Australasian Plant Pathol. (2011) 40:335338 DOI 10.

1007/s13313-011-0052-z

Biosecurity, microbial forensics and plant pathology: education challenges, overlapping disciplines and research needs
Keynote paper APPS 2011
Francisco M. Ochoa-Corona

Received: 8 April 2011 / Accepted: 10 April 2011 / Published online: 8 June 2011 # Australasian Plant Pathology Society Inc. 2011

Abstract This article comments on aspects of functional overlapping of biosecurity, biosafety, bioterrorism, microbial forensics, plant pathology and education in a changing world showing the impact of increasing international trading, globalization and anthropogenic interventions. This commentary also introduces current research approaches such as, rapid collection devices, massive parallel systems and helicase dependent amplification applied to problems in biosecurity and microbial forensics. Keywords Biosafety . Massive parallel sequencing . Helicase dependent amplification The sciences are constantly moving forward, and it seems a number of scientists are starting to conduct scientific research in a different way, adopting novel perspectives and/or using simultaneously multiple approaches due the volume and complexity of the questions given to them. A brief examination of the time lapsed between basic discoveries in genetics, molecular genetics, and factors and biochemistry of human blood shows the time between cornerstone discoveries and inventions has been recently reduced to approximately 1/3 after comparing dates registered from 1865 to the last 60 years (Table 1) (Krmczi and Mayr 2005). Today conditions are set for the scientific community to wonder whether are they surfing on a new

F. M. Ochoa-Corona (*) Department of Entomology and Plant Pathology, and National Institute for Microbial Forensics & Food and Agricultural Biosecurity (NIMFFAB), Oklahoma State University, 127 NRC, Stillwater, OK 74078 USA e-mail: francisco.ochoa_corona@okstate.edu

wave of next generation discoveries. Therefore, it would be relevant to examine new technology needs, who will be the users, who are the associated stakeholders, which are the hot areas of research, and the technological approaches now available to use. I will briefly describe some of these aspects that relate to science, biosecurity, education, microbial forensics and plant pathology. The increase in global trade, international and domestic passenger traffic and natural atmospheric disturbances associated with climate change favor the continental and intercontinental movement of pathogenic microbes and insects (Stack 2006; Fletcher and Stack 2007) that are impacting nature and the course of natural evolution in a number of ecosystems worldwide. New introductions of unwanted organisms associated with these globalized human driven pathways can eventually lead to complex incursions of emerging diseases and pests that are costly to control and/or eradicate (Lebas et al. 2004, 2005, 2006, 2007, 2009; Ochoa Corona et al. 2007). Moreover, during the last 10 years it has become evident that changes in world-geopolitical scenarios have also increased the risk of terrorism in general and bioterrorism against relevant agricultural commodities and the associated industries. This new scenario poses risks to agriculture due to inadvertent, illegal or criminal introductions of exotic pathogens. It seems scientific attention is being diverted from studying natural processes to study anthropogenic interventions that are not only accelerating the incorporation of new unwanted elements into our social and biological systems, but also giving rise to serious threats to the economy, environment, human health, culture and heritage of nations (Fletcher and Stack 2007; Stack 2006; Stack and Fletcher 2007). As a result, the need for novel rapid collection systems, detection and diagnostics approaches and solutions is increasing.

336 Table 1 Time lapsed between relevant discoveries in genetics, immunology and biochemistry Discovery/Invention Heredity and rules of inheritance Heat-sensitive substance in the plasma responsible for lysis of erythrocytes or bacteria by heat-stable antibodies Identification of antibodies as gammaglobulins DNA structure Chemical structure of antibodies Monoclonal antibodies PCR Metagenomics Pyrosequencing Adapted from Krmczi and Mayr 2005 Scientist (s) Mendel Bordet Tiselius Watson & Crick Edelman & Porter Khler & Milstein Saiki & Mullis Handelsman et al. Edwards et al. Year 1865 1899 1939 1953 19611963 1975 1985 1998 2006

F.M. Ochoa Corona

Years between discoveries

34 40 14 810 12 10 13 8

Teaching biosecurity has proven to be a complex task, either for undergraduates or graduate students. The goal is to communicate critically unbiased, not too ecological nor too policy oriented, concepts that ponder elements from plant pathology, entomology, and microbial forensics with policy and guidelines. A new lexicon with agro, bio, terror and security wording is creating unwanted analogies, expectations and misunderstanding among scientists, governments and the public driving a number of academics to face and confront concepts believed to be universal. For example, the United Nations Food and Agriculture Organization (FAO) had defined biosecurity in a broad context. Biosecurity encompasses science, policy and regulatory frameworks (including instruments and activities) to react and manage risks associated with food, agriculture, and animal health (United Nations Food and Agriculture Organization 2002). The concept has been progressively adopted by numerous countries. However, it differs from the concept commonly applied in the U.S. where the term biosecurity is mainly associated with counter-bioterrorism measures, particularly after the terrorist attack of September 11, 2001. Hence, defining biosecurity in these terms is incomplete in both of these cases. Biosecurity goes beyond food, agriculture, and animal health and must include other environmental areas of risks such as aquatic systems, forestry, and strongly associated sectors such human health, justice and defense. An additional complication to the broad and diverse use of the term biosecurity is that in most romance languages, particularly in Spanish, French and Italian, there are no different words to differentiate between biosecurity from biosafety (The sunshine project 2003; Torres 2003). Biosafety, instead refers to the application of practices, guidelines, techniques and equipment to prevent personal, laboratory and environmental exposure to potentially infectious agents or biohazards and is paramount for the proper enforcement of biosecurity, accreditation to interna-

tional standards and performance in microbial forensics (The sunshine project 2003; Torres 2003) (Fig. 1). A new field, microbial forensics, is not overlapping with biosecurity and biosafety only, but also with microbiology and plant pathology. Microbial forensics is a scientific discipline dedicated to analyzing microbial activity as evidence for attribution purposes and/or tracing back to a point of origin. Microbial forensics performs into a rigid legal frame and demands a rigorous (accredited) and unbiased performance (Budowle et al. 2011). The methods and techniques used are similar to those applied in plant pathology, but microbial forensics demands further exhaustive testing and supportive unbiased accreditation systems. In microbial forensics maximum sensitivity is necessary as well as specificity. In order to reach high specificity and discrimination capability, all new assays need to be challenged through broad inclusivity and exclusivity panels seeking unequivocal identification of the target. The new techniques to be proposed for investigative purposes have to be robust enough to pass the strict scrutiny at the court of law. From a biosecurity perspective, bioterrorism is the deliberate threat or use of biological agents to cause harm and intimidate governments and societies by destroying or attacking life, including plant and animal life, and is perpetrated by individuals or groups motivated by political, religious, ecological or other ideological purposes (Carus 2002). Along these lines, bioterrorism can also be seen as pathway through which exotic and harmful organisms are introduced into a biological system or country (Fig. 1). Scientists in microbial forensics and biosecurity share common goals with regulatory, law enforcement and government agencies. All of them aim for continuously improving international safe-trading and minimizing the impact of unwanted movement of microbial threats into the agricultural sector. The inadvertent introduction of exotic pathogens and pests is the most common scenario but

Biosecurity, microbial forensics and plant pathology Fig. 1 Interacting elements of a biosecurity system showing natural and globalized anthropogenic pathways through which exotics are introduced into a biological system, region or country. This diagram also shows scientific output moving toward policy and biosafety systems from scientific disciplines to encompass science, policy and other instruments and activities that allow biosecurity to react, remediate and manage risks through deliverables to different sectors of the society

337

special consideration is given today to intentional criminal escalation. In both of these situations multidisciplinary teams are required to work together and in some instances their overlapping functions are creating scientific and communication challenges that are calling for innovation not only regarding sciences, but also in relation to vertical and horizontal communication among and within agencies. Enzyme linked Immunorbent assay (ELISA) and Polymerase Chain Reaction (PCR) have been dominant technologies for microbes and insect detection and identification during the last 20 years (Lequin 2005; Bartlet and Stirling 2003) and questions have been posed about whether these two consolidated technologies have reached the limit for all detection and diagnostic possibilities. Microbial forensics is focusing on achieving extreme sensitivity and specificity standards, and is also requiring easy and user friendly procedures that require little manipulation and optimization. These are important challenges for the PCR technique today in addition to greater reliability, speed and reduced costs (May 2010). Biotechnology companies are offering a variety of reliable, cost effective PCR kits and master mixes that are streamlining laboratory processing of samples. The same advances are happening in the field of immunology and immuno-detection assays. However, for scientists interested in monitoring, gene surveys or large scale collection of genes sequenced from the environment (metagenomics)

(Handelsman et al. 1998), current end point PCR, real time PCR, and sequencing technologies fall short. Pyrosequencing (Edwards et al. 2006), in its multiple formats, offers today generating shorter sequence reads than conventional sequencing procedures, however this limitation is compensated by the immense numbers of overlapping short sequence reads that can be compiled in huge sequence contigs with software assistance. In addition, this technique does not require cloning of the DNA before sequencing, removing one of the main obstacles of handling high volumes of samples. The National Institute for Microbial Forensics and Food and Agricultural Biosecurity (NIMFFAB) at Oklahoma State University is focusing on refining the detection of a pre-determined number of fungi, bacteria and viruses of national importance using Massive Parallel Sequencing (MPS) (Rogers and Venter 2005). This procedure combines pyrosequencing and reverse Blasting to generate a high volume of overlapping short sequence reads that contain sequences for all DNA sources in a sample, both host and pathogen sequences. This project is seeking to streamline the metagenomic capacity for use in detection, identification and forensics by eliminating contig assembly and simply performing reverse search of MPS databases using only the key diagnostic sequences of interest (Fletcher et al. 2009). The possibility of periodically updating the databases makes MPS dynamic and effective through time.

338

F.M. Ochoa Corona Global infectious disease surveillance and detection: assessing the challenges finding solutions. The National Academies Press, Washington, D.C. pp. 8694 Fletcher J, Schneider W, Melcher U, Ochoa Corona F (2009) Massively Parallel Sequencing (MPS) as a diagnostic and forensic analysis tool for plant pathogens. http://bioinfosu. okstate.edu/MPS_site/index.html Handelsman J, Rondon MR, Brady SF, Clardy J, Goodman RM (1998) Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products. Chem Biol 5:245249 Krmczi GF, Mayr WR (2005) Milestones in immunohematology. Transpl Immunol 14:155157 Lebas BSM, Ochoa-Corona FM, Elliott D, Tang Z, Alexander BJR (2004) Apple chlorotic leaf spot virus infection induces Plum Pox virus-like symptoms on plum in New Zealand. Acta Horticulturae 657, ISHS. pp 121-125 Lebas BSM, Clover GRG, Ochoa-Corona FM, Elliott DR, Tang Z, Alexander BJR (2005) Distribution of Potato spindle tuber viroid in New Zealand glasshouse crops of capsicum and tomato. Australas Plant Pathol 34(2):129133 Lebas BSM, Ochoa-Corona FM, Elliott DR, Double B, Smales T, Wilson JA (2006) Control and monitoring: quarantine situation of Plum pox virus in New Zealand. Bulletin OEPP/EPPO Bulletin 36:296301 Lebas BSM, Ochoa-Corona FM, Tang ZJ, Thangavel R, Elliott DR, Alexander BJR (2007) First report of Spinach latent virus in tomato in New Zealand. Plant Dis 91:228 Lebas BSM, Ochoa-Corona FM, Elliott DR, Tang J, Blouin AG, Timudo OE, Ganev S, Alexander BJR (2009) Investigation of an outbreak of Soil-borne wheat mosaic virus in New Zealand. Australas Plant Pathol 38:8589 Lequin R (2005) Enzyme Immunoassay (EIA)/Enzyme-linked immune sorbent assay (ELISA). Clin Chem 51(12):2415 2418 May M (2010) Pushing new capabilities in PCR. Bioscience Technology 34(8):1011 Ochoa Corona FM, Lebas BSM, Elliott DR, Tang JZ, Alexander BJR (2007) New host records and new host family range for Turnip mosaic virus in New Zealand. Australasian Plant Disease Notes 2:127130 Rogers Y, Venter JC (2005) Genomics: massive parallel sequencing. Nature 437:326327 Stack JP (2006) Challenges to crop biosecurity. In: Crop Biosecurity. Assuring our global food supply. NATO Science for peace and security series-C: environmental security. Pag. 15-23 Stack JP, Fletcher J (2007) Plant biosecurity infrastructure for disease surveillance and diagnostics. pp. 95106. In: Institute of Medicine. 2007. In: Institute of Medicine (eds) Global infectious disease surveillance and detection: assessing the challenges finding the solutions. The National Academies Press, Washington, D.C. pp. 95106 The sunshine project (2003) Biosafety, biosecurity, and biological weapons. Backgrounder #11, October. 22 pages. http://www. sunshine-project.org/ Torres A (2003) A new international theme: BIOSECURITY IN FOOD AND AGRICULTURE. Discussions at the FAO. http://www. animalagriculture.org/proceedings/2003%20Proc/Torres.htm United Nations Food and Agriculture Organization. Biosecurity in food and agriculture: scope and relevance. Report of the Expert Consultation on Biosecurity in Food and Agriculture (TC/BRM 03/2), Rome, Italy, September 10-13, 2002

Moreover, MPS has the potential for creating a diagnostic/ forensics assay capable of detecting common landmarks of genetically modified organisms. However, despite the attractiveness of new genomic technologies such as high throughput 454 sequencing and/ or micro-arrays, concerns about sensitivity, high costs and requirements for highly trained personnel will require further assessment before they are broadly implemented by biosecurity or border patrol agencies. In this regard, our laboratory is addressing developing PCR based diagnostic assays using a minimum of resources. To that end, we confronted the application of 5A/T rich overhangs sequences on primers, and optimal thermodynamic parameters during primer design for increased PCR yields using DNA helicase dependant amplification (HDA). HDA requires no thermocycler and allows enhanced isothermal DNA amplification. HDA is easy to use and has the potential of being applied by non specialized personnel instead of end point PCR or real time PCR detection. Citrus leprosis virus C (CiLV-C) and Tobacco mosaic virus (TMV) were used as models for developing primers having both minimal and optimal Delta G values and 5 AT-rich overhangs, features shown to enhance total PCR yields and accuracy. RT-PCR results following these procedural changes indicate the described approaches have the potential to improve the efficiency of existing PCR based assays and detection methods that frequently use suboptimal primers. However, there are yet unresolved issues that are requiring further experimentation. In general, there is potential to increase the sensitivity for detection of predetermined targets of phytopathogens for agricultural, biosecurity and microbial forensics applications with low equipment investment.

Acknowledgment The author is grateful to Dr. Astri Wayadande for reviewing this manuscript.

References
Bartlet JMS, Stirling D (2003) A short history of the polymerase chain reaction. Methods Mol Biol 226:36 Budowle B, Schutzer SE, Breeze RG, Keim PS, Morse SA (Eds) (2011) Microbial forensics, 2nd edn. Academic Press-Elsevier. 722 pages Carus WS (2002) Bioterrorism and biocrimes: the illicit use of biological agents since 1900. Fredonia Books. ISBN 1410100235 Edwards RA, Rodriguez-Brito B, Wegley L, Haynes M, Breitbart M, Peterson DM, Saar MO, Alexander S, Alexander EC, Rohwer F (2006) Using pyrosequencing to shed light on deep mine microbial ecology. BMC Genomics 7:57 Fletcher J, Stack JP (2007) Agricultural biosecurity: threats and impacts for plant pathogens. In: Institute of Medicine (eds)