Plant /Plant Pathogen Research

This topic can be divided into two areas which may overlap: research with plant and/or plant pests and research with transgenic plants.

The two cornerstones of biosafety are risk assessment and containment. Both of these will be discussed.

Risk Assessment

A risk assessment starts with identifying all the hazards associated with the project and determining the likelihood that it will occur. A hazard is defined as a source of harm or danger. Risk is the likelihood that it will occur. So you are determining what can go wrong, the probability of that occurring and what the effects might be.

In classic biosafety risk assessment the focus was on the prevention of laboratory-acquired infections with human pathogens. Research dealing with nonhuman pathogens/transgenic plants must focus on the effect the biohazard will have on the environment and the resulting economic impact.

The risk assessment sequence is:

identify all risk factors or hazards
review guidelines, regulations, publications to determine current standard practice
assess available data, both field and laboratory
consult subject matter experts if necessary
design your mitigation strategy using administrative, work practice and engineering controls.

Questions to Ask

For Plant Pathogens/Pests (bacteria, viruses, fungi, arthropods, nematodes, etc.)

What is the nature of the agent?

Is the organism pathogenic for plants, animals, humans and how virulent (aggressive) is it?
Is it endogenous or exotic to the United States/ Mississippi?
What is the route of transmission? air-borne, water-borne, soil-borne, direct contact etc.
What is the host range? a single species of plant or many
Are local hosts present?
How communicable is it? How easily is it transmitted from plant to plant?
What is the infectious dose?
How stable is it in the environment?
What is the capacity to control or eradicate the organism if it escapes?
What is the history of the organism in other new environments?
Is there risk information already available for the organism?
What are the location and proximity of suitable hosts and the time of year of the proposed work?
What is the potential rate of local and long distance spread?
Are there any vectors present (e.g. arthropods, fungi, nematodes)?
Are there vectors present in or near the containment facility?
How persistent is the organism in the environment and what is its potential for overwintering?
What are the environmental requirements for establishment and spread?
Will it be cultured? If so, how large of a volume and at what concentration?
Will it be restricted to the lab or disseminated in a greenhouse or field plot?
What measures will be used to avoid dissemination of the organism (recombinant or wild type) beyond the laboratory or greenhouse?
What is the economic and environmental significance of potential pest organisms and their host plants?
Are there any biosecurity related risks (e.g. potential for theft and misuse)?

For a Plant:

What is the nature of the plant?

Is it endemic or exotic?
Is it a noxious weed?
Is it parasitic on other plants?
What impact will an accidental release have on agricultural production, forestry or natural environments?
Can the plant interbreed with local noxious weeds?

For Transgenic Plants/Recombinant Organisms:
A transgenic plant/organism contains a gene or genes which have been artificially inserted instead of through natural acquisition such as pollination in the case of plants or conjugation in the case of bacteria. The inserted gene sequence (known as the transgene) may come from another unrelated plant, or from a completely different species: transgenic Bt corn, for example, which produces its own insecticide, contains a gene from a bacterium. Plants containing transgenes are often called genetically modified or GM crops, although in reality all crops have been genetically modified from their original wild state by domestication, selection and controlled breeding over long periods of time. On this web site we will use the term transgenic to describe a plant which has transgenes inserted.

Describe the genes that will be introduced into the plant/microorganism. What is the source and nature of the DNA?
Is it a complete genome or a fragment of DNA?
What is the vector that will be used to transport the DNA?
What is the nature of the expressed protein? Is it a vertebrate toxin or allergen? Is it toxic to other organisms in the local environment?
How will the transgenic plants/recombinant microorganism be contained?
Will the resulting altered plant be considered a noxious weed?
Will the altered plant be able to interbreed with noxious weeds?
What impact will an accidental release have on agricultural production, forestry or natural environments?
What is the local environment? What is the nature and importance of nearby crops? Are sexually compatible wild or weedy species in the vicinity?
What are the experimental procedures for transferring: to and from greenhouse etc.
What is the recipient organism and its mode of dissemination?

Containment

Plants and plant pests rarely infect or infest healthy humans and therefore pose little direct risk to lab personnel. Some, however, can pose a significant threat to agricultural production, forests or natural ecosystems. As a result it is important that personnel working with plants and plant pests or facilities housing these organisms take steps to prevent the accidental escape of potentially damaging plants or plant pests into the environment. The containment requirements for a particular plant or plant pest are often project-specific and are determined after assessing the risk factors associated with the biology of the plant/plant pest and the impact an escape might have. The risk model in Table 1 demonstrates the general principle of requiring increased levels of containment with increasing risk of escape/establishment and the impacts on the economy, environment, agriculture, forests and trade in the event of an escape.

The U.S. government and other entities have published sets of guidelines for containment of plants and plant pests. The most common guidelines cover the following:

noxious weeds and parasitic plants
nonindigenous, phytophagous arthropods and their parasitoids and predators
plant pathogenic nematodes
bacterial plant pathogens
fungal plant pathogens
viral plant pathogens and their vectors

United States Department of Agriculture Animal and Plant Health Inspection Service
Containment Facility Guidelines for Noxious Weeds and Parasitic Plants
http://www.aphis.usda.gov/plant_health/permits/downloads/noxiousweeds_containment_guidelines.pdf

Containment Facility Guidelines for Arthropods
http://www.aphis.usda.gov/plant_health/permits/downloads/arthropod_biocontrol_containment_guidelines.pdf

Containment Facility Guidelines for Nematodes
http://www.aphis.usda.gov/plant_health/permits/downloads/nematodes_containment_guidelines.pdf

Containment Facility Guidelines for Non-indigenous Snails
http://www.aphis.usda.gov/plant_health/permits/downloads/snails_containment_guidelines.pdf

Containment Facility Guidelines for Bacterial Plant Pathogens
http://www.aphis.usda.gov/plant_health/permits/downloads/bacteria_containment_guidelines.pdf

Containment Facility Guidelines for Viral Plant Pathogens
http://www.aphis.usda.gov/plant_health/permits/downloads/plant_viral_pathogens_containment_guidelines.pdf

Containment Facility Guidelines for Fungal Plant Pathogens
http://www.aphis.usda.gov/plant_health/permits/downloads/plant_fungal_pathogens_containment_guidelines.pdf

A Practical Guide to Containment: Greenhouse Research with Transgenic Plants and Microbes. Information Systems for Biotechnology, Virginia Tech University 2001. A copy of these guidelines can be found at www.isb.vt.edu.

Descriptions of the Plant Biosafety Levels

BL1-P: This designation provides for a low level of containment with transgenic plants in which there is no evidence that the modified organism would be able to survive and spread in the environment and, if accidentally released, would not pose an environmental risk. An example would be an experiment with transgenic potato plants containing cloned genes for insect resistance obtained from primitive potato cultivars.

This biosafety level also applies to DNA-modified microorganisms that cannot spread rapidly and are not known to have any negative effects on either natural or managed ecosystems. This would include experiments using a transgenic Rhizobium strain containing Agrobacterium genes known to affect root colonization.

BL2-P: This level is assigned to experiments with transgenic plants and associated organisms, which if released from the greenhouse, could be viable in the surrounding environment but would have a negligible impact or could be readily managed. BL2-P is required for transgenic plants that may exhibit a new weedy characteristic or may be capable of interbreeding with weeds or related species growing in the vicinity. An example would be greenhouse tests with transgenic sunflower containing wheat genes able to confer resistance to the fungus Sclerotinia. This would be classified as BL2-P because sunflower is capable of hydridizing with wild relatives and becoming established as a volunteer weed.

BL2-P containment is also assigned to transgenic experiments that use the entire genome of an indigenous infectious agent or pathogen. This level is also appropriate for transgenic plant-associated microorganisms that are either indigenous to the area and potentially harmful to the environment but manageable or are exotic but have no potential for causing serious harm to managed or natural ecosystems. The BL2-P classification also applies to experiments with plant-associated transgenic insects or small animals as long as they pose no threat to managed or natural ecosystems.
Mississippi State University does not have the facilities for BL3-P and BL4-P work. These levels deal with exotic agents that can have a detrimental impact on the local environment. For more specific information, consult one of the above references.

Table 1 Suggested Criteria for Assigning Biosafety Levels

CRITERIA	TRANSGENIC PLANTS	TRANSGENIC MICROBES Exotic Non-exotic		INSECTS/ANIMALS/ASSOCIATED MICROBES WILD TYPE OR TRANSGENIC
Not a noxious weed or cannot outcross with one	BL1-P
Not easily disseminated			BL1-P
No detriment to environment		BL2-P or BL1-P +	BL1-P	BL2-P or BL1-P +
Noxious weed or can interbreed with weeds	BL2-P or BL-1P+
Contains complete genome of non-EIA*	BL2-P or BL-1P+
Contains genome of EIA	BL3-P or BL2-P+
Treated with an EIA	BL3-P or BL2-P+
Detriment to environment		BL3-P or BL4-P**	BL2-P or BL-1P+	BL3-P or BL-2P+
Involves EIA with detriment to environment	BL3-P or BL-2P+
May reconstitute genome of infectious agent in planta	BL3-P or BL-2P+
Contains vertebrate toxin	BL3-P	BL3-P	BL3-P

*EIA = Exotic Infectious Agent
** BL4-P containment is recommended only for experiments with readily transmissible exotic infectious agents whether transgenic or not, such as airborne fungi or viruses in the presence of their arthropod vectors that have the potential of being serious pathogens of major US crops.

Table taken from “A Practical Guide to Containment: Greenhouse research with Transgenic Plants and Microbes, Information Systems for Biotechnology, Virginia Tech”.

Table 2 Standard Practices for BL1-P and BL2-P Containment in Greenhouses

BIOSAFETY LEVEL 1-P	BIOSAFETY LEVEL 2-P
Discretionary access	Access limited to individuals directly involved with experiments
Personnel must read and follow instructions	Personnel must read and follow instructions
Procedures followed are appropriate for organisms	Greenhouse manual to advise of consequences: give contingency plans
Record kept of experiments in facility	Record kept of experiments and movement in/out of facility
	Containment required for movement in/out of greenhouse
Biologically inactivate experimental organisms at end of experiment	Biologically inactivate experimental organisms at end of experiment; decontaminate gravel periodically
Pest control program	Pest control program
Appropriate caging and precautions for escape of motile organisms	Appropriate caging and precautions for escape of motile organisms
	Sign for restricted experiment in progress with plant names, responsible person and special requirements

Table taken from “A Practical Guide to Containment: Greenhouse research with Transgenic Plants and Microbes, Information Systems for Biotechnology, Virginia Tech”.

Permits

Permits are required under specific USDA Animal and Plant Health Inspection Service Regulatory Authorities to import designated plants, plant products and soil into the U.S., transport designated plants and plant products through the U.S., import plant pests and biological control organisms into the U.S., and move plant pests and biological control organisms between states. View this page for more information about permitting. The FDA and EPA may also be involved in the regulation of transgenic plants. See Table 3.

http://www.aphis.usda.gov/plant_health/permits/index.shtml

Table 3 Interagency Permitting

NEW TRAIT/ORGANISM	REGULATED REVIEW CONDUCTED BY:	REVIEWED FOR:
Viral resistance in food crop	USDA EPA FDA	Safe to grow Safe for the environment Safe to eat
Herbicide tolerance in food crop	USDA EPA FDA	Safe to grow New use of companion herbicide Safe to eat
Herbicide tolerance in ornamental crop	USDA EPA	Safe to grow New use of companion herbicide
Modified oil content in food crop	USDA FDA	Safe to grow Safe to eat
Modified flower color in ornamental crop	USDA	Safe to grow
Modified pollutant degrading soil bacteria	EPA	Safe for the environment

Table taken from “A Practical Guide to Containment: Greenhouse research with Transgenic Plants and Microbes, Information Systems for Biotechnology, Virginia Tech”.