progression are unique to each pathogen-host pathosystem. .... extension and education projects directed at assuring a safe, high-quality, affordable ... organization dedicated to plant health, has been a significant resource and scientific voice ...
Microbial Forensics and Plant Pathogens: Attribution of Agricultural Crime Wiley Handbook of Science & Technology for Homeland Security J. Fletcher, D. Luster, U. Melcher and J. Sherwood ABSTRACT New awareness of the vulnerability of a nation's agricultural infrastructure to the intentional introduction of pathogens or pests has led to the enhancement of programs for prevention and preparedness. A necessary component of a balanced biosecurity plan is the capability to determine whether an outbreak may have been deliberate, to trace that outbreak to its source, and to identify those responsible for it. Microbial forensics is an emerging discipline that blends elements of numerous disciplines including microbiology, forensic science, and agricultural sciences. The discipline of plant pathology offers much knowledge, as well as many technologies and resources, developed for peaceful purposes that can be adapted and applied to the development of the new sub-discipline of plant pathogen forensics. Targeted research and education programs will be needed to meet this need. The United States food system is among the safest and most secure, worldwide. Yet, because plantbased systems are essential components of the nation’s farm-to-table food supply system they may be potential targets for the deliberate introduction of pathogens or pests by those intending harm (American Phytopathological Society 2002, Casagrande 2000, Madden and Wheelis 2003, Wheelis et al. 2002, Whitby 2001, 2002). In addition, plants comprise our fiber, forests and rangelands – and may become a primary source of biofuels. Possible impacts of intentional tampering include loss of product quality or availability, economic hardship for farm and market sectors and rural communities, and distrust of U.S. produce and value-added products. National biosecurity capabilities must include scientific knowledge, technologies and procedures to effectively trace the origin, timing and site of introduction of a plant pathogen or pest, and the collection of evidence that ultimately will allow successful criminal prosecution of those responsible. The general application of science to legal practices is forensic science, and if the activity focuses on pathogenic agents it is forensic microbiology. We will discuss the application of microbial forensic principles and activities to plant-based agricultural systems, and introduce the emerging sub-discipline of forensic plant pathology. Scientific Overview Agriculture as a target The vulnerability of agricultural production systems to natural or intentional threats has long been recognized (American Phytopathological Society 2002; see also Stack, this volume). Plant-based agriculture in the U.S. includes 382 million acres in crop production, 737 million acres in forests, and 525 million rangeland acres for grazing (U.S. Environmental Protection Agency ___). Complete security for such a vast area is not feasible. Biowarfare programs against plants and livestock were sponsored by governments of several countries during a period encompassing and following World Wars I and II, and deployment of animal pathogens including the agents of glanders and tularemia have been documented (Sutton and Bromley 2005). The purposeful introduction of plant pathogens to negatively affect crop production has not been confirmed, but some government sponsored programs included plant pathogens in their bioweapons development programs (Sutton and Bromley 2005). Although most such programs were terminated after the 1972 Biological Weapons Convention, signed by 142 countries, the potential for application of agricultural pathogens for harmful purposes remains. The U.S. security community has called for focused consideration of our capabilities to attribute a purposeful event that could threaten U.S. agriculture, including our critical plant-based resources. Impacts on various sectors Millions of dollars are lost as a result of plant diseases each year, including loss of the commodity
itself as well as downstream impacts and the costs of preventative measures. Loss estimates for new or emerging diseases can vary widely. For example, possible losses due to soybean rust, calculated before its arrival into the U.S., were estimated at $240 million to $2 billion (Livingston 2004). Fortunately, actual losses to date from the establishment of this pathogen have been at the low end of this range. Introductions of foreign crop pathogens inevitably impact domestic commodity markets and futures. In some cases impacts may be positive for producers, as anticipated supply shortages can result in higher prices. However, impacts on export markets are often negative, possibly including temporary halts in exports of produce or commodities or imposition of costly phytosanitary inspections. Negative impacts may also reach the consumer, with adverse public reaction to diseased commodities and concomitant drops in food product sales. Such events tend to promote tighter information management policies during future episodes. Resolution and recovery Recovery from a new introduction of a foreign crop pathogen is generally time-consuming and costly. Site recovery may include quarantine regulations to limit spread of the new pathogen to nearby regions and states, implemented by state authorities or by the United States Department of Agriculture (USDA), although many pathogens have proven impossible to contain once introduced. Actual decontamination of agricultural biocrime sites is feasible only in a limited number of scenarios such as storage or transportation facilities. Actual field sites may remain infested or contaminated for long periods of time, depending upon the climate and biological cycle of the microbe. The USDA has invested much effort into the development of National Plant Disease Recovery System (NPDRS) Plans for high threat pathogens (http://www.ars.usda.gov/research/docs.htm?docid=14271). Each Plan includes methods, practices and recommendations for short and long term management of a specific disease. Many of the NPDRS plans identify critical gaps in knowledge that must be filled before recovery would be likely to be successful, recognizing that true recovery often requires years of research toward the development of customized disease management and control practices, with the eventual deployment of genetic host resistance, where feasible.
Detection of a deliberate event The first question facing law enforcement personnel investigating a potentially suspicious plant disease outbreak is whether a crime actually has occurred. Responsibility lies with the USDA Animal and Plant Health Inspection Service (APHIS) and the Federal Bureau of Investigation (FBI) to decide quickly whether to sequester the site and surrounding evidence as a crime scene. If an outbreak has been threatened or announced, the question becomes whether the situation is real or a hoax. Hoaxes may have impacts as serious as those of a verified disease. While obvious physical evidence, such as microbiological or laboratory materials, may be present at an agricultural scene, early clues to discriminate between an intentional plant disease introduction and one that is natural or accidental often come from spatial or epidemiological patterns or signatures. Because the recognition of anomalous patterns requires baseline data on temporal and spatial patterns of disease incidence for all diseases, research on rapid recognition of deliberate events will require a prioritization of pathogens for targeted study and a focused, large-scale research effort to map worldwide disease outbreaks and determine “normal” baseline patterns for high priority diseases (Nutter 2006, Nutter 2007). Responses to first detection Immediate challenges would ensue as a result of declaring an agricultural field or storage site as a crime scene. The extent or scope of the delineation must be estimated rapidly, and sampling strategies
developed to cover the affected areas. Genetic background information on relevant pathogens must be gathered or accessed, and a careful plan developed for preservation of evidence. Microbial sampling may be driven to larger scale by factors of pathogen epidemiology and population dynamics, but limited by practical forensic sample preservation capacities. Critical factors and limitations in sampling schemes devised for agricultural settings include the advantages of maintaining live samples, the requirement for stringent chain of custody, and the limitations of sampling time frames. Research to improve statistical pathogen sampling with applications in natural, accidental and deliberate outbreak scenarios has been conducted and disease parameters modeled based upon modes of dissemination (fungi- air and soil-borne, bacteria –wind, rain, insect vectors, viruses, vector –borne) (Madden and Hughes 1999, Hughes et al. 2004), environmental factors, cropping practices (spacing, canopy parameters) and genetic susceptibility of the host. However, more research is needed, with emphasis on high-threat plant pathogens and on specific applications for forensic investigation. The USDA APHIS, the FBI and the Department of Homeland Security (DHS) will share responsibility for providing and implementing Incident Command at an agricultural crime scene, although it is not clear whether an Incident Command structure has been fully optimized and coordinated among the three federal agencies. Clearly, all agencies would benefit from field simulation exercises focusing on deliberate introductions. Crop disease outbreak simulation exercises have been, and continue to be planned and overseen by the National Plant Diagnostic Network (NPDN) Exercises Committee (Stack et al., 2006; personal communication, Carla Thomas). While crop disease scenarios have been carried out with or by the NPDN in all 50 states, involving county, state, and federal agricultural and law enforcement officials, an exercise has yet to focus on issues and problems related to conducting a criminal investigation in an agricultural setting. Major issues for such an exercise include information management, media briefings and access, and decisions on levels of access and knowledge. Decisions on “right to know” are relevant to local, state and national interests such as County and State agricultural extension specialists and commercial crop consultants (who may be the first detectors), State Plant Health Responsible Officials (SPRO), State Plant Health Directors (SPHD), and diagnosticians at NPDN labs, who may see similar or associated diseased plant samples for diagnosis during the crisis. The site may be subjected to quarantine or regulation by APHIS Plant Protection and Quarantine (PPQ) and/or state officials, introducing a second level of issues related to site accessibility and information management. The emerging discipline of forensic plant pathology How does forensic plant pathology differ from what plant pathologists do every day? The science of plant pathology is the study of plant diseases and their causal agents, which include fungi, oomycetes, bacteria, viruses and viroids, nematodes, and a few protozoa and parasitic plants. Most plant pathology research is directed at understanding the relationships between pathogens and their plant hosts and, if involved, their insect vectors, with the goal of identifying potential disease management strategies as well as points in the disease cycle at which such strategies are likely to be most effective. Among the most important components of any plant disease management program is early diagnosis, because of its potential to limit the spread and impact of a disease. However, due to the vast and widely-distributed nature of U.S. agricultural production, plant diseases may not be noticed for days or weeks. Early diagnosis often is unlikely and when a disease is finally noticed it may be difficult to identify the site and means of initial pathogen introduction. Fortunately, the plant disease diagnostician focuses primarily on pathogen identification, and selection of the best control strategy does not usually require complete knowledge of the disease event or fine discrimination among plant pathogen strains. In contrast, a forensic scientist investigating a potential crime involving a plant pathogen in a natural or field setting will need to understand as much as possible about the crop or landscape history and management, pathogen introduction and establishment, disease development and spread, and pathogen identification. Although much knowledge, technology and expertise in plant pathogens already exists, and can be accessed in a forensic investigation, special features characterize their application in a forensics setting. Because a criminal case is likely to be tried in a court of law, where testimony is subject to vigorous
cross-examination, all evidence and information must be collected using highly standardized and validated procedures. Elements of a Strong Microbial Forensics Capability Sampling and evidence handling Although many features of sampling for investigation of microbial incidents will be similar among incidents involving humans, livestock, or plants, details will differ. After initial infection and before symptoms are evident sick people and animals are likely to move (or be moved), interact with one another, and, once symptomatic, encounter health professionals. Plant production systems are immobile and involve huge acreages and very large numbers of individual plants. Significant time may pass during pathogen establishment and spread before a plant health care professional is involved. Crime investigators, who may have little training in, or understanding of, agricultural systems, must be prepared to sample large land areas and know what constitutes a useful sample and how such samples should be collected, packaged, transported and stored. Sampling techniques must be reliable, standardized, and validated, and their limits known. Stringent chain-of-custody records are necessary to document sample handling and movement from the field to the laboratory to the courtroom. The precise identity of the pathogen, and its relationship to all known microbial relatives, becomes critically important. Careful preservation of the evidence, including molecular signatures, and its suitability for downstream re-examination, are all required. Yet, very little research has been done to establish optimal sampling strategies in agricultural settings. Needed are specific data on optimal field sampling patterns, sampling tools such as swabs, knives, bags or containers, storage temperatures (on ice, room temperature, etc) and humidity, and other parameters of sampling. The role of plant disease epidemiology The processes and time course of progression and spread of diseases are diverse and complex. Pathogen generational cycling, dispersal, transport, deposition on the host, and disease initiation and progression are unique to each pathogen-host pathosystem. Fungal, oomycete, virus, bacterial and nematode pathogens occupy unique niches within the agroecosystem, so disease initiation and spread may be influenced by completely unique combinations of macro- and microclimatic factors (air, soil and/or leaf moisture, temperature, solar radiation), arthropod vectors, and/or cropping and cultural practices. The genetic structure and ecology of pathogen populations in their crop hosts and weedy relatives play an important role in the epidemiology of each associated disease. Modern plant pathogen epidemiology focuses on the development of models that describe and predict disease based upon measurements of the above mentioned factors. To identify anomalous patterns in the introduction and spread of plant pathogens, baseline data on pertinent epidemiological factors and fully developed disease models will be necessary for each high-priority pathosystem in each major climatic region of the U.S. Additionally, the development of statistically sound sampling strategies for forensic analysis will require detailed knowledge of epidemiological factors, particularly disease dispersal, and subsequent predictive models to identify the potential scale of an agricultural crime scene. Application of baseline epidemiological data to a suspicious event could, for example, recognize that multiple widespread simultaneous disease foci for a pathogen normally spread naturally by wind represent an anomalous pattern, indicative of suspicious origins. Current microbial forensic identification and typing methods applied to plant pathogens While evaluations of the event site and disease epidemiology are critical, identifying the pathogen and discriminating among very similar strains are generally among the most important components of a forensic investigation. Analysis beyond visual or microscopic inspection will likely result in the destruction of at least part of the sample for analysis of its component macromolecules, primarily nucleic acids or proteins. If the sample is very small, choice of analysis for one macromolecule may preclude the ability to analyze the
sample for another macromolecule. For example, RNA, the infectious molecule of many plant viruses, is more labile in plant samples than DNA and requires conversion to DNA before PCR can be utilized. Test validation is necessary to assure repeatability, efficacy and reliability. To date, such standards for plant pathogen diagnostics have generally been determined individually by the developer of the test or reagent, and “affirmed” by the procedure being published in a peer reviewed journal. Only recently have steps been taken to certify labs or individuals for specific tools and procedures. Genome dynamics, phylogeny and systematics Ideally, a standard operating procedure (SOP) for identifying and typing plant pathogens would be applicable to any microbe. Unfortunately, because microbes vary greatly in gene content, in the mechanisms by which genes change sequence and in rates of sequence change, it is impossible to choose a single small set of genes shared among all microbes as targets for forensic analysis. Insertion and deletion of large or small DNA fragments and nucleotide substitutions occur at different rates in different organisms and under different conditions. Thus, while substitutions may be better indicators in some organisms, in others examination of the sizes of genomic regions may yield more reliable information. Microbial forensics research and technology have focused on identifying suspect pathogens to strain level, to establish with reasonable certainty that the pathogen that caused an outbreak came from pathogens in the hands of the suspect. Often this is done by examining one or a limited number of genetic loci. However, the recent demonstration that the entire genome of an organism can be replaced with that of another (Lartigue et al. 2007) means that it may be possible to create designer pathogens by fusing genome parts of several different organisms, possibly with some totally synthetic DNA. As a result, the forensic microbiology investigator must be able to survey the causative agent’s entire genome to identify the sources of each of its parts. Considering the diversity of microbes, SOPs must be devised for multiple narrow groupings of microbes and/or pathogenicity components, and they must address multiple genomic regions. Currently preferred methods of testing matches between DNA from a crime scene and that in a suspect’s hands are based upon nucleotide sequences. Whether they can demonstrate absolute identity between two samples depends on the rate at which that DNA changes. Because rates of change are typically high for viruses, due to the error-prone nature of replicative enzymes and the rapid replication of many viral genomes, achieving absolute virus identity is not practical. For bacteria, as demonstrated by research on the anthrax incident (Read et al., 2002), the rate of strain divergence is much lower. However, detailed investigations of genomic changes over time are available for only a handful of organisms, and it is likely that many bacterial species undergo mutation at rates higher than those seen for anthrax. Changes in genomes during laboratory propagation also have been reported (Ye et al., 1996). Better understanding of the rates of change and the mechanisms that generate them (substitutions, insertions, deletions) is needed to judge which changes might be expected in short time frames and which are highly unlikely (Jordan et al., 2002). Multiple locus approaches to microbial typing include complete genome sequencing, multiple locus sequence typing (MLST), multiple locus variable number of tandem repeats analysis (MLVA), random amplification of polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), PCR approaches targetting repeated sequences, DNA-DNA quantitative hybridization and microarray methods involving SNPs, whole genome chips, and pathochips. MLST involves determining the nucleotide sequence of a few genes conserved within the taxon being tested (Wassenaar, 2003). Absence of an expected gene in a sequencing attempt may reveal man-made recombination events. A disadvantage of this approach is that different gene sets appear to be ideal for different species. In MLVA, (for example, Monteil et al. 2007) the genome sequence of one strain is searched for simple sequence tandem repeats, and primers designed to amplify across the region of repeats are then tested on a panel of strains to reveal which loci naturally exhibit polymorphism. If enough such polymorphic loci can be identified, they will likely be distributed over the whole genome and can thus also be identifiers for recombination events. The disadvantage is that development of such typing systems takes time and would need to be developed for every potential problem
microbe. RAPD and AFLP methods (Baransell et al., 2004), though useful in individual laboratories, are difficult to standardize for routine validated use in multiple laboratories. Rep-PCR methods, to amplify repeated sequences in bacteria, are limited to certain species. Single nucleotide polymorphisms (SNPs), detectable by DNA synthesis methods from small primers, such as SNaPshot (Makridakis et al., 2001), are useful when dealing with genomes with known fixed polymorphisms, particularly for rapidly-evolving viruses having small genomes. DNA-DNA hybridization will readily reveal large replacements (insertions and deletions) in genomes. Microarray applications (Bodrossy& Sessitsch, 2004; Kingsley et al., 2002; Wilse et al., 2004), include those in which whole, or pathogenically important regions, of select agents’ genomes are arrayed on slides. Arrays useful for detecting and characterizing synthetic genomes also could be developed. SNPs can also be surveyed by microarray methods (Xiao et al., 2006). Gene expression and protein modification – Plant pathogen and plant host An organism's genome provides insight into the potential transcripts that can be produced by an organism for subsequent translation, but the final complement of transcriptional and translational products in a pathogen or host can be significantly influenced by the interaction of organisms and the environment. The field of proteomics is rapidly expanding our ability to assess the impact of such regulation. In addition, as the cost of DNA sequencing declines and more sequences become available, their analysis can inform the characterization of genome transcription, providing information about environmentally influenced gene expression. High throughput microarrays can yield transcriptional signatures for both host and pathogen. Antibody microarrays and application of mass spectrometry to small samples have permitted significant advances. Thus, obtaining a “pathoprint” of a specific disease, i.e. all the bio-molecules in a specific hostpathogen interaction, may be possible, providing greater precision in attributing or excluding a specific pathogen isolate as the cause of the disease. Informatics and data analysis Informatics plays a large role in forensic microbiological investigation, and agricultural crimes are no exception. At the level of genomes and genome sequences, although many useful comparative tools and databases have been developed, further refinement may be required. Major developments are needed at the organismal and ecosystem levels to interrelate information about pathogen distribution, vector distribution, and weather patterns into easily accessible models that allow comparison of natural vs. man-made outbreaks. Current Contributions of the Field to Homeland Security and Critical Needs Analysis Recent and current Federal initiatives The United States’ bioforensics capacity in the U.S. has been substantially enhanced in recent years. A significant new component is the Department of Homeland Security’s (DHS) National Bioforensic Analysis Center (NBFAC), established at Ft. Detrick, MD as part of the National Biodefense Analysis and Countermeasures Center (NBACC). The NBFAC mission is to be the lead federal agency in conducting forensic analysis on materials recovered following a biological attack, and to provide data from analyses to law enforcement agencies for prosecution of biocrimes. The creation of NBFAC presents an opportunity to develop a cooperative federal program addressing forensics gaps for high priority microbial plant pathogens. The Laboratory Division of the Federal Bureau of Investigation (FBI) has played a central role in the development of the emerging discipline of microbial forensics (Budowle et al. 2004; Budowle 2003). FBI scientists organized and lead a productive Scientific Working Group on Microbial Genomics and Forensics, which has brought together individuals from various federal agencies involved in national security, the National Laboratories, academia and industry. The SWGMGF identified research and training needs, set priorities, and provided information to agency administrators and funding units. The USDA also has created new infrastructure in response to concerns about the security of U.S.
plant production systems. In 2002 the USDA Cooperative State Research, Education and Extension Service (CSREES) established the NPDN, a collective network of Land Grant University plant disease and pest diagnostic facilities located in each state (Stack 2007), enhancing national agricultural security by facilitating rapid detection of introduced pests and pathogens. CSREES’ National Research Initiative, a competitive grants program, created a small new, targeted program in Plant Biosecurity that supports integrated projects to facilitate research, extension and education projects directed at assuring a safe, high-quality, affordable food and fiber for consumers in the U.S. and its international trade partners. USDA APHIS considers itself “on the job 24 hours a day, 7 days a week working to defend America’s animal and plant resources from agricultural pests and diseases”. APHIS emergency response is outlined in a National Response Plan, released in 2004 as directed in Homeland Security Presidential Directive (HSPD)-5. APHIS responsibilities, as outlined in the Plan, include: “i) Implement an integrated national-level response to an outbreak of an economically devastating or highly contagious animal/zoonotic exotic plant disease, or plant pest infestation, and ii) In response to a biohazardous event, the decontamination and/or destruction of animals and plants as well as associated facilities (e.g., barns, processing equipment, soil, and feeding and growing areas) may be required.” As of 2007, a National Response Framework is under development to replace the National Response Plan. Finally, in addition to conducting its own research relevant to plant biosecurity, the USDA’s Agricultural Research Service (ARS) is responsible for implementing the new NPDRS, which was mandated under HSPD -9 (Agricultural Biosecurity). Plant Disease Recovery Plans are being developed for plant pathogen "Select Agents" listed under 7 CFR part 331 (7 plans developed to date) and for selected other high threat agents. These plans have been developed by teams of plant pathologist experts in government, academic and private companies, under the direction of the USDA Office of Pest Management Policy. NPDRS resources are also directed at research gaps on new and emerging pathogens by the ARS National Program Staff. Recent and current academic efforts Plant pathologists in several of the USDA-supported system of Land Grant Universities, as well as researchers in other academic institutions, have contributed significant research relevant to the emerging field of forensic plant pathology, addressing issues of plant disease epidemiology, diagnostics and pathogen strain discrimination, pathogen evolution and microbial background, and basic pathogen and vector biology. Such efforts are generally independent and focused within the investigator’s specific area of expertise. The National Institute for Microbial Forensics and Food and Agricultural Biosecurity (NIMFFAB), established recently at Oklahoma State University to be a component of an overall national effort to safeguard plant and food resources, will provide a synergistic focal point to conduct interactive research, address policy issues, provide cross-disciplinary education and training and participate in outreach activities, in plant pathology and forensic sciences. Rapid progress by either independent investigators or collaborative ventures has been constrained by the extremely limited amount of targeted funding, both federal and local, for plant biosecurity and agricultural bioforensics initiatives. The role of professional societies The American Phytopathological Society (APS), a 5000-member international professional organization dedicated to plant health, has been a significant resource and scientific voice in U.S. plant pathology research, education, outreach, and policy-making. APS utilizes its strong publication house and press, interactions with related scientific societies and coalitions, and value-rich annual meetings that serve to bring plant pathology professionals together for scientific exchange. Since 2002, the APS Plant Pathogen Forensics Interest Group, composed of members from various Federal agencies, academic institutions, industry and others, has met annually in conjunction with the APS Annual Meeting to review and plan U.S. and global initiatives related to forensic plant pathology.
Future Research – Building Capacity and Science Gaps assessment and recommendations Prioritization of plant pathogens and threats With dozens of potential high value crop targets capable of hosting thousands of pathogens (Madden 2001) the amount of information required to develop a comprehensive forensics information baseline in plant pathology vastly exceeds the capacity of the federal agricultural research funding base. Highest priority pathogens must be identified and prioritized. Criteria for plant pathogen prioritization have been developed using external funding (Schaad et al. 2006), and refined in workshops sponsored by USDA and hosted by the American Phytopathologial Society, under the NPDRS. Future prioritization efforts will focus on pathogens most likely to be applied in a biocrime, requiring a unique subset of criteria and focusing on deliberate introduction scenarios. The resulting list of highest priority plant pathogens should be forwarded to agricultural and biosecurity research program leaders for their use in developing and applying research priorities for forensics applications.
Training and education Education and training are critical to a strong capability in the emerging discipline of forensic plant pathology (Fletcher et al 2006). Extant graduate plant pathology curricula are strong in molecular technology and basic sciences, both of which are necessary components of forensic science, but many have moved away from the field-based and applied plant pathology that must underpin targeted forensic investigations. A new paradigm of education, blending the disparate disciplines of plant pathology and forensic sciences, is needed to prepare a new cadre of plant pathologists having the range of expertise necessary for attribution of agricultural crime. Such a program will require close collaborations among educators, and must incorporate epidemiology, botany, biochemistry, microbial ecology and phylogeny, and other relevant disciplines. Training cannot stop with graduate student education. Extension educators and staff, crop consultants, Master Gardeners, and others who may be called first in the case of a new and threatening agricultural emergency, also must learn key indicators of intentional pathogen release and appropriate reporting steps. Law enforcement personnel need to know how to recognize an agricultural crime and respond appropriately. These education needs pose challenges at a time when the Land Grant University system, primarily responsible for education in agriculture-relevant disciplines, are suffering from financial cutbacks, reduced faculty numbers, and falling research budgets. Forensics tools and procedures Better knowledge of plant pathogen background information. Important to decisions as to whether outbreaks are man-made or natural is knowledge of whether the pathogen was present in the vicinity of the outbreak before the outbreak occurred. For that reason it is important to consolidate scattered world-wide information on the occurrence of pathogens and potential pathogens in a structured easily searchable database. Further surveys of what potential pathogens exist in natural and managed ecosystems, is also needed to obtain a complete picture of the background against which outbreaks occur. The decision tree. Deciding whether a crop disease is natural or man-made may be very challenging for law enforcement personnel. A series of criteria for making such a decision, or“decision tree,” could facilitate this crucial early step, as happened during the investigation of a tularemia outbreak among residents of Kossovo, a nation in strife from 1999-2000 (Grunow and Finke, 2002). To assess allegations that the outbreak was the result of biowarfare, a series of twelve non-conclusive questions was devised. Answers were rated to reflect characteristics such as “uncertain and indistinct” “peculiarities or suspicions”; “obvious peculiarities or indications”; “considerable peculiarities or deviations”, or “no data available.” Formulas converted the data to conclusions having a specified degree of confidence. A current NIMFFAB initiative is
to develop a similar decision tree for use in plant disease outbreaks, and to test the tree using as a model a common, endemic plant disease for which a direct comparison can be made between a natural outbreak and an intentional, man-made event (such as those commonly created for assessment of disease-resistance or pesticide efficacy among crop cultivars).
Dedicated funding for targeted research and education /training Unlike diseases of humans and production animals, those of plants are usually managed within large host populations. Since eradication of infected host plants is generally unsuccessful, most of the limited funding for plant biosecurity related research and education has targeted the development of new approaches for pathogen detection, understanding plant pathogen epidemiology, or identification of disease resistant plant material. That a plant disease could result from a criminal act has been a new perspective within the plant pathology community. The plant pathology and U.S. security communities now recognize and support the emergence of a new discipline of plant pathogen forensics, but the lack of targeted funding for research and education efforts directly related to forensics for plant diseases has constrained progress. The focused adaptation and extension of our national plant pathology capabilities to meet the needs of microbial forensic investigation will require the attention of Federal funding agency decision-makers and the creation of targeted funding opportunities for development of technologies and resources to enhance national capability to identify, trace, and attribute criminal assaults on plant-based resources. Cross-communication among plant, veterinary and human systems Although specific case details and appropriate responses will differ, the needs of the plant pathologist-forensic scientist will be very similar to those of forensic investigators in public health and veterinary sciences with respect to disease epidemiology, molecular biology and detection technologies, sample collection and handling, and other forensic tools, as both seek to determine the origin and source of an outbreak (Fletcher et al 2006). Close communication among microbial forensics investigators in all three areas will promote synergy and optimization in the use of limited resources. Encouragement of collaborative research, especially by the creation of targeted funding programs, the development of blended and forensicsfocused scientific meetings, and other cross-disciplinary activities will facilitate effective communications. References American Phytopathological Society Public Policy Board. 2002. The American Phytopathological Society, first line of defense. APSnet. http://www.apsnet.org [online]. Baransel, A., Dulger, H.E. and Tokdemir, M., 2004. DNA amplification fingerprinting using 10 x polymerase chain reaction buffer with ammonium sulfate for human identification. Saudi Med J 25, 741-5. Bodrossy, L. and Sessitsch, A., 2004. Oligonucleotide microarrays in microbial diagnostics. Current Opinion in Microbiology 7, 245-54. Casagrande, R. 2000. Biological terrorism targeted at agriculture: The threat to U.S. national security. The Nonproliferation Rev./Fall-Winter: p. 92-105. http://cns.miis.edu/pubs/npr/vol07/73/73casa.pdf. [Online.] Dallot, S, Gottwald, T, Labonne, G, Quiot, J-B. 2003. Spatial pattern analysis of Sharka disease (Plum pox virus strain M) in peach orchards of southern France. Phytopathology 93:1543-1552. Grunow R, Finke E-J. 2002. A procedure for differentiating between the intentional release of biological warfare agents and natural outbreaks of disease: its use in analyzing the tularemia outbreak in Kosovo in 1999 and 2000. Clin Microbiol Infect 8:510–521. Hughes, G, Madden, L, Gottwald, TR 2004. Strategies of sampling for detection. Phytopathology. 94:S137. Jordan, I.K., Rogozin, I.B., Wolf, Y.I. and Koonin, E.V., 2002. Microevolutionary genomics of bacteria. Theor Popul Biol 61, 435-47.
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