Forensic Plant Pathology

89

Microbial Forensics. DOI:

© Elsevier Inc. All rights reserved.2011

10.1016/B978-0-12-382006-8.00007-4

Jacqueline Fletcher,a Neel G. Barnaby,b James P. Burans,c Ulrich

Melcher,d Forrest W. Nutter Jr.,e Carla Thomas,f and

Francisco M. Ochoa Coronaa

aDepartment of Entomology and Plant Pathology, National Institute for Microbial Forensics

and Food and Agricultural Biosecurity, Oklahoma State University, Stillwater, OklahomabFBI Laboratory, Quantico, Virginia

cNational Bioforensics and Analysis Center, Ft. Detrick, MarylanddDepartment of Biochemistry and Molecular Biology, Oklahoma State University,

Stillwater, OklahomaeDepartment of Plant Pathology, Iowa State University, Ames, Iowa

fDepartment of Plant Pathology, University of California, Davis, California

INTRODUCTION

Plant resources in the United States, which include crops, forests, range, nurs-

eries, and orchards, as well as natural and landscaped spaces, are essential for

human and animal life. In addition to providing food, feed, fiber, and recrea-

tional opportunities they harness sunlight energy, utilize carbon dioxide, and

recycle oxygen. Plants are affected naturally by a host 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, and trigger or suppress their gene activity. The science and practice

of plant pathology are targeted to the prevention, detection and diagnosis,

response, and recovery from such naturally induced disease outbreaks.

Heightened biosecurity concerns in the early 2000s brought focus to the pos-

sibility that crops and other plant resources could be targeted directly by indi-

viduals or groups motivated to cause harm. Intentional targeting of plants

by the release of significant pathogens could not only reduce crop yield and

quality, but also could erode consumer confidence, affect economic health

and the environment, and possibly impact human nutrition and interna-

tional relations (1–3). Since that time a number of countries have imple-

mented steps to enhance agricultural biosecurity. In the United States, new

Forensic Plant Pathology

CHAPTER 7

CHAPTER 7: Forensic Plant Pathology90

programs in microbial forensics and criminal attribution have strengthened

national security capabilities (4).

NATURALLY CAUSED VERSUS INTENTIONAL INTRODUCTION?

Farmers, foresters, and other plant producers know that the vast majority of

plant disease outbreaks are incited through sequences of natural events. In

most cases, a familiar set of diseases for any given crop will appear repeat-

edly in a given location, depending upon weather and cropping conditions.

However, even an unfamiliar set of symptoms is unlikely to cause alarm—a

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

or—more likely—in combination, are most likely to trigger a consideration

that a disease should be examined more closely, and that a criminal investiga-

tion is appropriate (5,6). Factors such as a new geographical location (disease

not seen in this area before), absence of an insect vector required for natural

introduction, presence of a pathogen not seen before in this location, unu-

sual pattern of disease in the field, 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.) are all potential indicators of human involvement in a pathogen release.

To assist law enforcement personnel in determining if an agricultural crime

has occurred, a decision tool was developed (6) in which criteria were

assigned weights and values to assess the probability of intent. An accom-

panying worksheet and fact sheet aid inexperienced users to apply the tool.

Evaluations in both natural and intentional field settings in Oklahoma show

promise for the utility of the tool in a field investigation to support decision

making related to criminal activity (Figure 7.1).

HISTORY OF AGRICULTURAL BIOWEAPONS

Motives for intentional plant pathogen introduction could include eco-

nomic 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

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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 into Florida

were likely to have occurred due to the illegal importation of citrus planting

stock from canker-affected countries; those responsible probably knew that

bringing the plant stock into the United States was illegal, but it is unlikely

that they knew that the plant pieces carried bacterial inoculum.

The history of state-sponsored programs to develop and weaponize biologi-

cal agents for use against agricultural targets is well documented (3,7–9). The

Germans are believed to have used biological weapons in World War I against

History of Agricultural Bioweapons

FIGURE 7.1

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 fleld information and test results inform a flnal

diagnosis. A “presumptive diagnosis” is sometimes made when circumstances require a quick response

(before or in the absence of conclusive diagnosis), allowing responders to act based on the evidence at

hand until a deflnitive diagnosis is completed.


the United States, inoculating horses with Burkholderia mallei, which causes

the disease glanders (8). During and after World War II, research was con-

ducted on the efficacy of various bioagents, optimal dissemination methods,

and defensive countermeasures. The United States, Russia, and other coun-

tries are known to have generated weapons against numerous crop species,

including corn, rice, wheat, potatoes, soybeans, sugar beets, and cotton (8,9).

Because most antiplant biological weapons are not harmful to humans and

animals, they are therefore safer than zoonotic pathogens to handle, develop,

and deploy. In most state-sponsored programs that developed biological

weapons, they were considered to serve more as deterrents than as actual

offensive weapons (9). However, in the wake of the 2001 anthrax mailings,

the use of biological weapons for nonstate-sponsored terrorism was brought

to the forefront. An attack on a nation’s agricultural systems in the furtherance

of political or social objectives, known as agroterrorism, was suddenly consid-

ered a real possibility. Because the goals of a terrorist group or lone individual

are often different from those of a nation, there is no need to reproduce an

extensive bioweapons program. Simple introduction of a foreign disease agent

to a nation’s agricultural enterprise could produce economic destruction or

panic in a population as confidence in the food supply is lost (2,7,10).

THE NEED FOR FORENSIC PLANT PATHOLOGY

If plant pathogens or their products are used deliberately to cause social or eco-

nomic damage or are introduced inadvertently by illegal actions, law enforce-

ment officials are responsible for determining the source, method, and time of

the introduction and for identifying those responsible by forensic investigation

and analysis (11–17). Forensic science provides scientific analytical support for

the ultimate goal of attribution of a criminal act (11–13,18,19). The significant

legal ramifications resulting from criminal attribution and prosecution neces-

sitate higher degrees of scientific validation and stringency than those normally

used in disease diagnosis and plant pathogen identification (18,20).

The ideal bioforensic investigation will support the identification and charac-

terization of a specific microbe, determinations of how the microbe was pro-

duced, and reconstruction of its method of introduction, thereby providing

scientific data that will be useful to investigators to link it to the perpetrator(s).

The bioforensic investigation should consist of a collection of defined and vali-

dated techniques that minimize the time between on-site sample collection and

arrival at a forensics laboratory and the time required for controlled laboratory

analysis. It may be easier to generate data that an investigator can use to estab-

lish exclusion (that a particular pathogen or person is not involved in the inci-

dent) than absolute attribution (evidence that uniquely associates a particular

pathogen or person to the incident).

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Although a subdiscipline of forensics targeted specifically toward microbial

pathogens and toxins associated with bioterrorism and biocrimes involv-

ing humans and animals has been developing over the past several years, few

specific methods or standard operating procedures (SOPs) have been devel-

oped and validated rigorously for application to plant pathogens. The emerg-

ing science of forensic plant pathology requires the adaptation and validation

of protocols for crime scene sampling, evidence handling, laboratory testing,

and analysis. As plant pathogen forensics takes shape, existing methods, SOPs,

and protocols are being assessed, standardized, and validated so that their use

will be defensible in a criminal investigation. Plant pathologists and forensic

scientists (especially those in microbial forensics) are working together closely

in both group environments (such as the American Phytopathological Society’s

Microbial Forensics Interest Group) and in small collaborative projects with the

National Bioforensics Analysis Center (NBFAC).

PATHOGEN DETECTION AND DIAGNOSTICS

Detection of a microbe in a plant sample by observation of disease symptoms or

pathogen signs or by a molecular assay establishes that an organism is present but

implies nothing about a causative role for that microbe in the disease. Detection

technologies based on symptoms are relatively simple, but 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,

causing different symptoms in each, or act synergistically with another pathogen,

producing a distinctive and sometimes more severe disease than either pathogen

alone (21–23). Plant disease diagnosis is establishment of the cause of observed

damage, generally accomplished by a combination of careful observations of plant

and pathogen growth, signs and symptoms, soil, water and environmental con-

ditions, seasonality, host and pathogen diversity, epidemiological data, and sero-

logical, DNA- or RNA-based assays. For new diseases, an additional requirement is

the fulfillment of Koch’s postulates (24). In the past three decades, serological and

nucleic acid-based 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, Figure 7.1) (21), a service frequently offered by plant diag-

nostic clinics and used at the farm level for making crop management decisions.

Presumptive diagnosis is insufficiently rigorous for applications in agricul-

tural biosecurity and forensic plant pathology, in which sample handling fol-

lows a chain of custody and each sample has a legal identity. Diagnostic and

detection procedures for agricultural biosecurity and forensics should include

multiple methods: light and/or electron microscopy, biological assays (cultur-

ing, indexing, and mechanical transmission), and serological and molecular

tests (25). The number of methods applied in a given case will depend on the

Pathogen Detection and Diagnostics


pathogen type, the availability of validated methodologies, and the genomic

stability of the pathogen (26).

Diseases reported most often are those occurring in plant populations that

have monetary value to humans: crops, orchards and vineyards, nurseries,

forests, rangelands, or ornamental landscapes. Also of concern are pathogens

that slip across a nation’s borders (ports of entry) during international trade

of produce, bulbs, ornamentals, seeds, and wood or other biological products,

or that are found at quarantine transitional facilities or mail centers. Cases of

biocrime or agroterrorism also would require forensic analysis. In all cases,

rapid detection is critical to effective response and timely mitigation (27,28).

Symptomatology alone is too variable for reliable diagnosis. Data from

biological assays or indexing can be highly accurate, but also costly, time-

consuming, and unsuitable for high throughput (27). ELISA, polymerase

chain reaction (PCR) (and sequence validation), and microarrays allow rapid

and sensitive detection and timely decision making (27,29–32). ELISA and

PCR are economical, and ELISA allows high numbers of predetermined tests

to be processed. Although not high throughput, PCR, real-time PCR, and

their variants provide high sensitivity with limited capability for multiplex

applications (27,28,30–32). The high sensitivity of PCR makes it the preferred

method for samples collected out of season or carrying pathogens in low tit-

ers. Microarray sensitivity is comparable to that of ELISA and for the method

can provide high throughput and high specificity (29).

Despite recent impressive advances in diagnostic technologies, accurate and

timely plant disease diagnosis—in the end—is a human interpretation of a

preponderance of evidence. No technology can replace the hands-on experi-

ence of a diagnostician, information available from databases and journals,

and consultation and validation with external laboratories (22).

EPIDEMIOLOGY IN FORENSIC INVESTIGATION

Plant disease epidemiology can provide objective, quantitative data, data

analyses, and science-based data interpretation for the attribution of bio-

crimes involving plant pathogens (7,33–35). A critical early decision during

a new plant disease incident is whether the pathogen was introduced deliber-

ately (i.e., a biocrime). The integration of global positioning systems (GPS),

geographic information systems, and satellite imagery can provide valuable

data to make such decisions in near real time. A 10 10-km, high-resolution

( 1 m2) satellite image, taken as soon as an outbreak is confirmed, can pro-

vide the following forensics-relevant information:

A permanent “fixed” record of a suspect field that can be digitally stored,

retrieved, and analyzed years after the event.

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Detection and geospatial analysis of pathogen-specific anomalies in fields

of the same crop within the same 10 10-km scene (e.g., what other

fields are likely to be affected and should be examined). These analyses

can help investigators determine if the incident was a natural event or a

deliberate attack.

Within-field anomalies (such as primary and secondary disease foci)

can be geospatially referenced and analyzed to determine if their

spatial pattern is indicative of a natural or deliberate introduction.

Ground crews could then be directed to sample at the exact GPS

coordinates of primary disease foci to assess whether the pathogen

genetic structure is typical or atypical of the population (i.e., whether

the pathogen is a natural population or an artificial mixture of

multiple biotypes).

Analysis of spatial patterns of disease foci using spatial statistics applied

at within-field and multifield scales. Precise GPS coordinates for the

epicenters of primary disease foci could inform ground investigators

where to look for physical evidence of a deliberate introduction. For

example, image intensity contour maps generated using ArcGIS (ESRI,

Redlands, CA) were used to locate the exact GPS epicenter of disease

foci of Asian soybean rust (Figure 7.2). Such maps depict areas of

lower image intensities (i.e., crop canopies showing severe soybean

rust symptoms in the center of the disease focus) relative to areas with

higher image intensities (healthier areas of the crop canopy). Using

the contour map method, the location of nine focal epicenters was

predicted within 1.5 0.92 m of the actual locations where soybean

rust point sources were introduced by researchers into soybean field

plots (35).

MUTATION, EVOLUTION, AND FORENSIC PLANT PATHOLOGY

Like human and other animal pathogens, plant pathogens undergo mutations

that, when they are not repaired, become variations on which selection acts to

produce evolution (7). Such mutations are at once a boon to and a problem for

the microbial forensic investigator. On the one hand, evolution means that dif-

ferences between 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,

evolution may be so rapid that genomes of the crime scene and suspect source

organisms are not identical due to changes occurring since their derivation from

their most recent common ancestor or to selection of different individuals from

the pool that is the source strain. Such differences make reliable attribution

Mutation, Evolution, and Forensic Plant Pathology


more difficult and suggest that methods beyond those based on DNA need also

be pursued in an investigation.

The considerations given earlier apply when deciding on which method to

use to compare a crime scene organism with a suspect phytopathogen. The

effects of mutation and evolution on results are strongly method depen-

dent. In important enough cases, the ultimate analysis, from which data on

all the other kinds of DNA tests can be derived, and the one recommended

for courtroom presentation, is nucleotide sequencing of the entire genome

of crime scene, suspect, and control organisms. However, in initial investiga-

tions where exclusion is the principal objective, less sensitive, but less expen-

sive, methods to survey the phytopathogen genomes are useful. These include

allele-specific PCRs, single strand conformation polymorphism, multilocus

variable number tandem repeat analysis, amplified fragment length polymor-

phism, and restriction fragment length polymorphism of PCR products.

FIGURE 7.2

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 August 27, 2006, over Quincy, Florida (35). Image

consists of 22 22 pixels, each providing 1-m2 resolution. (See Color Insert.)

97

Investigators must keep in mind that mutation and evolution do not stop

after a crime is committed or discovered. They continue as organisms con-

tinue to live and replicate their genomes. 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 genomic

analyses. Such propagation is often done in laboratory hosts whose selective

environment is sufficiently different from the original that multiple adaptive

mutations occur. Such genome changes are particularly well documented for

many phytopathogens. Many plants, particularly perennials, can harbor mul-

tiple microbes and multiple strains of individual phytopathogens. For exam-

ple, grapevines carry multiple strains distinguishable by restriction (36). The

population composition of such mixtures changes drastically during as few as

three propagation cycles. Even triply cloned isolates of bacterial phytopatho-

gens can alter their genomes drastically during prolonged passage (37).

INVESTIGATION

Forensic investigation of a plant disease outbreak requires careful assessment

of disease characteristics, sample collection, identification of the pathogen,

identification of likely pathogen sources, and attribution or exclusion of

pathogen(s) as the causal agent (33).

“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; http://npdn.ppath.cornell.edu) may become involved if

tissue samples are sent there for diagnosis (38).

Initial disease assessment should be done prior to any field disturbance and

should include the pattern of disease occurrence and any relevant or unusual

field characteristics. Forensically relevant SOPs may include collection of whole

plants, plant parts, plant swabs, soil, insect vectors, water, air samples, and/

or biological samples, such as alternate weed hosts. Documentation should

include an administrative log, a sample log, complete chain of custody, col-

lection site map(s), and detailed information on the crop, field history, and

environment. Photographs, GPS, and other aids are useful supplements to this

documentation.

What constitutes a “good” sample depends on the disease incidence, the patho-

gen, and the host. Samples should be collected from a representative number

Investigation


of disease foci (see earlier discussion), from outside the focal areas, and from

different plants and plant parts (39). Pooling samples from several sources

allows a larger proportion of the plant population to be tested and improves

the detection limit (40); positives can then be tested individually if appropri-

ate. Sampling of necrotic lesions is from their edges, as the centers may be

invaded by saprophytic microbes. Seeds are a good source for seed-borne path-

ogens, whereas underground stems and tubers are suitable for other pathogens.

Specialized pathogen structures, such as galls or tumors, may also be collected.

Sampling for pathogen detection (i.e., presence or absence) requires different

sampling patterns and sample size than that to determine disease incidence

or severity. Presence–absence data can be more important than incidence or

severity data for forensic purposes, for example, to determine the geographi-

cal 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 applica-

tions, disease incidence or severity data will be needed to develop spatial dis-

ease intensity maps to identify the potential point(s) of inoculation.

Sample integrity and security must be preserved during collection, movement,

storage, and analysis (41). Storage conditions must be documented, and

chain-of-custody records must reflect all aspects of exposure to the environ-

ment and records of individuals having access.

ROLES AND RESPONSIBILITIES

A successful response to a plant health event involving a criminal investiga-

tion 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 signficant ways

(Table 7.1), 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 regu-

latory 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 states, the State Department of Agriculture

(SDA) has the authority to conduct an agriculture investigation in the field.

Most SDAs have investigative services units that will investigate cases where

plant health regulatory statutes and laws may have been violated. The SDA

also has the authority to implement a 90-day stop movement on plant mate-

rials and to implement quarantines with the assistance of local law enforce-

ment and/or the National Guard.

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The federal plant regulatory authority belongs to the U.S. Department of

Agriculture (USDA) Animal and Plant Health Inspection Service (APHIS)

Plant Protection and Quarantine (PPQ). In each state, the State Plant Health

Director (SPHD) is an APHIS employee and has the highest level of fed-

eral authority for that state. The SPHD and the SPRO work together to lev-

erage 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 clin-

ics or SDA laboratories, usually members of the National Plant Diagnostic

Network (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 those

laboratories that have been vetted to handle evidence.

Initially, the intent of the criminal act may be unknown, therefore requiring

multiple law enforcement agency participation 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 criminal acts involving agricul-

ture of a nonterrorism nature. The Department of Homeland Security (DHS)

agencies Customs and Border Protection (CBP), Immigrations and Customs

Enforcement, and Coast Guard were assigned authority for incident management

Roles and Responsibilities

Table 7.1 Comparison of Objectives of Agricultural (Ag) and Law

Enforcement (LE) Specialists in a Plant Disease Emergency

Ag Objectives LE Objectives

Damage assessment Security

Economic impact Investigation

Potential for spread Perimeter control

Impact to market/populations Surveillance

Delimited area Profiling

Trace in/out or forward/back Trace in/out or forward/back

Personal safety, responders and public Catch the perpetrator

Outreach, education, public information

Containment/control

Evidence security/collection

Stop the epidemic


and resource coordination. Regardless of which agency serves as the investiga-

tive 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

dissemination of the anthrax letters in 2001, with incorporation of new and

more discriminatory scientific technologies. The U.S. Homeland Security

community recognized the need for a broad capability in forensic microbiol-

ogy, including pathogens of humans, animals, and plants (7). Because new

homeland security initiatives require capable, well-trained professionals to

carry them out, that capability must include provision for the education of

young scientists and for training of those already working in homeland secu-

rity roles.

New career roles require scientists trained and experienced in both agricul-

tural and forensic sciences, and both knowledgeable and appreciative of

the concerns of homeland security. Traditional academic units (i.e., depart-

ments of plant pathology and similar disciplines) at several U.S. universities

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 agri-

cultural sciences (available in existing, traditional strong programs) and sub-

stantive new coursework and applications in forensic sciences and homeland

security. Because new security-focused careers in the FBI, the DHS, the Central

Intelligence Agency, and even in the USDA’s regulatory agency, APHIS, are

unfamiliar to students, it is important also to provide opportunities for them

to learn about these careers through (i) interactions with agency personnel

at meetings and seminars and (ii) internships in which students experience

agency operations and receive hands-on experience. A program that incor-

porates 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 out-

reach 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 investi-

gation (sampling, chain of custody, and site preservation) will facilitate attri-

bution and assure that justice is done. Audiences targeted by NIMFFAB for

training exercises include agricultural specialists, plant disease diagnosticians,

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extension educators, Master Gardeners, and security and law enforcement

officers of the FBI and the DHS, as well as state and local law enforcement

officers, regulatory officials, and others.

RESOURCES AND INFRASTRUCTURE

Preparedness for a criminal event involving a plant pathogen includes preven-

tion, detection and diagnostics, response, and recovery (42,43). The responsibil-

ity for protecting U.S. crops, rangelands, forests, and other plant resources

from introduced pathogens and pests is shared by the USDA (especially the

APHIS-PPQ), the DHS (through CBP), and the NBFAC, the FBI, and local law

enforcement. In a prevention strategy, focus is on agents having a high proba-

bility 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.

Because huge numbers and volumes of plants and plant products move

through our ports and borders we cannot completely exclude the introduction

of new agents that arrive accidentally or intentionally, and we must be prepared

at all times to respond to the introduction of pathogens that threaten our plant

systems. The principal capabilities of the United States in plant pathogen identi-

flcation and disease diagnostics center in 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 by the USDA into a highly effective and coor-

dinated network that works with APHIS to monitor, diagnose, and report plant

diseases in the United States (38).

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.

Diagnosis is provided primarily by the NPDN, which has developed a triage

system for rapid and accurate diagnosis of introduced plant pathogens and

insect pests (38). The NPDN sends diagnostic data collected at network labo-

ratories to a national database; tools for data and syndromic analyses are cur-

rently under development to enhance the usefulness of the collected data.

Response to plant disease outbreaks resulting from new pathogen introductions

is a responsibility of USDA APHIS, which provides leadership for a coordi-

nated response that often includes APHIS-led rapid deployment teams, SDAs,

industry, and NPDN laboratories. Response elements include surveillance,

Resources and Infrastructure


epidemic delimitation, application of disease control or management strate-

gies, 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 devel-

oped, but few 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 by APHIS and Agricultural

Research Service (ARS), the DHS NBFAC, and the NIMFFAB at Oklahoma

State University (44).

Recovery is intended to restore pre-event status or establish a new, but stable, sta-

tus. Effective recovery, which must include both short- and long-term plans, gen-

erally 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 Homeland Security Presidential Directive 9

(HSPD-9), is managed by the USDA ARS. NPDRS has involved other federal

agencies [e.g., APHIS and Cooperative State Research, Education, and Extension

Service (now National Institute of Food and Agriculture)], SDAs, scientific soci-

eties, and universities in the development of national response plans for the

select agents and other plant pathogens of high consequence.

GAPS

Forensic plant pathologists may arise not only from within the discipline of

plant pathology, but also from related disciplines such as microbiology, molec-

ular biology, and genetics. These scientists must accommodate the needs and

stringent requirements of forensic science while adapting some of the exist-

ing tools, knowledge, and resources in plant pathology, which were developed

for peaceful purposes and natural disease outbreaks, as well as by developing

targeted new technologies. It is not enough to identify a pathogen to genus

and species; we also must discriminate among highly similar pathogen strains.

We need to know the confidence levels of our tests. For many plant patho-

gens, detection and identification tools are not optimized, standardized, or

validated. Some still-used traditional methods, such as host range studies and

use of sets of “differential” cultivars, are tedious. Tools based on DNA typing

and genomics are highly promising, but new, rigorous, and reliable analytical

methods are needed. Priority should be given for development of technolo-

gies applicable to high-priority plant pathogens, such as those on the “Select

Agent” list, for multiplex tests, and for assays that are portable and rapid.

We need to better understand the mutation rates of threatening pathogens in

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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 and influence sample characterization.

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 exten-

sion personnel and law enforcement officials are excellent, few address law

enforcement issues. Security and law enforcement training, similarly, rarely

provides exposure to agricultural issues and threats. More training opportuni-

ties 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 host of disciplines. The tar-

geted stakeholders of forensic plant pathology are members of the law enforce-

ment and security communities, whose immediate goals are to identify the

source of a criminally introduced pathogen and to attribute responsibility to

the perpetrator(s) so that they are brought to justice. For this emerging disci-

pline to function optimally, the law enforcement community must commu-

nicate their needs to plant pathologists effectively. Similarly, forensic plant

pathologists must design their work based on regular interaction and commu-

nication with members of the security community so as to assure its relevance

and utility in solving real problems.

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Forensic Plant Pathology