Ross, 2015). In some cases, presenceeabsence data can be more important than incidence or severity data, e.g., to determine the geographical extent of the disease or to decide whether a field should be quarantined. In such cases, sampling can concentrate on high-risk areas in a field, such as borders or wet areas, depending on the pathogen. In most forensic applications, however, disease incidence or severity data will be needed to develop spatial disease intensity maps to identify potential point(s) of inoculation. TABLE 5.2

Sample integrity and security must be preserved during collection, movement, storage, and analysis. Storage conditions, such as the starting material, suspending medium, and temperature, must be documented, and chain-of-custody records must reflect all aspects of exposure to the environment (temperature and length of time before placement on ice in the field, length of time on ice before final storage) and records of individuals having access.

Roles and responsibilities A successful response to a plant health event involving a criminal investigation requires extensive collaboration, coordination, and communication between numerous agencies and organizations at the local, state, federal, and potentially international level. Because the primary interests and goals of the agricultural and law enforcement communities differ in some significant ways (Table 5.2), it is important that the groups are able to work in a coordinated manner. Most states have laws requiring the reporting of any diseases of regulatory significance to regulatory officials. At the state level, the State Plant Regulatory Official (SPRO) is the highest-level plant health official and serves the State Secretary of Agriculture or State Agriculture Commissioner. In most

Comparison of objectives of agricultural and law enforcement specialists in a plant disease emergency.

Objectives of agricultural specialists

Objectives of law enforcement specialists

Damage assessment Economic impact Potential for spread Impact to market/populations Delimitated area Trace in/out or forward/back Personal safety, responders, and public Outreach, education, public information Containment/control Evidence security/collection Stop the epidemic

Security Investigation Perimeter control Surveillance Profiling Trace in/out or forward/back Catch the perpetrator

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Education and outreach

states, the State Department of Agriculture (SDA) has the authority to conduct an agricultural investigation. Most SDAs have investigative services units to investigate cases in which plant health regulatory statutes and laws may have been violated. The SDA also has the authority to implement a 90-day stop movement order on plant materials and to implement quarantines with the assistance of local law enforcement and/or the National Guard. The federal plant regulatory authority belongs to the USDA APHIS Plant Protection and Quarantine unit (PPQ). In each state, the State Plant Health Director (SPHD) is an APHIS employee and has the highest level of federal authority for that state. The SPHD and the SPRO work together to leverage state and federal roles and authorities in a complementary manner to respond optimally to an event. The SPHD has the authority to implement a local mitigation measure (quarantine, crop destruction, sanitation, etc.) as an Emergency Action Notice. Agriculture diagnostic laboratory testing is conducted by university plant clinics or SDA laboratories, usually members of the NPDN, which coordinates and collaborates with the APHIS National Identification Service, the national confirmatory authority. However, evidentiary samples collected by law enforcement will be analyzed by labs that have been vetted to handle evidence. If the intent of a criminal act is unknown, a number of law enforcement agencies will participate in the response until the lead agency can be identified. The Federal Bureau of Investigation (FBI) is designated as the lead authority for the investigation of domestic terrorism, as outlined in Homeland Security Presidential Directive/ HSPD-5. However, the USDA’s Office of the Inspector General will be the lead agency for nonterrorist criminal acts involving agriculture. The Coast Guard, along with DHS CBP and Immigrations and Customs Enforcement agencies, were assigned authority for incident management and resource coordination. Regardless of which

agency serves as the investigative lead, coordination with the response and recovery agencies will be crucial for the preservation of evidence, both microbial and traditional.

Education and outreach The discipline of microbial forensics was purposefully expanded, following the dissemination of the anthrax letters in 2001, with the incorporation of new and more discriminatory scientific technologies. The US homeland security community recognized the need for a broad capability in forensic microbiology including pathogens of humans, animals, and plants (Fletcher et al., 2006). Because new homeland security initiatives must be implemented by capable, well-trained professionals, that capability must include provision for the education of young scientists and for training of those already working in homeland security roles. New career roles require scientists trained and experienced in both agricultural and forensic sciences, and both knowledgeable and appreciative of the concerns of homeland security. Traditional academic units (i.e., Departments of Plant Pathology and similar units) at numerous US universities as well as at least one medical school have developed new coursework at the graduate and/or undergraduate levels on biosecurity, agricultural biosecurity, plant health, and plant biosecurity. Although these programs introduce students to important new issues in plant health, they are limited in coverage of related security areas. An ideal training program for agricultural forensics would provide both a strong footing in agricultural sciences (available in existing, traditional strong programs) and substantive new coursework and applications in forensic sciences and homeland security. Because securityfocused careers in the FBI, the DHS, the Central Intelligence Agency, and even in the USDA’s regulatory agency, APHIS, are generally unfamiliar to students, it is important also to provide

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opportunities for them to learn about these careers through interactions with agency personnel at meetings and seminars, and through internships in which students experience agency operations and receive hands-on experience. A program that incorporates all of these elements has been established at the National Institute for Microbial Forensics & Food and Agricultural Biosecurity (NIMFFAB) at Oklahoma State University (http://www.ento.okstate.edu/nimffab/). In addition to targeted educational programs for students, training and outreach to career specialists, who might be first on the scene or involved in the response, are also critical. Specific training on recognition of intentional pathogen introductions, and on the appropriate conduct of a criminal investigation (sampling, chain of custody, and site preservation), will facilitate attribution and help to assure that justice is done. Audiences targeted by NIMFFAB for training exercises include agricultural specialists, plant disease diagnosticians, Extension educators, Master Gardeners; decision makers including politicians, advisors, and intelligence analysts; and security and law enforcement officers of the FBI, DHS, and state and local law enforcement officers, regulatory officials.

Resources and infrastructure Preparedness for a criminal event involving a plant pathogen includes prevention, detection and diagnostics, response, and recovery (Fletcher and Stack, 2007; Stack and Fletcher, 2007). The responsibility for protecting US crops, rangelands, forests, and other plant resources from introduced pathogens and pests is shared by the USDA (especially APHIS-PPQ), DHS (through CBP), the National Bioforensic Analysis Center (NBFAC), the FBI, and local law enforcement.

Prevention In a prevention strategy, focus is on agents having a high probability of introduction and

establishment. Because threat characterizations and determinations of vulnerability to a specific plant pathogen and, ultimately, the risk are imprecise, prioritization is based on perceived potential to cause persistent, wide-scale damage.

Detection and diagnostics Because huge numbers and volumes of plants and plant products move through our ports and borders, we cannot completely prevent the introduction of new agents that arrive accidently or intentionally. Therefore, we must be prepared to respond to the introduction of pathogens that threaten our plant systems. The US principal capabilities in plant pathogen identification and disease diagnostics center on the NPDN, an interconnected network of plant disease diagnostic laboratories, generally one per state. In 2002, these formerly independent laboratories, affiliated either with the state’s Land Grant University or SDA, were organized (as called for in HSPD-9 with joint authority to USDA (lead) and DHS (supportive)) into a highly effective and coordinated network that works with APHIS to monitor, diagnose, and report plant diseases in the US (Stack et al., 2006). The NPDN has developed a triage system for rapid and accurate diagnosis of introduced plant pathogens and insect pests (Stack et al., 2006, 2014). Diagnostic data collected at the network labs are submitted to a national database, and tools for data and syndromic analyses are currently under development to enhance the usefulness of the collected data. Our surveillance and detection systems vary significantly with the plant system, target pathogen or pest, and geographic region. Because surveillance usually targets specific agents of concern, programs are concentrated in “at-risk” areas. For some plant systems, industry also conducts effective surveillance programs and provides data to APHIS. However, new unmanned systems technologies offer sensing and tracking capabilities across broad geographic regions.

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Gaps

Response Response to plant disease outbreaks resulting from new pathogen introductions is a responsibility of USDA APHIS, under the authority of the Plant Protection Act of 2004, providing leadership for a coordinated response that often includes APHIS-led rapid deployment teams, state departments of agriculture, industry, and NPDN labs. Response elements include surveillance, epidemic delimitation, application of disease control or management strategies, and other actions to minimize both spread and damage. Forensic capability is another important response element in cases in which intentional introduction is suspected. Bioforensic analyses for a number of human and animal high consequence biological agents have been developed, but fewer similar bioforensic analyses/assays exist for plant pathogens. The need for this capability is now well recognized and efforts are moving forward through the development of new assays, particularly real-time PCR protocols, by APHIS and the USDA Agricultural Research Service (ARS), the DHS NBFAC and the NIMFFAB at Oklahoma State University (Fletcher et al., 2010). Recovery is intended to restore preevent status or to establish a new, but stable, status. Effective recovery, which must include both short- and long-term plans, generally focuses on local- and system-level issues and considers ecological impacts, production declines, and downstream effects on transportation systems, trade, market reentry, and replacement systems. The National Plant Disease Recovery System (NPDRS), mandated by HSPD-9 issued in February 2004, is managed by the USDA Agricultural Research Service (ARS). NPDRS has involved other federal agencies (e.g., APHIS and the National Institute of Food and Agriculture, NIFA), state departments of agriculture, scientific societies, and universities in the development of national recovery plans for the Select Agents and other plant pathogens of high consequence. The

recovery plans, which support the efforts of USDA and others to prepare for and recover from new plant diseases in the US, include brief reviews of key disease and pathogen features, disease impacts, actions needed for management and recovery, resources and scientific expertise available, and gaps in research, extension, and education. At the time of this writing (August 2018), more than 20 plant disease recovery plans have been completed. McRoberts et al. (2016) reviewed the evolution of the process for selecting and prioritizing plant diseases for recovery plans, including the concept of generic recovery plan templates for groups of pathogens and diseases with similar biological characteristics.

Gaps Future forensic plant pathologists, who may arise not only from within the discipline of plant pathology but also from related disciplines such as microbiology, molecular biology, and genetics, must accommodate the stringent needs and requirements of forensic science. We are currently adapting some of the existing tools, knowledge, and resources in plant pathology, which were developed for peaceful purposes and natural disease outbreaks. However, targeted new technologies are still needed. It is not enough to identify a pathogen to genus and species, we also must discriminate among highly similar pathogen strains. Secure, curated collections of pathogen reference strains are a critical resource required as a genotypic and phenotypic baseline against which new strains, variants, and mutants are compared. We need to know the degree of uncertainty of our test results. For many plant pathogens, detection and identification tools have not been optimized, standardized, or validated. Some traditional methods still in use, such as host range studies and use of sets of “differential” cultivars, are laborious. Tools based on DNA typing and genomics are highly promising, but

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new, rigorous, and reliable analytical methods are needed. Priority should be given for development of technologies applicable to high-priority plant pathogens, such as those on the “Select Agent” list (Table 5.1), for multiplex tests, and for assays that are portable and rapid. We need to better understand the mutation rates of threatening pathogens in natural settings and in culture, and how they affect a forensic investigation. It is important also to better understand the microbial communities that make up natural environments (microbiomes and phytobiomes) and influence sample characterization. New models are needed to couple scales of atmospheric transport (Schmale and Ross, 2015) and technologies with unmanned systems need to move toward realtime, onboard detection methods that drive smart, autonomous tracking of agents. There continues to be a need for education and training at several levels. Bright, well-trained scientists having experience in both plant pathology and forensic sciences are needed to fill new positions in federal agencies. Yet few graduate programs provide coursework relevant to both disciplines. Although existing training programs for plant disease diagnosticians and for extension personnel and law enforcement officials are excellent, few address law enforcement issues. Security and law enforcement training, similarly, rarely provide exposure to agricultural issues and threats. More training opportunities are needed in which law enforcement and agricultural experts are brought together to address not only the scientific aspects of an incident but also the unique roles and responsibilities of various agencies and responders so that actions at the crime scene are seamless and that appropriate follow-up occurs.

Summary Forensic plant pathology combines elements of a wide range of disciplines. The targeted stakeholders of forensic plant pathology are members of the law enforcement and security

communities, whose immediate goals are to identify the source of an intentionally introduced pathogen and to attribute responsibility to the perpetrator(s) so that they are brought to justice and to serve as a deterrent against the commission of additional attacks or crimes. For this emerging discipline to function optimally, the law enforcement community must effectively communicate its needs to plant pathologists. Similarly, forensic plant pathologists must design their work based on regular interaction and communication with members of the security community, so as to assure its relevance and utility in solving real problems.

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