Forensic Plant Pathology

Cha pt er 7

Forensic Plant Pathology 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 a

Department of Entomology and Plant Pathology, National Institute for Microbial Forensics and Food and Agricultural Biosecurity, Oklahoma State University, Stillwater, Oklahoma b FBI Laboratory, Quantico, Virginia c National Bioforensics and Analysis Center, Ft. Detrick, Maryland d Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma e Department of Plant Pathology, Iowa State University, Ames, Iowa f Department of Plant Pathology, University of California, Davis, California

Introduction Plant resources in the United States, 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 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 possibility that crops and other plant resources could be targeted directly by individuals 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 international relations (1–3). Since that time a number of countries have implemented steps to enhance agricultural biosecurity. In the United States, new Microbial Forensics. DOI: 10.1016/B978-0-12-382006-8.00007-4 © 2011 Elsevier Inc. All rights reserved.

89

90

CHAPTER 7:  Forensic Plant Pathology

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 repeatedly 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 investigation 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, unusual 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 accompanying 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 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

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 field information and test results inform a final 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 definitive diagnosis is completed.

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 biological 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

91

92

CHAPTER 7:  Forensic Plant Pathology

the United States, inoculating horses with Burkholderia mallei, which causes the disease glanders (8). During and after World War II, research was conducted on the efficacy of various bioagents, optimal dissemination methods, and defensive countermeasures. The United States, Russia, and other countries 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 considered 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 economic damage or are introduced inadvertently by illegal actions, law enforcement 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 necessitate 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 characterization of a specific microbe, determinations of how the microbe was produced, 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 validated 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 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).

Pathogen Detection and Diagnostics

Although a subdiscipline of forensics targeted specifically toward microbial pathogens and toxins associated with bioterrorism and biocrimes involving humans and animals has been developing over the past several years, few specific methods or standard operating procedures (SOPs) have been developed and validated rigorously 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 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 conditions, seasonality, host and pathogen diversity, epidemiological data, and serological, 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 diagnostic clinics and used at the farm level for making crop management decisions. Presumptive diagnosis is insufficiently rigorous for applications in agricultural biosecurity and forensic plant pathology, in which sample handling follows 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 (culturing, indexing, and mechanical transmission), and serological and molecular tests (25). The number of methods applied in a given case will depend on the

93

94

CHAPTER 7:  Forensic Plant Pathology

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, timeconsuming, 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 titers. 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 experience 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 biocrimes involving plant pathogens (7,33–35). A critical early decision during a new plant disease incident is whether the pathogen was introduced deliberately (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 provide 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.

l

Mutation, Evolution, and Forensic Plant Pathology

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. l 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). l 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).

l

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 differences 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

95

96

CHAPTER 7:  Forensic Plant Pathology

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.)

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 dependent. 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 investigations where exclusion is the principal objective, less sensitive, but less expensive, 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 polymorphism, and restriction fragment length polymorphism of PCR products.

Investigation

Investigators must keep in mind that mutation and evolution do not stop after a crime is committed or discovered. They continue as organisms continue 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 multiple microbes and multiple strains of individual phytopathogens. For example, 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 phytopathogens 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, collection 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 pathogen, and the host. Samples should be collected from a representative number

97

98

CHAPTER 7:  Forensic Plant Pathology

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 appropriate. 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 pathogens, 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 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, disease incidence or severity data will be needed to develop spatial disease 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 environment 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 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 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 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 materials and to implement quarantines with the assistance of local law enforcement and/or the National Guard.

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 Economic impact Potential for spread Impact to market/populations Delimited 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

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 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 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 agriculture 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

99

100

CHAPTER 7:  Forensic Plant Pathology

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 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 microbiology, 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 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 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 agricultural sciences (available in existing, traditional strong programs) and substantive 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 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 assure that justice is done. Audiences targeted by NIMFFAB for training exercises include agricultural specialists, plant disease diagnosticians,

Resources and Infrastructure

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 prevention, detection and diagnostics, response, and recovery (42,43). The responsibility 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 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. 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 identification 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 coordinated 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 laboratories to a national database; tools for data and syndromic analyses are currently 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 coordinated response that often includes APHIS-led rapid deployment teams, SDAs, industry, and NPDN laboratories. Response elements include surveillance,

101

102

CHAPTER 7:  Forensic Plant Pathology

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 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, 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 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 societies, 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, molecular biology, and genetics. These scientists must accommodate the needs and stringent requirements of forensic science while adapting some of the existing 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 pathogens, 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 technologies 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

References

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 extension 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 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 host 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 a criminally introduced pathogen and to attribute responsibility to the perpetrator(s) so that they are brought to justice. For this emerging discipline to function optimally, the law enforcement community must communicate their needs to plant pathologists effectively. 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.

References   [1] R. Casagrande, Biological terrorism targeted at agriculture: the threat to U.S. national security, The Nonproliferation Rev. Fall-Winter (2000) 92–105. Available from: http://cns.miis .edu/pubs/npr/vol07/73/73casa.pdf.   [2] L. Madden, M. Wheelis, The threat of plant pathogens as weapons against U.S. crops, Annu. Rev. Phytopathol. 41 (2003) 155–176.   [3] S.M. Whitby, Biological Warfare Against Crops, Palgrave, Basingstoke, UK, 2002.   [4] American Phytopathological Society Public Policy Board. 2002. The American Phyto­ pathological Society, first line of defense. APSnet. http://www.apsnet.org.   [5] Food and Drug Administration, U.S. Department of Agriculture, and the Federal Bureau of Investigation. Undated. Criminal Investigation Handbook for Agroterrorism, U.S. Government Publication.

103

104

CHAPTER 7:  Forensic Plant Pathology

  [6] S.M. Rogers, R. Hunger, J. Fletcher, An agricultural biosecurity decision tool: Is it natural or intentional? Phytopathology 99 (2009) S110.   [7] J. Fletcher, C.L. Bender, B. Budowle, W.T. Cobb, S.E. Gold, C.A. Ishimaru, et al., Plant pathogen forensics: capabilities, needs and recommendations, Microbiol. Mol. Biol. Rev. 70 (2006) 450–471.   [8] R. Harris, J. Paxman, A Higher Form of Killing, Random House, Inc., New York, 2002.   [9] D.M. Huber, Anti-crop bioterrorism (Chapter 7), in: S.A. Amas (Ed.), The Science of Homeland Security, vol. 1, Purdue University Press, W. Lafayette, IN, 2006. [10] M. Wheelis, R. Casagrande, L.V. Madden, Biological attack on agriculture: Low-tech, high impact bioterrorism, Bioscience 52 (2002) 569–576. [11] B. Budowle, R. Chakraborty, Genetic considerations for interpreting molecular microbial forensic evidence, in: C. Doutremepuich, N. Morling (Eds.), Progress Forensic Genet. 10, Elsevier, Amsterdam, 2004, pp. 56–58. [12] B. Budowle, M.D. Johnson, C.M. Fraser, T.J. Leighton, R.S. Murch, R. Chakraborty, Genetic analysis and attribution of microbial forensics evidence, Crit. Rev. Microbiol. 31 (2005) 233–254. [13] B. Budowle, R.S. Murch, R. Chakraborty, Microbial forensics: The next forensic challenge, Int. J. Leg. Med. 119 (2005) 317–330. [14] M.A. Cooper, Label-free screening of bio-molecular interactions, Anal. Bioanal. Chem. 377 (2003) 834–842. [15] P.G. Jones, A. Gladkov, FloraMap. A Computer Tool for Predicting the Distribution of Plants and Other Organisms in the Wild, Centro Internacional de Agricultura Tropical (CIAT), Cali, Colombia, 1999. [16] F. Kaffarnik, P. Muller, M. Leibundgut, R. Kahmann, M. Feldbrugge, PKA and MAPK phosphorylation of Prf1 allows promoter discrimination in Ustilago maydis, EMBO J. 22 (2003) 5817–5826. [17] J. Fletcher, The need for forensic tools in a balanced national agricultural security program, in: Crop Biosecurity: Assuring Our Global Food Supply, Proceedings of a NATO Project, Springer Science  Business Media B.V., 2008, pp. 93–101. [18] J. Fletcher, U. Melcher, D. Luster, J.L. Sherwood, Microbial Forensics and Plant Pathogens: Attribution of Agricultural Crime, in: Handbook of Science & Technology for Homeland Security, Ed. J. G. Voeller, John Wiley & Sons, Inc. 2008. [19] B. Budowle, J. Burans, R.G. Breeze, M.R. Wilson, R. Chakraborty, Microbial forensics, in: R.G. Breeze, B. Budowle, S.E. Schutzer (Eds.), Microbial Forensics, Elsevier Academic Press, San Diego, CA, 2005, pp. 1–26. [20] B. Budowle, Defining a new forensic discipline: Microbial forensics, Profiles in DNA 6 (2003) 7–10. Available from: http://www.promega.com/profiles/601/ProfilesInDNA_ 601_07.pdf. [21] B. Nyvad, Diagnosis versus detection of caries, Caries Res. 38 (2004) 192–198. [22] F.M. Ochoa-Corona, J. Tang, B.S.M. Lebas, L. Rubio, A. Gera, B.J.R. Alexander, Diagnosis of Broad bean wilt virus 1 and Verbena latent virus in Tropaeolum majus in New Zealand, Australasian Plant Pathol. 39 (2) (2010) 120–124. [23] F.M. Ochoa Corona, B.S.M. Lebas, D.R. Elliott, J.Z. Tang, B.J.R. Alexander, New host records and new host family range for Turnip mosaic virus in New Zealand, Australasian Plant Dis. Notes 2 (2007) 127–130. [24] R.G. Grogan, The science and art of plant-disease diagnosis, Annu. Rev. Phytopathol. 19 (1981) 333–351.

References

[25] B.S.M. Lebas, F.M. Ochoa-Corona, Impatiens necrotic spot virus, in: G.P. Rao, C. Bragard, B.S.M. Lebas (Eds.), Characterization, Diagnosis and Management of Plant Viruses, vol. 4, Studium Press LLC, Houston, TX, 2007, pp. 241–243. [26] R.R. Martin, J. Delano, A.C. Lévesque, Impacts of molecular diagnostic technologies on plant disease management, Annu. Rev. Phytopathol. 38 (2000) 207–239. [27] S.A. Miller, R.R. Martin, Molecular diagnosis of plant disease, Annu. Rev. Phytopathol. 26 (1988) 409–432. [28] N.W. Schaad, R.D. Frederick, J. Shaw, W.L. Schneider, R. Hickson, M.D. Petrillo,  et al., Advances in molecular-based diagnostics in meeting crop biosecurity and phytosanitary issues, Annu. Rev. Phytopathol. 41 (2003) 305–324. [29] N. Boonham, J. Tomlinson, R. Mumford, Microarrays for rapid identification of plant viruses, Annu. Rev. Phytopathol. 45 (2007) 307–328. [30] M.F. Clark, Immunosorbent assays in plant pathology, Annu. Rev. Phytopathol. 19 (1981) 83–106. [31] E.L. Halk, S.H. De Boer, Monoclonal antibodies in plant-disease research, Annu. Rev. Phytopathol. 23 (1985) 321–350. [32] J.M. Henson, R. French, The polymerase chain reaction and plant disease diagnosis, Annu. Rev. Phytopathol. 31 (1993) 81–109. [33] F.W. Nutter Jr., Developing forensic protocols for the post-introduction attribution of threatening plant pathogens, Phytopathology 94 (2004) S77. [34] F.W. Nutter Jr., L.V. Madden, Plant pathogens as biological weapons against agriculture, in: L.I. Lutwick, S.M. Lutwick (Eds.), Beyond Anthrax: The Weaponization of Infectious Disease, Springer, New York, 2008, pp. 335–363. [35] F.W. Nutter Jr., N.S. Holah, S.K. Eggenberger, E. Byamukama, D.L. Wright, J. Marois, N. Van Rij, Emerging GPS, GIS, and remote sensing technologies for improved crop biosecurity, in: D.M. Gadory, R.C. Seem, M.M. Moyer, W.E. Fry (Eds.), Proceedings of the 10th International Epidemiology Workshop, New York Agricultural Experiment Station, Geneva, NY, 2009, pp. 116–117. [36] P. Naraghi-Arani, S.D. Daubert, A. Rowhani, Quasispecies nature of the grapevine fanleaf virus genome, J. Gen. Virol. 82 (2001) 1791–1795. [37] F. Ye, U. Melcher, J.E. Rascoe, J. Fletcher, Extensive chromsome aberrations in Spiroplasma citri strain BR3, Biochem. Genet. 34 (1996) 269–286. [38] J. Stack, K. Cardwell, R. Hammerschmidt, J. Byrne, R. Loria, K. Snover-Clift, W. Baldwin, G. Wisler, H. Beck, R. Bostock, C. Thomas, E. Luke, The National Plant Diagnostic Network, Plant Dis. 90 (2006) 128–136. [39] F.W. Nutter Jr., Post-introduction mapping of new and emerging agricultural pathogens in real-time using GPS and GIS technologies, Phytopathology 94 (2004) S130 (Abstract). [40] G. Hughes, T.R. Gottwald, Survey strategies for citrus tristeza virus disease assessment, Phytopathology 88 (1998) 715–723. [41] FBI Scientific Working Group on Forensic Analysis on Chemical, Biological, Radiological, and Nuclear Terrorism. Best practices for the collection of chemical, biological, radiological or nuclear evidence, Forensic Sci. Commun. (in press). [42] J.P. Stack, J. Fletcher, Plant biosecurity infrastructure for disease surveillance and diagnostics, in: Microbial Threats, National Academy of Sciences, Institute of Medicine, Washington, DC, 2007. [43] J. Fletcher, J. Stack, Agricultural Biosecurity: Threats and Impacts for Plant Resources, in: Microbial Threats, National Academy of Sciences, Institute of Medicine, Washington, DC, 2007. [44] J. Fletcher, D. Luster, R. Bostock, J. Burans, K. Cardwell, T. Gottwald, et al., Emerging infectious plant diseases (in press), in: J. Hughes (Ed.), Emerging Infectious Diseases, ASM Press, 2010.

105

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.