Response to the Upjohn Company/Asgrow Seed Company Petition 92-204-01 for Determination of Nonregulated Status for ZW-20 Squash
Prepared by: United States Department of Agriculture,
Animal and Plant Health Inspection Service,
Biotechnology, Biologics, and Environmental Protection
List of Abbreviations
BWYV Beet western yellows virus
CaMV Cauliflower mosaic virus
CMV Cucumber mosaic virus
CP Coat protein
ELISA Enzyme-linked immunoabsorbent assay
FLCP Free-living Cucurbita pepo
mRNA Messenger RNA
PRSV Papaya ringspot virus (previously watermelon mosaic virus 1)
PVY Potato virus Y
TMV Tobacco mosaic virus
TVMV Tobacco vein mottling virus
WMV2 Watermelon mosaic virus 2
ZYMV Zucchini yellow mosaic virus
Table of Contents
I. Summary 1
II. Background 3
Regulatory Authority 3
Oversight by Other Federal Agencies 5
III. Response to Comments 6
The Taxonomy of C. pepo in the United States 7
Issue 1. Will the Introduction of ZYMV and WMV2 Coat Proteins Increase the Likelihood of Creating New Plant Viruses? 9
Issue 2. Could the Introduction of Two New Virus Resistance Genes Cause Squash to Become a Weed? 16
Issue 3. Will the Virus Resistance Genes Move to FLCP Plants, and Will This Have a Detrimental Impact? 23
Issue 4. Are FLCP Plants Serious Weeds? Will Virus-Resistant FLCP Plants Be More Difficult to Control Than Virus Susceptible Plants? 25
Issue 5. Will Hybrids Between ZW-20 Squash and FLCP Plants Persist in the Environment and Become Weeds? 27
Other Comments to the Draft EA/Determination 28
IV. Analysis of the Properties of ZW-20 Squash 28
The Introduced Genes, Their Products, and the Added Regulatory Sequences Controlling Their Expression Do Not Present a Plant Pest Risk in ZW-20 28
The ZW-20 Squash is No More Likely to Become a Weed Than a Virus-Resistant Plant Developed by Traditional Breeding Techniques 35
The ZW-20 Squash is Unlikely to Increase the Weediness Potential for Any Other Cultivated Plant or Native Wild Species With Which the Organism Can Interbreed 35
The ZW-20 Squash Should Not Cause Damage to Processed Agricultural Commodities 35
The ZW-20 Squash Should Not Increase the Likelihood of the Emergence of New Plant Viruses 35
The ZW-20 Squash Should Not be Harmful to Beneficial Organisms, Including Bees 36
V. Conclusion 37
VI. References 32
VII. Appendices 38
Appendix I. Distribution of WMV 2 and ZYMV, Their Plant Hosts and Insect and Leading Cucurbit Production States
Table 1. Prevalence of WMV2 and ZYMV by State
Table 2. Host Plants of WMV2
Table 3. Host Plants of ZYMV
Table 4. Select List of Aphids That Transmit ZYMV, WMV2, PRSV, and CMV
Table 5. Acreage of Cucurbit Crops In States Containing FLCP Plants
Table 6. Results From Survey Performed by Upjohn of FLCP Plants for Plant Viruses
Appendix II. Free-Living Cucurbita pepo in the United States. Viral Resistance, Gene Flow, and Risk Assessment. Report to USDA, Biotechnology, Biologics, and Environmental Protection, by Dr. Hugh Wilson
I. Summary
Based on a review of scientific data and public comments, the Animal and Plant Health Inspection Service (APHIS) has determined that the genetically engineered, virus resistant line of yellow crookneck squash (Cucurbita pepo subsp. ovifera var. ovifera) designated ZW-20 does not represent a plant pest risk and is therefore not a regulated article under the regulations found at 7 CFR Part 340.6. As a result of this determination, permits under those regulations will no longer be required from APHIS for field testing, importation, or interstate movement of ZW-20 squashes or their progeny.
This determination by APHIS has been made in response to a petition received from Asgrow Seed Company, a subsidiary of the Upjohn Company, Inc., Kalamazoo, Michigan, dated July 13, 1992. The petition requested a determination from APHIS that the ZW-20 squash does not present a plant pest risk and is therefore not a regulated article. On September 4, 1992, APHIS announced receipt of the Upjohn/Asgrow petition in the Federal Register (57 FR 40632) and stated that the petition was available for public view. In that notice, APHIS also announced its intent to issue an interpretive ruling that the ZW-20 squash does not present a plant pest risk and would therefore no longer be considered a regulated article under its regulations. APHIS invited written comments on this proposed action, to be submitted on or before October 19, 1992. On March 22, 1993, APHIS published a second Federal Register notice (58 FR 15323) requesting additional information on eight issues raised by commenters to the first Federal Register notice. Briefly, the issues raised included the weediness potential of squash and its taxonomic relatives, the distribution of zucchini yellow mosaic virus (ZYMV) and watermelon mosaic virus 2 (WMV2) in the United States, and the likelihood of creating new plant viruses. Concurrently, APHIS commissioned Dr. Hugh Wilson of Texas A&M University, an expert in cucurbit taxonomy and ecology, to prepare a report (see Appendix II) addressing issues raised by commenters to the first Federal Register notice. On May 23, 1994, APHIS published a notice in the Federal Register (59 FR 266l9-26620) announcing the availability of an environmental assessment (EA) and preliminary finding of no significant impact (FONSI) for comment at a public meeting and for written comment during a 45-day comment period, which ended July 7, 1994. At the public meeting held June 21, 1994, only two individuals spoke, one in favor of the EA and FONSI, and one against.
The ZW-20 squash, as defined by its developer, the Asgrow Seed Company, is a squash line that is designed to resist infection by two plant viruses that frequently infect squash, namely ZYMV and WMV2. ZW-20 squash has been modified with genes that express the coat proteins of ZYMV and WMV2. Expression of these coat protein (CP) genes does not cause plant disease, but rather confers resistance to infection by ZYMV and WMV2. The introduced DNA that encodes the CP genes also has accompanying DNA regulatory sequences that modulate their expression. The DNA regulatory sequences were derived from three plant pathogenic organisms: the bacterium Agrobacterium tumefaciens, cauliflower mosaic virus (CaMV), and cucumber mosaic virus (CMV). Although the regulatory sequences were derived from plant pathogens, the regulatory sequences cannot cause plant disease by themselves or in conjunction with the genes that they regulate in these squash. With respect to A. tumefaciens, the genes that cause disease have been removed. The sequences derived from the two plant viruses are only small portions of their genomes and do not encode any pathogenic properties.
APHIS regulations at 7 CFR Part 340, which were promulgated pursuant to authority granted by the Federal Plant Pest Act (FPPA) (7 U.S.C. 150aa-150jj) as amended, and the Plant Quarantine Act (PQA) (7 U.S.C. 151-164a, 166-167) as amended, regulate the introduction of certain genetically engineered organisms and products. An organism is no longer subject to the regulatory requirements of 7 CFR Part 340 when it is demonstrated not to present a plant pest risk. Section 340.6 of the regulations, entitled "Petition Process for Determination of Nonregulated Status," provides that a person may petition the agency to evaluate submitted data and determine that a particular regulated article does not present a plant pest risk and should no longer be regulated. ZW-20 squash has been considered a "regulated article" for field testing under Part 340 of the regulations in part because ZW-20 squash has been engineered with CP genes derived from the plant pathogenic viruses ZYMV and WMV2. Field testing of the ZW-20 squash has been done under APHIS permits in 1990, 1991, 1992, and 1993, and is continuing in 1994. All field trials were performed essentially under conditions of reproductive confinement.
APHIS has determined that the ZW-20 squash does not pose a direct or indirect plant pest risk and, therefore, will no longer be considered a regulated article under APHIS regulations at 7 CFR Part 340. Permits under those regulations will no longer be required from APHIS for field testing, importation, or interstate movement of ZW-20 squash or their progeny. (Importation of ZW-20 squash [and nursery stock or seeds capable of propagation] is still, however, subject to the restrictions found in the Foreign Quarantine Notice regulations at 7 CFR Part 319.) This deter- mination has been made based on an analysis that revealed that ZW-20 squash: (1) exhibits no plant pathogenic properties; (2) is no more likely to become a weed than a virus-resistant plant developed by traditional breeding techniques; (3) is unlikely to increase the weediness potential for any other cultivated plant or native wild species with which the organisms can interbreed; (4) should not cause damage to processed agricultural commodities; (5) should not increase the likelihood of the emergence of new plant viruses; and (6) is unlikely to harm other organisms that are beneficial to agriculture, such as bees. APHIS has also concluded that there is no reason to believe that new progeny ZW-20 squash varieties bred from these lines will exhibit new plant pest properties, i.e., properties substantially different from any observed for the ZW-20 squash lines already field tested, or those observed for squashes in traditional breeding programs.
The potential environmental impacts associated with this determination have been examined in accordance with regulations and guidelines implementing the National Environmental Policy Act of 1969 (42 U.S.C. 4331 et seq.; 40 CFR 1500-1509; 7 CFR Part 1b; 44 FR 50381-50384; and 44 FR 51272-51274). An Environmental Assessment (EA) was prepared and a Finding of No Significant Impact (FONSI) was reached by APHIS for the determination that ZW-20 squash is no longer a regulated article under its regulations at 7 CFR Part 340.
The body of this document consists of the following two parts: (1) background information, which provides the legal framework under which APHIS has regulated the field testing, interstate movement, and importation of ZW-20 squash, and a summary and response to comments provided to APHIS on its proposed action during the public comment periods; and (2) analysis of the key factors relevant to APHIS' decision that the ZW-20 squash does not present a plant pest risk.
II. Background
Regulatory Authority
APHIS regulations, which were promulgated pursuant to authority granted by the Federal Plant Pest Act (FPPA), (7 U.S.C. 150aa-150jj) as amended, and the Plant Quarantine Act (PQA), (7 U.S.C. 151-164a, 166-167) as amended, regulate the introduction (importation, interstate movement, or release into the environment) of certain genetically engineered organisms and products.
Under § 340.0 of the regulations, a person is required to obtain a permit before introducing a regulated article. A genetically engineered organism is deemed a regulated article either if the donor organism, recipient organism, vector or vector agent used in engineering the organism belongs to one of the taxa listed in the regulation and is also a plant pest; or if APHIS has reason to believe that the genetically engineered organism presents a plant pest risk. Permission to conduct a field trial with an article regulated under 7 CFR Part 340 is granted when APHIS has determined that the conduct of the field trial, under the conditions specified by the applicant or stipulated by APHIS, does not pose a plant pest risk.
Before the introduction of a regulated article, a person is required under § 340.0 of the regulations to either (1) notify APHIS in accordance with § 340.3 or (2) obtain a permit in accordance with § 340.4. Introduction under notification (§ 340.3) requires that the introduction meets specified eligibility criteria and performance standards. The eligibility criteria impose limitations on the types of genetic modifications that qualify for notification, and the performance standards impose limitations on how the introduction may be conducted. Under § 340.4, a permit is granted for a field trial when APHIS has determined that the conduct of the field trial, under the conditions specified by the applicant or stipulated by APHIS, does not pose a plant pest risk.
The FPPA gives USDA the authority to regulate plant pests and other articles to prevent direct or indirect injury, disease, or damage to plants and plant products. In addition, the PQA provides an additional level of protection by enabling USDA to regulate the importation and movement of nursery stock and other plants that may harbor injurious pests or diseases. Some imported plant material must be grown under confined conditions after importation and certified as free of pests before it can be released from oversight by USDA.
An organism is not subject to the regulatory requirements of 7 CFR Part 340 when it is demonstrated not to present a plant pest risk. Section 340.6 of the regulations, entitled "Petition Process for Determination of Nonregulated Status," provides that a person may petition the agency to evaluate submitted data and determine that a particular regulated article does not present a plant pest risk and should no longer be regulated. If the agency determines that the regulated article does not present a risk of introduction or dissemination of a plant pest, the petition will be granted, thereby allowing for unregulated introduction of the article in question. A petition may be granted in whole or in part.
Section 340.6 of the regulations was published on March 31, 1993 (58 FR 17044), after publication of the initial notice of receipt of this petition. It is our intent that the interpretive ruling under which this petition was submitted utilizes standards equivalent to those for the petitioning procedure subsequently adopted. These standards include the opportunity for public comment on the petition and a reasoned consideration of the relevant scientific information and the comments.
ZW-20 squash has been considered a "regulated article" for field testing under Part 340 of the regulations in part because the CP genes were from plant viruses and the vector system used to transfer the viral CP genes was derived from A. tumefaciens, all of which are on the list of organisms in the regulation and are widely recognized as plant pathogens. In addition, certain noncoding regulatory sequences were derived from plant pathogens, i.e., from CaMV, CMV, and A. tumefaciens.
APHIS believes it prudent to provide assurance before commercialization that organisms such as the ZW-20 squash, that is derived at least in part from plant pests, do not pose any potential plant pest risk. Such assurance may aid the entry of new plant varieties into commerce or into breeding and development programs. The decision by APHIS that the ZW-20 squash is not a regulated article is based in part on evidence provided by Upjohn/Asgrow concerning the biological properties of the ZW-20 squash and its similarity to other varieties of squash grown using standard agricultural practices for commercial sale or private use. Through the end of 1993, the ZW-20 squash has been field tested under 14 APHIS permits at 46 sites in 10 States.
The fact that APHIS regulates genetically engineered organisms having plant pest components does not carry with it the presumption that the presence of part of a plant pest makes a whole plant a pest or that the plants or genes are pathogenic. The regulations instead have the premise that when plants are developed using biological vectors or material from pathogenic sources, or when pathogens are used as vector agents, they should be evaluated to assure that there is not a plant pest risk (McCammon and Medley, 1990). APHIS performs a review that allows a verification of the biology and procedures used; assesses the degree of uncertainty and familiarity; and allows the identification of any hazards, should they be present and predictable. The overall aims of APHIS' regulations in the Code of Federal Regulations at 7 CFR Part 340 are to allow for the safe testing of genetically engineered organisms under an appropriate level of oversight, and to enable any issues of potential or hypothetical risks to be addressed early enough in the development of the new organisms to allow for the safe utilization of the technology in agriculture.
A certification that an organism does not present a plant pest risk means that there is reasonable certainty that the organism cannot directly or indirectly cause disease, injury, or damage either when grown in the field, or when stored, sold, or processed. APHIS' approach to plant pest risk is considerably broader than a narrow definition that encompasses only plant pathogens. Other traits, such as increased weediness, and harmful effects on beneficial organisms, such as earthworms and bees, are clearly subsumed within what is meant by direct or indirect plant pest risk. In APHIS' regulations at 7 CFR Part 340, a "plant pest" is defined as: "Any living stage (including active and dormant forms) of insects, mites, nematodes, slugs, snails, protozoa, or other invertebrate animals, bacteria, fungi, other parasitic plants or reproductive parts thereof; viruses; or any organisms similar to or allied with any of the foregoing; or any infectious agents or substances, which can directly or indirectly injure or cause disease or damage in or to any plants or parts thereof, or any processed, manufactured, or other products of plants."
A determination that an organism does not present a plant pest risk can be made under this definition, especially when there is evidence that the plant under consideration: (1) exhibits no plant pathogenic properties; (2) is no more likely to become a weed than a virus-resistant plant developed by traditional breeding techniques; (3) is unlikely to increase the weediness potential for any other cultivated plant or native wild species with which the organisms can interbreed; (4) should not cause damage to processed agricultural commodities; (5) should not increase the likelihood of the emergence of new plant viruses; and (6) is unlikely to harm other organisms, such as bees, which are beneficial to agriculture. Evidence has been presented by Upjohn/Asgrow that bears on these topics. Upjohn/Asgrow has also presented data that ZW-20 may alter current methods used for the control of ZYMV and WMV2. In addition, because the Upjohn/Asgrow petition seeks a determination regarding new squash varieties containing the virus resistance genes, it should be established that there is no reason to believe that any new squash varieties bred from ZW-20 squash lines will exhibit plant pest properties substantially different from any observed for squash in traditional breeding programs or as seen in the development of the ZW-20 squash lines already field tested.
Oversight by Other Federal Agencies
The Environmental Protection Agency (EPA) regulates the use of pesticide chemicals in the environment. Under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136 et seq.), the EPA has the authority to regulate the development, sale, distribution, use, storage, and disposal of pesticides. The EPA has published its proposal rule for plant pesticides including an proposed exemption for viral CP produced in plants (59 FR 60495-60547). Their draft proposal has been the subject of three scientific advisory meetings. The material presented at these meetings is available from the EPA's Office of Pesticide Program's Public docket. The proposed exemption of viral CP was supported by EPA's scientific advisory panel.
The USDA Food Safety Inspection Service (FSIS) is responsible for regulation of genetically engineered meat and poultry products (59 FR 12582-83; 56 FR 67054-55). Food safety in the United States, for products other than meat and poultry, is assured by regulation under the Federal Food, Drug and Cosmetic Act (FFDCA) (21 U.S.C. 201 et seq.). The FDA's policy statement concerning the regulation of foods derived from new plant varieties, including genetically engineered plants, was published in the Federal Register on May 29, 1992, and appears at 57 FR 22984-23005. Regulatory oversight for the safety of any food or feed products derived from ZW-20 squash is under the jurisdiction of the FDA, shared with the EPA when pesticides are involved. The FDA has completed its consul- tation and has concluded that ZW-20 squash is just as safe to consume as any other squash variety. On November 2 and 3, 1994, the FDA Food Advisory Committee concurred with the process used by FDA to arrive at this position. Under the FFDCA, the EPA has responsibility for establishing tolerances or exemptions from the requirement of tolerance for pesticide residues on food or feeds, including viral CP.
III. Response to Comments
On September 4, 1992, APHIS announced receipt of the Upjohn/Asgrow petition in the Federal Register (57 FR 40632) and announced its intent to issue an interpretive ruling that the ZW-20 squash does not present a plant pest risk and would no longer be considered a regulated article under its regulations. During the 45-day comment period, APHIS received 17 comments regarding its proposed interpretive ruling in response to Upjohn/Asgrow's petition. Of the 17 comments, 7 were generally supportive of APHIS' proposed action and 10 expressed serious reservations or disapproval of it.
On March 22, 1993, APHIS published a second Federal Register notice (58 FR 15323) requesting additional information on eight issues raised by commenters to the first Federal Register notice. Concurrently, APHIS commissioned Dr. Hugh Wilson of Texas A&M University, an expert in cucurbit taxonomy and ecology, to prepare a report (see Appendix II) related to issues raised in comments to the first Federal Register notice. Of the 12 comments to the second notice, 10 (none of whom commented to the first Federal Register notice) were generally supportive of APHIS' action and two expressed serious reservations. (The latter two commenters had previously commented negatively in the first Federal Register notice.)
After the close of the official comment period, APHIS received letters from two commenters urging the agency not to approve this petition because they believed that the agency would not fulfill its requirements under the National Environmental Policy Act (NEPA). One of the letters reiterated issues previously communicated in comments to the two previous Federal Register notices. The second letter reiterated issues dealing with gene movement also previously communicated in the two Federal Register notices and expressed the opinion that if the agency decides to prepare an EA that reaches a FONSI, the availability of such documents must be announced in the Federal Register with the opportunity to comment during a minimum 30-day comment period. The letter also requested that APHIS consider holding a public hearing during the 30-day comment period because of the "considerable controversy surrounding the Upjohn/Asgrow's petition." The plant pest risk issues raised in these two letters are addressed in this determination. Besides this determination document, the agency has prepared an EA in compliance with NEPA that addresses the environmental issues regarding the widespread use of virus-resistant squash in agriculture.
Most of the supportive comments in response to both Federal Register notices were based on scientific data concerning the lack of plant pest risk presented by ZW-20 squash and its plant pest-derived components. Several commenters mentioned that the A. tumefaciens derived transformation system is well-characterized, has been used extensively, and does not present any plant pest risk. Several commenters, including State agricultural officials, said that they think that the virus resistant squash plants would not become noxious weeds. Several plant breeders and State agricultural officials mentioned that the viruses ZYMV and WMV2 cause severe losses in squash production, and that a heritable form of viral resistance would be advantageous for producers and preferable to use of chemicals or other control strategies.
Four commenters (including plant virologists) discussed in detail why they believe that transencapsidation (i.e., the coating of one viral RNA partially or completely by the CP of another virus) and the possible appearance of new plant viruses by recombination between plant encoded viral CP and endemic plant viruses would not pose a plant pest risk significantly different than could occur in natural mixed viral infections. APHIS agrees with these comments.
Eight commenters noted that pest-resistant squash plants are used and have been used in commercial production without any significant increase in weediness of squash or its sexually-compatible relatives. One commenter (a plant breeder) noted that free-living Cucurbita pepo is limited not by infection by plant viruses but by dispersal of the seeds. The range of dispersal is limited by deposition of the buoyant fruits that are usually deposited between high water marks during spring flooding. APHIS agrees with these comments.
Of the 12 comments opposed to granting this petition, 6 said that the petition should be rejected because there is no strong Federal program to which all transgenic crops should be subjected to assess and minimize their risks. APHIS disagrees. The "Coordinated Framework for Regulation of Biotechnology" (51 FR 23302-50), developed by the Office of Science and Technology Policy (OSTP), establishes a clear system for product-based coordinated reviews of the products of agricultural biotechnology. Roles are set out for APHIS, FSIS, the Environmental Protection Agency (EPA), and the Food and Drug Administration (FDA), based on the existing legal authorities of the respective agencies for oversight over particular aspects of this economic sector. This system is entirely adequate to identify and address any significant potential risks that may be posed by any of the new products of agricultural biotechnology. One comment suggested that APHIS' authority to regulate these products under the FPPA is questionable. APHIS also disagrees with this comment. Our responsibilities under the Act to protect against the introduction or dissemination of plant pests provide broad authority over any products that may have potentially significant impacts on the environment, based on the broad definition of "plant pest" in the statute.
One commenter argued that if seeds of ZW-20 squash are to be exported, the U.S. Government should address the potential adverse impacts on transgenic squash in the other centers of genetic diversity for cucurbits, Mexico and Central America. APHIS disagrees with the apparent assumption of the commenters that this determination is likely to allow unregulated commerce across national borders of the seeds of ZW-20 squash. The comment is correct, however, in suggesting that this determination does not carry with it any foreign safety presumption, since our authority and our review only extend to the borders of the United States and its territories and possessions. APHIS is in frequent contact with agricultural officials from many foreign nations, including those with interest in the cultivation of genetically engineered squashes. We are actively involved with many countries to help them develop national scientific and regulatory frameworks that will enable them to make their own scientifically credible decisions about the safety of new crop varieties.
Most countries prohibit the unrestricted importation of seeds, and many have specific requirements to address any releases of transgenic plants in their environment. We believe that the issues raised about use of ZW-20 squash in Mexico can be analyzed by the same approach used in the determination. Many crucial scientific facts explained in this determination for the United States also apply in Mexico: (1) ZYMV and WMV2 are major problems in cucurbit production in Mexico (Delgadillo et al., 1988). (2) Many of the same aphid species in Mexico are also vectors in the southern United States (see Appendix I, Table 4, data from Perring et al., 1992). (3) The common free-living cucurbit C. pepo ssp. fraterna of Mexico and northern Central America is most likely susceptible to these viruses as are all other C. pepo subspecies. (4) C. pepo ssp. fraterna or ssp. pepo is not reported to be a serious weed in Mexico (Holm et al., 1979).
The three major plant pest risk issues raised by commenters to both Federal Register notices that had reservations or disapproved of APHIS' proposed action were: (1) Will the introductions of ZYMV and WMV2 CP increase the likelihood of new plant viruses (five comments)? (2) Could the introduction of two new virus resistance genes cause squash to become a weed (three comments)? (3) Will the virus resistance genes move to wild squash relatives and would this have a detrimental impact on these wild plants (four comments)?
On May 23, 1994, APHIS published a notice in the Federal Register (59 FR 266l9-26620) announcing the availability of an environmental assessment (EA) and preliminary finding of no significant impact (FONSI) for comment at a public meeting and for written comment during a 45-day comment period, which ended July 7, 1994. At the public meeting held June 21, 1994, only two individuals spoke, one in favor of the EA and FONSI, and one against. Each of the speakers at the public meeting submitted written comments as well. During the remainder of the 45-day comment period, APHIS received an additional 52 written comments from individuals. A total of 29 comment letters disagreed with APHIS' proposal to approve the subject petition, whereas 23 comments supported the APHIS findings in the EA and FONSI. The affiliations of the persons that commented were: private individuals (18); universities (12); agricultural experiment stations (11); public policy/public interest groups (6); industry (2); associations (1); cooperative extension service (1); and a Federal research laboratory (1). Approximately two-thirds of the respondents in both groups wrote relatively brief, general comments expressing their views about the petition and APHIS procedures for conducting an environmental analysis and making a final determination. The remaining one-third of the respondents, both in opposition to and in favor of approval of the petition, provided detailed, issue-specific comments on the EA and FONSI.
The five major plant pest risk issues raised by commenters in all three Federal Register notices that had reservations or disapproved of APHIS' proposed action were:
(1) Will the introduction of ZYMV and WMV2 CP increase the likelihood of appearance of new plant viruses?
(2) Could the introduction of two new virus resistance genes cause squash to become a weed?
(3) Will the virus resistance genes move to wild squash relatives and would this have a detrimental impact on these wild plants?
(4) Are free-living C. pepo (FLCP) plants serious weeds?
(5) Are hybrids between ZW-20 squash and FLCP plants weeds, and will they persist in the environment and become weeds?
APHIS addresses each of these questions following a brief description of the taxonomy of squash.
Each of the five issues mentioned in comments as relevant to potential plant pest risk for ZW-20 squash will now be addressed.
Issue 1. Will the Introduction of ZYMV and WMV2 Coat Proteins Increase the Likelihood of the Appearance of New Plant Viruses?
Conclusion: ZYMV and WMV2 and the aphids that vector the viruses are widely prevalent in the United States. Based on the known physical and biological properties of ZYMV and WMV2, the likelihood of the appearance of new plant viruses with novel biological properties through field cultivation of ZW-20 plants is no greater than in naturally occurring potyvirus-infected squash.
ZYMV and WMV2 are members of the potyvirus group, which is one of the largest groups of plant viruses. At least 40 different potyviruses are reported to be widely prevalent in at least 43 States. ZYMV produces a severe disease consisting of mosaic (patchwork of yellow chlorotic tissues and green uninfected tissues), yellowing, shoestringing, stunting, and fruit and seed deformations on zucchini squash, muskmelon, cucumber, and watermelon. WMV2 causes mosaic and mottle diseases of cantaloupe, pumpkin, squash, and watermelon. On a given cucurbit host, ZYMV usually causes more severe symptoms than WMV2. The complete nucleotide sequence of at least three potyviruses (Reichmann et al., 1992) and partial sequences of WMV2 and ZYMV have been published (Quemada et al., 1990).
ZYMV and/or WMV2 are reported to be widely prevalent in at least 34 States (see Appendix I, Table 1) including those where commercial squashes are grown and FLCP plants are found.
There are many known and potential plant hosts to provide ZYMV or WMV2 as a source of inoculum to infect C. pepo. WMV2 and ZYMV are apparently not seed transmitted in common virus-susceptible cucurbit crops (Purcifull et al., 1984). The source of these viruses for infection of commercial plantings must be other host plants. These plants may be either: perennials; other plants that transmit the virus in seed; or in warm climates where continuous cucurbit production occurs, other production fields. WMV2 is potentially harbored in many wild and cultivated crops through the winter months in the Imperial Valley of California. By contrast, all the known sources for ZYMV are in the Cucurbitaceae (Perring et al., 1992). The reservoirs for ZYMV are home garden plantings of squash or sponge gourd, or commercial plantings of melons or squash grown under plastic or in greenhouses. The overwintering host for ZYMV in Florida is the wild perennial cucurbit Melothria pendula (Adlerz et al., 1983), while the host in New York State has not been identified (Provvidenti et al., 1984). The source for WMV2 in Texas is probably M. pendula (Chala et al., 1986), and in Arizona the sources are mallow, sour clover, and sweet pea (Nelson and Tuttle, 1969). The overwintering hosts of WMV2 in Florida (Adlerz, 1978) and Massachusetts (Komm and Agrios, 1978) have not been identified. For most areas in the United States where cucurbits are grown, the specific overwintering host(s) has not been identified; however, many potential hosts have been identified (see Appendix I, Tables 2 and 3, data from Perring et al., 1992). Because of the extreme susceptibility of the FLCP plants to these viruses (Provvidenti et al., 1978), it is a reasonable assumption that FLCP plants would not be significant reservoirs for these viruses. APHIS could not identify evidence that FLCP plants have ever been naturally infected by ZYMV or WMV2.
The three aphid species involved in the dissemination of ZYMV and WMV2 are widespread in the United States. The viral CP is not the primary determinant of aphid transmission of potyviruses. Most potyviruses are transmitted by many aphid species in a nonpersistent manner (see Appendix I, Table 4, data from Perring et al., 1992). The most important and widespread of these aphid vectors are Myzus persicae, Aphis gossypii, and Macrosiphum euphorbiae (CMI/AAB Description of Plant Viruses, 1988; Perring et al., 1992). The main features of non- persistent transmission are that the virus can be picked up by the aphid after as little as 15 seconds on the infected plant and can transmit it immediately to one or only a few healthy plants. These brief acquisition and inoculation times limit the usefulness of insecticides to reduce the spread of these viruses. Most research suggests that viral infections generally originate locally (less than one-quarter of a mile distant) and that long-range emigration of viruliferous aphids is rare (Perring et al., 1992; Adlerz, 1978).
There is evidence that two virus-coded proteins, a noncapsid protein (called helper component) and the CP, play key roles in potyvirus transmission and vector specificity. The way in which helper component makes aphid transmission possible has not been established. The most likely effect is that the protein makes it possible for the virus to attach to sites within the aphid in a way that allows it to be transmitted. Although helper component appears essential for aphid transmission of potyviruses, its presence does not guarantee transmission (Matthews, 1991). Modifications to the CP can result in loss of aphid transmissibility (Atreya et al., 1990).
Coat proteins are the most extensively characterized potyviral gene products (Reichmann et al., 1992). The primary function of CP is to encapsidate viral RNA. Other CP functions reported for other plant viruses include host response determinant and cell-to-cell movement, but these functions have not been identified for potyviruses (Reichmann et al., 1992).
Upjohn/Asgrow's approach for achieving viral resistance is based on observations that plants expressing a viral CP gene are often resistant to infection by the virus from which the CP was derived (Powell Abel et al., 1986). Most evidence suggests that expression of viral CP by a plant interferes with one of the first steps in viral replication uncoating (removal of CP) from the incoming virus (for a review of this topic, see Register and Nelson, 1992).
ZW-20 does not exhibit stronger disease symptoms than its parental variety when infected with common cucurbit-infecting viruses. To determine the response of ZW-20 to infection with common cucurbit- infecting viruses, ZW-20 plants (and control plants) were inoculated with each tested virus. The symptoms that developed and the amount of CP produced were determined. The ZW-20 population used was segregating for resistance to WMV2 and ZYMV. The line used is fixed for one block of genes that provides moderate viral resistance and segregating for another block that confers strong resistance. All the nontransgenic control plants inoculated were susceptible to viral infection. ZW-20 plants were resistant to WMV2 infection and to ZYMV in ZW-20 plants containing the strong resistance gene block. ZW-20 plants containing the moderate viral resistance block showed milder symptoms than the nontransgenic controls (data reports for permit numbers 90-365-02 and 90-365-03).
The levels of ZYMV and WMV2 CP detected by ELISA (enzyme-linked immunoabsorbent assay) did increase when ZW-20 plants were challenged with ZYMV and WMV2 singly or in combination (data reports for permit numbers 90-365-02 and 90-365-03, tables IV and V) as compared to mock-inoculated ZW-20 plants. This increase may be due to limited replication of the viruses in ZW-20 plants, or that ZYMV and WMV2 CP produced by the plants are stabilized in the presence of replication of limited amounts of ZYMV and WMV2. Similar results have been reported for other potyviruses (Farnelli et al., 1992).
According to data provided by Upjohn/Asgrow, ZW-20 plants are as susceptible as the parental plants to CMV and papaya ringspot virus (PRSV), which are two of the most prevalent cucurbit viruses (data reports for permit numbers 90-365-02 and 90-365-03). As anticipated, the levels of ZYMV and WMV2 CP detected by ELISA did not increase when ZW-20 plants were inoculated with CMV (data reports for permit numbers 90-365-02 and 90-365-03, tables IV and V), as compared to uninoculated ZW-20 plants.
In contrast to the reported synergism between CMV and ZYMV infections in cucumber (Poolpol and Inouye, 1986), no synergism was detected in CMV-infected ZW-20 (data reports for permit numbers 90-365-02 and 90-365-03). The levels of ZYMV and WMV2 CP detected by ELISA did increase when ZW-20 plants were inoculated with PRSV, a closely related potyvirus that often infects cucurbits (data reports for permit numbers 90-365-02 and 90-365-03, tables IV and V), as compared to uninoculated ZW-20 plants. This increase may be due to the stabilization of two CP when the potyvirus PRSV is replicating. Similar results have been reported for other potyviruses (Farnelli et al., 1992). The levels of CP produced, in general, were still less than those detected in naturally-occurring plant infections. Infections of ZW-20 were no more severe than those of the parental variety.
Genomic masking in ZW-20 virus-infected plants should not increase the likelihood of the appearance of plant viruses with altered host specificities. When two viruses multiply together in the same tissue, some progeny particles may be formed that consist of the genome of one virus encased in a particle made partially or completely from the structural component of the other virus. Where the genome of one virus is encased in a protein shell made entirely of subunits of another virus (or strain), the phenomenon has been termed "genomic masking" or "transencapsidation." When the protein coat consists of a mixture of proteins from the two viruses, it has been termed "phenotypic mixing" (Matthews, 1991). This phenomenon occurs with potyviruses and is important in insect transmissibility of certain potyviral isolates (Bourdin and Lecoq, 1991). Since these three potyviruses (PRSV, WMV2, and ZYMV) are already endemic in the United States and infect many of the same crops (according to APHIS survey of endemic viruses, Appendix I, Table 1, and data reports for permit numbers 90-365-02 and 90-365-03); therefore, one would expect that significant amounts of masked virus particles are naturally present.
Genomic masking between CMV and potyviruses has not been reported and would not be expected because of the different architecture of the virions, icosahedral and flexuous (slightly curved) rods, respectively.
The likelihood of genomic masking is expected to be higher between another potyvirus and ZW-20-encoded CP. The most common potyvirus in squash (besides ZYMV and WMV2) is PRSV. There are two questions that need to be addressed in considering the likelihood and significance in any potential instance of genomic masking. First, is there a sufficient amount of CP produced by the transgenic plant to produce masked virus? Second, if a masked virus is produced, would it have any new biological properties, e.g., the ability to be transmitted by insect vectors, especially vectors different from those that transmit the parent virus?
The amount of CP produced is important in genomic masking. Often, in mixed infections where genomic masking occurs, there is an increase in the amount of the viral CP produced by one of the viruses. For example, co-replication of carmovirus-like ST9 RNA and the luteovirus beet western yellows virus (BWYV) results in a 10-fold increase in BWYV CP levels over luteoviral replication alone (Falk and Duffus 1984; Passmore et al., 1993). In addition, the CP produced by the ZW-20 plant under the direction of CaMV 35 S promoter is found in the same tissues (leaf mesophyll cells) (Benfey et al., 1990) that CP is found during natural infections (Matthews, 1991).
Generally, lower levels of ZYMV and WMV2 CP are produced by PRSV- infected ZW-20 (as detected by ELISA) than in PRSV-infected nontransgenic squash plants also infected with ZYMV or WMV-2 (data reports for permit numbers 90-365-02 and 90-365-03, tables IV and V). Thus, less ZYMV and WMV2 CP are available to potentially produce masked virus, so that production of masked virus in ZW-20 should be less efficient than in nontransgenic squash infected with combinations of the three viruses.
Even if a masked virus were generated, it would not pose a risk to the environment because it would not have any significant new biological properties. If masked virus arose in PRSV-infected (or any other cucurbit-infecting potyvirus) ZW-20 plants, the primary determinant for aphid transmissibility is not CP, but rather a noncapsid protein, the helper component of PRSV (Reichmann et al., 1992; Matthews, 1991). Thus, the primary determinant for transmission would be derived from the potyvirus that would infect ZW-20 squash (i.e., the helper component of the infecting virus), not the plant-encoded CP.
If masked PRSV, containing CP of ZYMV and/or WMV2, was produced, the masked virus would not gain any significant advantage in its ability to be transmitted by aphids or to be transmitted to new plant hosts since the three most common vectors for all three viruses are the same: A. gossypii, M. euphorbiae, and M. persicae (see Appendix 1, Table 4; data from Perring et al., 1992). In fact, these are the most common aphid vectors of potyviruses in temperate regions of the United States. One comment to the draft EA/Determination noted the existence of gaps in the list of aphids that transmit four cucurbit viruses (see Appendix I, Table 4). The data presented was tabulated from scientific literature (CMI/AAB Description of Plant Viruses and Perring et al. 1992). APHIS again notes that information provided in Table 4 is from published reports. Not every researcher could or would test every aphid vector for its ability to transmit each of the four viruses. APHIS stands by its earlier conclusion that the most economically important aphid vectors of ZYMV, WMV2, PRSV, and CMV in the United States are three aphid species listed above.
If masked virus were produced, it could only be maintained in the population as long as the virus replicated in ZW-20 plants or plants infected with ZYMV and/or WMV2. Once the masked virus was transmitted to a nontransgenic or an uninfected plant, only the original virus would be produced because the ZYMV and/or WMV2 CP would not be available for the production of masked virus.
Generation of recombinant virus in ZW-20 virus-infected plants should not increase the likelihood of the appearance of viruses with novel attributes. Recombination is defined as the formation of new genetic combinations by the exchanging of genes. In this case, the result of the recombination event would be that the plant-encoded CP from either ZYMV or WMV2 would replace the CP in a cucurbit-infecting virus (most likely a potyvirus).
First, recombination has not been demonstrated within the potyvirus group (Lai, 1992) although recombination has been seen with other plant viruses under high selection pressure (Bujarski and Kaesberg, 1986; Allison et al., 1990; Greene and Allison, 1994). In contrast, under weak selection pressure no recombination is seen (Angenent et al., 1989; Robinson et al., 1987; Falk and Bruening, 1994).
"New" potyviral strains or viruses apparently have not arisen via recombination. In the early 1980's, a new cucurbit-infecting potyvirus ZYMV appeared in Europe, the Middle East, and Northern Africa (Lecoq et al., 1981); and in Connecticut, New York, Florida, and California (Provvidenti and Gonsalves, 1984). Subsequent molecular analysis of the nucleotide sequence of ZYMV revealed that it apparently did not arise from recombination between "known" cucurbit-infecting potyviruses WMV2 and PRSV, but these three viruses probably evolved from a common progenitor (Quemada et al., 1990; Shukla and Ward, 1989). Recently, a necrotic strain of potato virus Y (PVY) has caused severe losses in tobacco plants. Nucleotide sequencing reveals that it differs from existing PVY strains by several bases that are distributed throughout the genome (Robaglia et al., 1989; Sudarsono, et al., 1993) and apparently did not arise from recombination gene cassettes. Many "new" viruses are only newly recognized because they impact forest, nursery, and crop plant production.
Why these three closely related potyviruses maintain their individual genome sequence may be a result of selection pressure in their over- wintering hosts and their reliance on aphids for transmissibility. In temperate regions of the United States where cucurbit crops are only grown during the summer months, these viruses must survive most of the year in other plant hosts. In Florida the overwintering host for ZYMV is the wild perennial cucurbit Melothria pendula (Adlerz et al., 1983), and for PRSV it is Momordica charantia (Adlerz, 1972). The overwintering host(s) for WMV2 in Florida has not been identified, although the above two plants have been ruled out (Adlerz, 1978). Thus, since each virus has a different overwintering host, this may be an important source of selection pressure on these viruses in maintaining their individuality. The viruses must maintain genome that replicates efficiently in the many commercially grown cucurbit crop plants (squash, cantaloupe, watermelon, and cucumber) and the different overwintering hosts, and be able to be transmitted by the aphid vectors between these plants. Another factor in maintaining separate identities of these three viruses may be that they replicate in different subcellular sites and that other viral RNAs cannot permeate the replication complexes, thus reducing viral RNA recombination frequency.
Recombination and ZW-20. For recombination to occur, at least two different viruses or viral strains must replicate in the same plant. In many field grown plants multiple viral infections (up to six viruses in a single plant) have been detected (Abdalla et al., 1985; Falk and Bruening, 1994; data report 93-365-02 and 93-065-03, table VI). Most evidence suggests that recombination occurs at a higher frequency between viruses and viral strains that share significant nucleotide homology (homologous recombination) than those that do not share nucleotide sequences (Lai, 1992). Thus, recombination is more likely to occur between the ZW-20 encoded CP mRNAs and potyviruses rather than other nonrelated viruses. The primary function of CP is to encapsidate viral RNA. Its other biological property is as a secondary determinant of aphid transmissibility. Because the aphids that are the most important vectors for transmitting these viruses are the same for the most widely prevalent potyviruses in cucurbits (see above), the recombinant virus would not have gained any new attributes.
Issue 1 was the subject of several comments: Two plant virologists (Drs. de Zoeten and Palukaitis) who commented in support of the Agency's FONSI have written publications on this general subject area that were used by other commenters to support their opposing views. None of the plant virologists who commented on the draft EA/Determination disagreed with the conclusions made by APHIS regarding this issue. APHIS concurs with the several comments that indicated that there may be certain crops and gene constructs in which the creation of new plant viruses that pose increased disease risk may be theoretically possible, but that ZW-20 squash is not one of those.
APHIS' conclusion that new viruses are no more likely to be produced through cultivation of ZW-20 squash than through cultivation of traditional varieties is based in part on the fact that the insect vectors of the relevant squash viruses are all the same and also in part on the fact that multiple infections by these viruses are common in commercial squash.
APHIS disagrees with one commenter who suggested that the data presented from a random sampling of cucurbits from grocery stores does not support the contention that multiple infections are common. In our analysis of the data, we had never concluded that all cucurbits are always infected with plant viruses every year. For a variety of reasons (e.g., reduced population of insect vectors), the frequency and severity of viral infections in a given location can vary from year to year. The survey showed that in a random sample of 35 fruits, 89 percent showed at least one viral infection, 55 percent were infected with at least two viruses, and 18 percent were infected with all four viruses tested. APHIS concludes that these viruses are widely prevalent in the environment and that most samples tested contained two or more viruses. Other reports confirming multiple viral infection of crop plants have been reported (Falk, 1994; Abdalla et al., 1985).
FLCP plants have survived since the arrival of the severe strain of ZYMV. APHIS would like to respond to a general concern about the occurrence of severe strains of viruses, given that ZYMV can kill FLCP plants when inoculated with human intervention. In his classic textbook Plant Virology (1991), Matthews discusses survival of severe strains.
"A virus that kills its host plant with a rapid developing systemic disease is much less likely to survive than one that causes only a mild disease that allows the host plant to survive and reproduce effectively. There is probably natural selection in the field against strains that cause rapid death of the host plant. Leafhoppers living on desert plants of the western United States are infective primarily with strains of beet curly top virus that cause mild symptoms in beet. Virulent strains of curly top virus kill certain desert plant species before they can allow a generation of leaf- hoppers to mature. Thus, the virulent strains in the desert tend to be self limiting. However, disease severity has a very different effect in beet plants. Sugar beet is a good host for the leafhopper if the plants are small and exposed to full sunlight. They are poor hosts when large, providing a lot of shade. For this reason severe strains of curly top virus facilitate their own spread in beet by producing small, stunted plants, which favor vector multiplication (Bennett, 1963). Leafhoppers over- wintering near beet fields carry strains of higher virulence than hoppers found in the desert."
It is true that young FLCP plants deliberately inoculated with ZYMV or WMV2 may die? What happens to the strain of potyviruses that killed the plant? Aphids do not feed on dead and dying plants (Matthews, 1991). Thus, a severe strain of a virus , ie., a virus that kills its host plant, will theoretically be transmitted to other plants less efficiently than less severe strains. Thus, virus strains tend to persist that cause mild symptoms and do not kill plants before seeds are produced. The survival of even severe strains is enhanced if there is any genetic variability in the hosts, i.e, if there are some host biotypes present that do not die upon infection and can produce seeds. The association of a moderation of symptoms coupled with maintaining the ability of a virus to be efficiently transmitted has been observed with barley stripe mosaic virus in barley and seed transmission (Timian, 1974), rice stripe virus in rice and leafhopper transmission and lettuce mosaic virus and seed transmission (Matthews, 1991).
Do severe viral strains exist? Yes, but they are predominantly seen in commercial plantings where plants are well maintained and are of one uniformly susceptible genotype, which greatly facilitates transmission via vectors (e.g., aphids) that travel a short distance. In unmanaged ecosystems, plants are not well maintained (i.e., no irrigation, fertilizer, or pesticides) and the aphid vectors may feed on nonhost plants, thus losing the virus.
APHIS believes that the recent history of the development of biological control agents illustrates the general (though not exclusive) trend that virulent viruses can evolve or be selected by the host to become less virulent strains. One classic example is myxoma (rabbitpox) in Australia and Great Britain. A severe strain of the virus was introduced into the country to control rabbits. From the initial very severe strain of virus (highly virulent with essentially no recovery of infected rabbits) researchers found in the field progressively less and less virulent strains of the virus with faster and faster recovery rates of infected rabbits (Morse, 1993). There is indirect evidence of a similar trend with plant viruses that have been considered for use in biological control. APHIS could find no evidence of the successful use of plant viruses to control weeds (i.e., no registration of plant viruses as biological control agents by EPA and no recent requests to APHIS for testing of plant viruses as biological control agents (C. Divan, USDA, BBEP, personal communication)). This has frequently been attributed by plant virologists to difficulty in finding strains that kill the plant, attenuation of the severe strain of the virus to a less virulent strain (less effective), and failure to obtain high infection rate. There are no specific literature citations for this finding because such negative data traditionally have not been judged suitable for publication.
Issue 2. Could the Introduction of Two New Virus Resistance Genes Cause Squash to Become a Weed?
Conclusion: Introduction of virus resistance genes is unlikely to increase the weediness of yellow crookneck squash.
A study (National Research Council, 1989) entitled "Field Testing Genetically Modified Organisms: Framework for Decisions," identified the potential to inadvertently produce a new weed or increase the aggressiveness of existing weeds as "perhaps the single most commonly voiced concern about the introduction of genetically modified plants."
A weed pest is a plant that grows persistently in locations where it is unwanted. Baker (1965) described the ideal characteristics of a weed that include: rapid plant growth to germination in many environments; internally controlled discontinuous germination; long-lived seeds; rapid growth to flowering; continuous seed production; use of wind or unspecialized insects for pollination if outcrossing occurs; high seed production; good competitiveness achieved through for example, allelochemicals or choking growth; and long-lived seeds. None of the characteristics described by Baker involved resistance or susceptibility to pathogens or insects. In 1989, Keeler considered in detail whether genetically engineered crops can become weeds. Her analysis of the closely-related squash, C. maxima, stated that squash possesses 3 out of the 15 characteristics of plants that are notably successful weeds. Those are: continuous production of seeds as long as growing conditions permit; use of unspecialized insects as pollinators; and strong competitiveness with other plants.
Several comments criticized APHIS' mention in the draft EA/Determination of Baker's list of characteristics of known weeds. Almost all definitions of weediness stress as core attributes the undesirable nature of weeds from the point of view of humans; from this core, individual definitions differ in approach and emphasis (Baker, 1965; de Wet and Harlan, 1975; Muenscher, 1980). Baker (1965) listed 12 common weed attributes, almost all pertaining to sexual and asexual reproduction, which can be used as an imperfect guide to the likelihood that a plant will behave as a weed. Keeler (1989) and Tiedje et al. (1989) have adapted and analyzed Baker's list to develop admittedly imperfect guides to the weediness potential of transgenic plants; both authors emphasize the importance of looking at the parent plant and the nature of the specific genetic changes. APHIS listed Baker's characteristics as a preamble to introducing Keeler's characterization (1989) of C. maxima, a close relative of yellow crookneck squash. Keeler's article listed the weedy characteristic of C. maxima. We believe that Keeler described several important growth characteristics of squashes. Although Baker's list has been criticized, no other universally acceptable characters have been defined by ecologists (Williamson, 1994) and in our view, there is no formulation that is clearly superior at this time.
Yellow crookneck squash is not listed as a weed in the Federal Noxious Weed Act (7 U.S.C. 2801-2813) and is not reported by the Weed Society of America to be a common or troublesome weed anywhere in the United States (Bridges and Baumman, 1992). Although squash volunteers are not uncommon in areas next to production fields, they do not readily establish feral or free-living populations. Volunteers can still be controlled by mechanical means or herbicides. The ZW-20 squash is likely to be grown mostly in areas that are currently under squash cultivation, i.e., in typical growing regions for the crop. Upjohn/Asgrow has reported that there are no major changes in seed germination, cucurbitin levels, seed set viability, susceptibility or resistance to pathogens or insects (except ZYMV and WMV2), and there are no differences in overwintering survivability between virus resistant transgenic squash and nontransgenic squash (data reports for permit numbers 90-365-02, 92-027-01, 90-365-03, and 93-053-02).
There is no evidence to support the conclusion that introduction of virus resistance genes into squash could increase its weediness potential. Many pathogen and insect resistance genes have been introduced into commercial varieties of squash by conventional means in the past without any reports of increased weediness of squash plants. These include genes for resistance to scab, powdery mildew, downy mildew, cucumber beetle, squashbug, and cucumber mosaic virus. Squash cultivars having ZYMV, PRSV, and CMV resistance genes introduced by conventional plant breeding techniques are soon to be sold by Upjohn/Asgrow (H. Quemada, personal communication).
There are no morphological or physiological characteristics of the ZW-20 squash that would entail the use of agricultural practices that vary from the traditional practices used today for the cultivation and propagation of squash except a reduced need for control of aphids that vector these viruses.
One commenter who took issue with our draft EA/Determination stated, ". . . there are examples where resistance to stress is suspected (emphasis added) of playing a role", and three references were cited. One, Darmency and Gasquez (1990), discussed the movement of a herbicide tolerance gene into a known common weed, lamb's quarters, and another was a review article covering the same subject (Darmency, 1994). The third paper (Burdon et al., 1981) dealt with biological control of an exotic weed in Australia. Several commenters cited a reference by Williamson (1994). This paper, which discusses the major invasive plant pests in Great Britain and their traits, is not relevant to the discussion. The paper does not address the introduction of resistance genes into indigenous plants of Great Britain. All of the 14 weedy plants described in the paper were foreign introductions (one from Australia, 5 from Asia, and 4 each from Europe and the Americas).
Even successful multiple virus infection of weed populations does not guarantee their poor growth and reproductive patterns. Plants from nonagricultural ecosystems are known to be infected and act as significant reservoirs for many viruses. Plantago species may be one of the most important potential virus reservoirs. These plants are efficient and adaptable perennial weeds with a worldwide distribution. They are listed as common or serious weeds in 37 countries (Holm, 1979). They have been found infected naturally with at least 26 viruses from 19 groups and families (Hammond, 1982). Although Plantago sp. are often infected with many viruses, they are still successful and troublesome weeds. Of course, viruses are not the only pathogens and pests of these plants that exert stress on the plant.
Issue 3. Will the Virus Resistance Genes Move to FLCP Plants, and Will This Have a Detrimental Impact?
Conclusion: The survey of FLCP plants for the presence of plant viruses was adequate and scientifically based. Many plants can be infected with a plant virus with direct human intervention but are never naturally infected. A survey for the presence of viral infections is an appropriate, adequate, and proven means for determining whether a plant is a significant natural host for a particular virus. APHIS believes that other experimental approaches suggested by commenters to determine impact of movement of virus resistance genes to FLCP plants are flawed.
The major scientific controversy for this issue was APHIS' reliance on a survey of FLCP plants for the presence of several plant viruses as a basis for determining whether the virus resistance genes would provide selection advantage to the FLCP plants. Two types of experiments were proposed as alternatives to survey approach. We will discuss the scientific merits of the survey approach and the deficiencies in the other experimental approaches before our analysis of the impact.
a. The survey for the presence of plant viruses in FLCP plants—To determine whether FLCP plants are natural hosts for ZYMV or WMV2 Upjohn/Asgrow surveyed at APHIS' request natural stands of FLCP plants in Arkansas, Louisiana, and Mississippi for the presence of ZYMV, PRSV, WMV2, CMV, squash mosaic virus, tomato ringspot virus, and tobacco ringspot virus. No FLCP plants were taken in Texas because the previously identified C. pepo ssp. ovifera var. texana stands could not be located and stands in Illinois were inundated by floods in 1993. Analysis of previous published data and anecdotal evidence strongly suggests that the FLCP plants are not infected by ZYMV and WMV2. The presence of viruses was assayed by double diffusion and ELISA tests. The plant samples were also visually inspected for symptoms, and plant extracts assayed for viruses on indicator plants (Chenopodium quinoa, Nicotiana benthamiana, cucumber, tobacco, and zucchini). Although a sample of FLCP plants from a single site showed symptoms consistent with viral infection, none of the above-listed viruses were detected in FLCP samples even when susceptible commercially-grown nontransgenic squash plants were growing less than 2,500 feet away (see Appendix I, Table 6).
The survey that was done to address the presence of these viruses in FLCP plants was performed by agricultural Extension agents and agricultural scientists who are trained to recognize and identify disease symptoms. These scientists were asked to survey the FLCP stands for plants showing symptoms of virus infection (e.g., necrosis, foliar mosaic, etc.) and collect representative plant tissue samples to conduct laboratory and greenhouse tests to confirm the presence of the viruses. The surveys were done in mid-July to late August 1993 to maximize the likelihood that infected FLCP plants would be present and detectable. APHIS wanted to ensure that commercial cucurbit production was well underway to increase the potential for viral inoculum from aphid populations in the commercial plantings of squash and other crops. Sampling later in the growing season also increases the likelihood of detecting FLCP plants that are infected with multiple viruses (Matthews, 1991). Provvidenti et al. (1978) have shown that infection of young FLCP plants with certain cucurbit viruses can yield severe symptoms, but if infection occurs when the C. pepo plants are older, the symptoms are less severe. This observation is true for many plant/virus combinations, i.e., that once seed production in a plant is initiated (therefore, the older the plant is) the less effect viral infections have on plant seed production and plant survival (Matthews, 1991). This is a trade-off for this sampling date. When a greater number of plants may potentially be infected, less severe symptoms may be observed.
The surveyors saw no clear evidence of viral infection in any FLCP plants. The only suggestion of any infection was slight chlorosis in a single plant. The vine-like nature of these plants renders it difficult to determine the number of FLCP plants growing in a given area, especially late in the growing season. However, because these viruses move systemically throughout the infected plants, sampling one or two leaves of a plant should be sufficient to detect the viruses in any size plant, assuming the initial infection started several weeks before the sampling date. (By this time the virus would have moved systemically throughout the plant from initial site of infection). Because the surveyors did not see any viral symptoms on the FLCP plants, they took only a limited number of samples at each site. There is a low probability of viral infection in plants that do not exhibit symptoms. The asymptomatic samples were tested for the presence of seven different viruses that infect cucurbits, and none was detected.
Occasionally virus concentrations in field grown plants are too low to allow easy detection. To increase the likelihood of detecting virus if it were present, sap from sampled FLCP plants was used to inoculate other plants (including cultivated squash) that are highly susceptible to the seven viruses. None of these plants showed viral symptoms, nor could any viruses be detected by any of the serological tests performed.
b. The first proposed alternative experiment to the survey— Several commenters to our draft EA/Determination stated that counting the number of plants that exhibit symptoms of infection by a virus does not necessarily give an adequate indication of the frequency of the presence of the virus in those plants. They mentioned the nonviral example of cactus moth and prickly pear in Australia. The commenters noted that in this instance the moth populations are low and the host plant prickly pear populations are small. They pointed out that even though the number of cactus moths that one could count is low, the moth has been effective in reducing cactus populations. APHIS believes that the Australian scenario is not comparable to FLCP plants and cucurbit viruses for the following reasons. First, the FLCP plants are native to the United States. By contrast, all cacti are native to the Americas and are exotics in Australia. Cactus moth was successfully introduced to Australia as a biological control agent for prickly pear. Second, the cucurbit viruses and their aphid vectors populations are not low in the United States. In fact, the viruses are widely prevalent and aphid populations high.
Several commenters to our draft EA/Determination suggested additional experimental approaches that they believed appropriate and/or necessary in order to study the impact of movement of virus resistance genes to FLCP plants. In a report prepared by Dr. Hugh Wilson for APHIS, Dr. Wilson described a series of experiments (see Appendix II, p. 17-19) to decide if the introduction of virus resistance gene(s) into FLCP plants would increase the weediness potential of FLCP plants. Briefly, he suggested that the following plants be inoculated with ZYMV: ZW-20; nontransgenic yellow crookneck squash; FLCP plants; and F1 and F2 hybrids between ZW-20 and nontransgenic parent and FLCP plants. Wilson suggested that the experiments should be performed in a greenhouse because: (1) FLCP plants are "unwieldy" and (2) pollen carrying the virus resistance gene from transgenic plants should not be allowed to fertilize FLCP plants. As part of the experiments to determine the response of the FLCP carrying the virus resistance gene, Wilson suggests that the listed genotypes be inoculated with ZYMV in the greenhouse.
APHIS does not believe that this greenhouse experiment will yield reliable or useful information, because the response of plants to viral infection under artificial conditions cannot predict their resistance or susceptibility under natural conditions. APHIS believes that viral inoculation of plants under artificial conditions may give rise to conclusions that are not substantiated under natural conditions. It is not uncommon for a crop plant to be susceptible under controlled conditions to a widely prevalent virus yet are rarely infected by that virus under natural conditions. One of the best documented examples is that of CMV infection of tobacco (Nicotiana tabacum). Tobacco plants are readily infected by all widely prevalent strains of CMV under artificial conditions (Kaper and Waterworth, 1981). All commercial tobacco cultivars grown in North Carolina and Kentucky are susceptible to CMV under greenhouse conditions with human intervention (E. Wernsman, North Carolina State University, personal communication). CMV is widely prevalent in North Carolina and Kentucky (APHIS List of Widely Prevalent Viruses). CMV is transmitted under field conditions by the aphid vector M. persicae, which is common in tobacco fields and is the same vector that transmits the major tobacco potyviral diseases: potato virus Y, tobacco etch virus, and tobacco vein mottling virus (Shew and Lucas, 1991). However, field grown tobacco plants are only rarely infected with CMV (Shew and Lucas, 1991; E. Wernsman, North Carolina State Univ., personal communication). Thus, reliance on greenhouse data in this instance would have led to incorrect conclusions, i.e., one would have predicted that CMV would be a major problem of tobacco plants but it is not. This observation is not an isolated case. APHIS has identified other virus-plant combinations (Jones et al., 1991; Hooker, 1981) similar to CMV-tobacco scenario described above: the aphid-transmitted potyvirus, tobacco vein mottling virus infecting tomato (confirmed by T. Pirone, University of Kentucky, personal communication); the aphid transmitted luteovirus, potato leafroll virus infecting tomato; the aphid transmitted potyvirus, tobacco etch virus infecting potato; and the aphid transmitted potyvirus, tobacco vein mottling virus infecting potato (confirmed by T. German, University of Wisconsin, personal communication).
APHIS concludes: (1) there is sufficient scientific evidence that surveying of host plants for the presence or absence of a particular virus is adequate to determine if a particular virus will have a significant impact on those plants; and (2) this approach will provide more reliable information than a greenhouse experimentation-based one.
c. The second proposed alternative experiment to the survey— A second experiment described in two comments entails exclusion of, "a particular natural enemy . . . as the way to determine the effect of that natural enemy on a wild population". APHIS believes that after careful consideration exclusion of the "natural enemy" is not feasible. First, although the details of this experiment were not given by the commenters, we assume that the commenters' experiment involved placing insect-proof cages over FLCP plants or using pesticides to eliminate the aphid vectors. Wilson noted the "unwieldy nature" of FLCP plants. Performing this experiment for an extended period under natural conditions would be difficult or impossible.
Second, what is the "natural" enemy? Aphids are the only important vectors for transmission of ZYMV and WMV2 under field conditions. However, the same aphids that vector these viruses are plant pests themselves and cause significant crop losses even when they do not vector viruses (Davidson and Lyon, 1987). Thus, placing insect-proof screens around plants would prevent not only virus-infected aphids but noninfected aphids from developing on the FLCP plants. Furthermore, screening or insecticide treatment to exclude the aphid vectors would also eliminate other insects, e.g., thrips, leafhoppers, beetles, vine borers, squash bugs, mites, and whiteflies, that are known pests of cucurbits (Davidson and Lyon, 1987). Several of these insects also vector serious viruses of cucurbits. For example, thrips transmit tomato spotted wilt virus; beetles transmit squash mosaic virus; and aphids transmit zucchini yellow fleck virus, CMV, and PRSV (Matthews, 1991); all of which would be eliminated.
Therefore, APHIS believes that the experiment described to exclude the "natural enemy" would not only eliminate the two viruses in question but many insect pests and the plant pathogens that they transmit. This type of experiment would not only eliminate selective pressure of aphids containing ZYMV or WMV2, but all aerial pests and the pathogens that they may vector. It would therefore potentially grossly overestimate any potential impact from the viruses in question. While APHIS believes that additional scientific research in these general directions could yield information about virus replication in FLCP plants and about total pest and disease pressure on FLCP plants, we believe that Upjohn/Asgrow's experiments are most appropriate for addressing the relevant concerns.
In conclusion, APHIS believes that the additional experiments proposed by commenters were neither appropriate nor necessary in order to study the impact of movement of virus resistance genes to FLCP plants.
The virus resistance genes in ZW-20 will be transferred via pollen to FLCP plants. APHIS assumes pollen from ZW-20 squash is likely to be carried by bees and successfully pollinate FLCP plants. Although Upjohn/Asgrow has presented data (data reports for permit numbers 89-300-01 and 90-365-03) showing that pollen movement declines rapidly at distances greater than 50 feet from a source plant, we assume that fertile hybrids between ZW-20 plants and FLCP plants will occur given a sufficient period of time and widespread use of ZW-20 squash in agricultural settings.
FLCP plants are susceptible to ZYMV and WMV2 infection but are not infected in the wild. In greenhouse and field tests, C. pepo spp. ovifera var. texana (seed source from Texas) was found to be highly susceptible to CMV, PRSV, and WMV2 when mechanically infected or grown in the presence of aphid vectors and infected plants (Provvidenti et al., 1978). Recently, several accession lines of C. pepo that have genes for resistance to CMV and WMV2 have been identified by the USDA Germplasm Information Network (Kyle et al., 1993). Thus, earlier reports that no sources of resistance to potyviruses and cucumoviruses have been reported in C. pepo are incorrect (Provvidenti, 1990). However, the survey of FLCP plants failed to detect seven important cucurbit-infecting viruses (see Appendix, Table 6).
APHIS believes that further reviews of the botanical record with respect to FLCP plants in Arkansas supports our previous decision that FLCP plants have not been impacted by ZYMV or other potyviruses. ZYMV first appeared in the mid-1980's in the United States and is now widely prevalent in Arkansas and Texas. It is the most devastating single virus in cucurbit production. Has the appearance of ZYMV in Arkansas affected the FLCP population in soybean fields? The evidence available is qualitative, but has been provided by agricultural experts who have continuously monitored cucurbits in these areas. Dr. Ford Baldwin (Weed Specialist of the USDA, Extension Service) said that FLCP populations in these fields have not noticeably changed since the arrival of ZYMV. Dr. Greg Weidemann (University of Arkansas), who worked during the 1980's to identify biological control agents for FLCP control, potentially including viruses, does not recall any evidence over several years of research of natural viral infections in stands of FLCP plants. (Instead, he selected the fungus Fusarium solani to test as a biological control agent (Weidemann and Templeton, 1988). Mr. Joe Vestle also did not recall the presence of viral-infected FLCP plants but did recall the presence of rust (fungus) infection of FLCP plants. Furthermore, these Arkansas scientists indicated that the appearance of ZYMV as a major pest of cucurbits did not affect FLCP populations enough to alter the rate of farmer requests for information on controlling FLCP plants in soybean and cotton fields during the 1980's.
The lack of infection of FLCP plants is not a result of absence of virus or aphid vectors. APHIS believes that the absence of potyviral infection in FLCP plants is not a result of the lack of inoculum for the following reasons: (1) ZYMV and WMV2 are widely prevalent in cucurbit crops in many of the major honeydew melon-, cucumber-, pumpkin-, and squash-producing States (see Appendix I, Tables 1 and 5); (2) many potential weed and annual plants that are hosts of ZYMV and WMV2 are present in these States (see Appendix 1, Tables 2, 3, and 5); and (3) many aphid vectors that can transmit ZYMV and WMV2 (see Appendix I, Table 4, and data from Perring et al., 1992) are widely distributed in the States where FLCP have been reported. Wilson (1993) states, "C. pepo populations during a given growing season would probably include every county with the 12 State FLCP distribution. . . ."
Why FLCP plants are not infected with common cucurbit-infecting viruses is unknown. If the aphids are to infect FLCP plants they must feed on a ZYMV- or WMV2-infected plant immediately before visiting the FLCP plants. The development cycle of the aphids and the maturation of plants in the spring where FLCP grow may not be favorable to viral infection of FLCP. Second, the FLCP plants could produce chemicals that make them unattractive to feeding by the aphid vectors. A Cucumis melo genotype has been identified that exhibits this type of resistance (Gray et al., 1986).
It should be noted that the fact that the two cucurbit viruses' ZYMV and WMV2 have the cucurbit names "watermelon" and "zucchini" in their titles does not imply that they have ever been pests of FLCP plants in nature. Viruses are given their names after the plant from which they were first isolated and characterized. If the only hosts of these viruses in the eastern United States were squash, these viruses would likely perish. Because these viruses are not seed-transmitted in squash and do not overwinter in squash detritus, squash by itself is a dead-end host. The critical host for survival of these viruses is their overwintering host (usually woody perennials or in dormant seeds). Since the overwintering host for ZYMV in Florida is the wild perennial cucurbit Melothria pendula (Adlerz et al., 1983), a more biologically appropriate name for the virus might be Melothria yellow mosaic virus.
The absence of (poty)viral infection of FLCP was not unexpected. FLCP plants have been reported to be highly susceptible to several viruses, yet FLCP plants have survived for decades in areas where these viruses and their aphid vectors are widespread. Therefore, if these viruses impacted FLCP populations, there should have been natural selection for resistance or symptomless infections. The virus survey of FLCP plants performed by Upjohn/Asgrow showed that the plants were not infected asymptomatic strains of the selected plant viruses since no viruses were detected. Therefore, FLCP plants are apparently not infected by common viruses that affect commercial cucurbit production (see Appendix I, Table 6).
The movement of the virus resistance genes from ZW-20 to FLCP plants should not have a significant negative impact on FLCP plants. Wilson (1992) states, ". . . any genetically transmissible trait that provides enhanced fitness in the wild is cause for concern". Foreign genes (e.g., virus resistance genes) are most likely to be retained in a population if they confer a reproductive advantage to the plant containing the foreign gene over other competitors in the population. Since all evidence supports the conclusion that FLCP populations are not under significant environmental stress from viral infection, the selective pressure to maintain the virus resistance genes in natural populations of FLCP plants should be minimal.
Issue 4. Are FLCP Plants Serious Weeds? Will Virus- Resistant FLCP Plants Be More Difficult to Control Than Virus Susceptible Plants?
Conclusion: FLCP plants are not serious weeds in unmanaged or agricultural ecosystems. No scientific or anecdotal evidence was presented in comments or uncovered in APHIS review establishing that FLCP plants are weeds in unmanaged ecosystems. Although FLCP plants were reported to be weeds in cotton and soybean fields during the 1970's, registration of new herbicides now allows effective management of these plants.
There are no reports of FLCP plants as significant weeds in any unmanaged ecosystems, but rather, they stably occupy only a particular biological niche along riverbanks. FLCP plants have only been reported to be a serious problem in soybean and cotton fields in the Red River Valley of Arkansas. These reports date from the 1970's, but the FLCP plants continue to be an occasional problem in soybean and cotton fields that are located in flood-prone areas today (F. Baldwin, personal communication). Dr. Baldwin was the representative from Arkansas on the Weed Society of America's 1992 publication entitled "Crop Losses Due to Weeds in the United States" in which FLCP plants were listed as serious weeds in soybean fields. Dr. Baldwin stated that he believed that FLCP plants were not as serious a problem currently as in the past, and he provided APHIS with the names of other persons with up-to-date familiarity with FLCP plants occurrence in Arkansas. A summary of APHIS' discussions with these scientists follows.
Dr. Greg Weidemann (University of Arkansas) conducted research during the 1980's to identify biological control agents to eliminate FLCP plants from soybean fields. He said that the FLCP problem in soybean fields has been controlled in recent years by new herbicides (e.g., Cobra®) that were not available in the 1980's, so that FLCP plants are only a minor problem in soybean fields in the Red River Valley. Joe Vestle, a County Extension agent who works in areas where the FLCP plants were previously serious problems, agreed with Drs. Baldwin and Weidemann that the FLCP plants are less a problem in 1994 than during the 1980's. They also noted that pending registrations of the herbicide bromoxynil for use in conjunction with bromoxynil-tolerant cotton and of the herbicide glyphosate for use in conjunction with glyphosate-tolerant soybeans will further expand the tools for effective control of FLCP plants. They noted that with these additional options FLCP plants should not become a significant weed problem in soybean or cotton fields in Arkansas.
If FLCP plants acquire resistance to WMV2 and ZYMV from ZW-20 squash, the control of the virus-resistant wild plants in soybean or cotton fields should not be more difficult or require new measures than of their nonengineered counterparts. Incorporation of the ZYMV and WMV2 resistance genes into FLCP plants growing in cotton or soybean fields would not make the control of these plants more difficult. Soybean and cotton crops are not affected by these two viruses (or PRSV and CMV) and no viruses are known that cause both squash diseases, in either of these crops (Matthews, 1991). ZYMV and WMV2 resistance genes will not confer any resistance to any soybean or cotton virus. Thus, even if the soybean or cotton plants were severely infected by a plant virus, the virus resistant FLCP plants would not have any selective advantage over their nonengineered counterparts with respect to viruses present in soybean or cotton plants. The most effective means of controlling FLCP plants are herbicide application, rogueing, and collection of the gourds at the end of the season to eliminate the seed source. The effectiveness of all of these methods would be uncompromised by the presence of virus-resistant FLCP plants.
Issue 5. Will Hybrids Between ZW-20 Squash and FLCP Plants Persist in the Environment and Become Weeds?
Conclusion: There is no scientific or anecdotal evidence that supports the contention that hybrids between yellow crookneck squash and FLCP plants are weeds and are persistent.
Several comments criticized APHIS' draft EA/Determination for not adequately addressing whether there might be a risk of a new weed pest arising after the pollination of FLCP plants by ZW-20 squash. A number of comments suggested that APHIS should require greater testing of actual hybrids to determine if there are any potential risks. APHIS agrees that the petition does not contain extensive data to address definitively the potential "weediness" of such hybrids under a large variety of environmental conditions. However, APHIS believes it is possible to reach some valid conclusions based on our current knowledge of FLCP plants in managed and unmanaged ecosystems.
Traditional plant breeding methods have been used for centuries to develop squash varieties that exhibit improved ability to resist environmental stresses, both biotic and abiotic. During the past century many disease resistant varieties have been developed and cultivated throughout the world. FLCP plants have grown in proximity to new, improved cultivars of squash, and yet there have been no reports in the scientific literature to suggest that disease resistance traits have introgressed into FLCP plants to produce hybrid populations that pose increased problems as weeds. There is no reason to believe that the viral resistance associated with ZW-20 squash will impact FLCP populations differently from viral resistance introduced into squash cultivars by traditional breeding. This includes the new Harris Moran zucchini squash, developed by traditional breeding techniques, that is phenotypically identical to ZW-20.
Upjohn/Asgrow has supplied to APHIS additional information of ongoing field tests with hybrid plants derived from controlled crosses of ZW-20 with FLCP plants. Based upon the limited observations in field tests during the past 2 years, the FLCP x ZW-20 hybrids do not appear to be strong competitors when growing in fields that have not been tilled to remove competing wild plants based on survival of plants and seed set. These field tests have been conducted with several hundred hybrid progeny growing at a single site (interim data report 93-041-01). Whereas these results cannot predict the behavior of any future hybrid progeny when FLCP plants are pollinated by ZW-20 plants, the evidence supports APHIS' contention that the introgression of the virus resistance from ZW-20 into FLCP plants does not appear to pose a risk of developing a weed pest.
By contrast, unlike squash and FLCP plants, there are several well-known interbreeding crop-weed complexes that are serious pests. One example is the shattercane that is derived from crosses of wild and cultivated sorghum. Shattercane is a serious pest in the United States and world-wide. Other hybrid complexes that are serious pests outside the United States include rice, barley, wheat, and corn (Harlan, 1992).
Other Comments to the Draft EA/Determination
One commenter asked APHIS to clarify its statement, "Genetically engineered crops are comparable to traditionally bred resistant varieties." This statement was meant to convey a limited meaning: that blockage of viral replication results in a similar phenotype whether produced by genetic engineering, traditional breeding, or whether the plant is tolerant via natural cross protection. It is true that the nature and mechanism of only one plant virus resistance gene (N gene for resistance to tobacco mosaic virus in tobacco) has been identified (Whitmam et al. 1994). However, it has been established that plants bred via traditional means do not achieve that resistance by means of expressing a gene identical to, or homologous, to any viral CP genes (Matthews, 1991). By contrasts, genetically engineered cross protection is likely mediated directly by CP, as is the phenomenon of natural cross protection. In the latter cross protection, the production of CP by a mild strain of virus has been suggested as the means by which the challenge virus replication is blocked. In fact, this hypothesis was one of the bases for the landmark research by Dr. Roger Beachy (Scripps Research Institute), that determined that expression of CP genes in plants results in resistance. Thus, when ZYMV or WMV2 infect a ZW-20 plant or another cross protected squash plant, replication is blocked. All the virus "sees" is that CP is present and does not "recognize" whether it was produced by another strain of the virus or the transgenic plant. APHIS acknowledges that other modes of action of cross protection have been suggested (Matthews, 1991). APHIS also notes one significant difference between traditional breeding and genetically engineered resistance, i.e., the presence of viral sequences in the plant. Issues dealing with that difference have already been addressed above.
Genes from nonsexually compatible plants have been introduced into C. pepo. Genes from nonsexually compatible plants have been previously introduced into C. pepo. CMV, WMV2, ZYMV, and PRSV resistance genes from C. ecuadorensis, C. martinezii, and C. moschata (Gilbertalbertini et al., 1993); fruit fly resistance from C. maxima (Nath, 1975); trifluralin herbicide tolerance from C. moschata (Adenijji and Coyne, 1981); powdery mildew and scab resistance from C. martinezii (Kyle et al., 1993) have been or are being introduced into C. pepo by classical breeding techniques. C. martinezii and C. ecuadorensis are not sexually compatible with C. pepo, but through a series of bridging crosses (i.e., crosses with other species compatible with both) the genes have been moved into domestic squash plants. The risk of gene pool corruption from ZW-20 is no greater than has been accepted without alarm in the past with no noted ill effects.
The use of ZW-20 squash provides an alternative method for the control of ZYMV and WMV2. Currently, chemical, physical, biological, and genetic methods are used to control these viruses. Attempts to reduce virus spread through vector control by insecticides are largely ineffective but are still used in limited situations. Although these insecticides can kill the aphids, the aphids transmit these viruses efficiently throughout the field before their death. Insecticides registered for control of aphids include diazinon, lannate, metasystox-R, phosdrin, and thiodan. Many insecticides are toxic to nontarget organisms. Many growers now use mineral oils (often supplemented with insecticidal soaps or insecticides) to control both aphid and non-aphid insect pests. These chemicals are effective, but the oil sprays need to be applied frequently, every 3 to 5 days, to effectively control aphids.
Physical measures, like reflective surfaces and sticky yellow sheets, can also diminish vector spread. These measures are expensive and cumbersome to use on a large scale.
Another approach for protecting plants from viral infection requires inoculation of the plants with an asymptomatic (mild) strain of the virus. Infection of a plant by the mild strain of a virus protects the plant from the effects of subsequent inoculation with another severe strain of the same virus (Matthews, 1991). This phenomenon is called cross protection. It has been field tested successfully to control ZYMV in Taiwan (Wang et al., 1991) and Hawaii (Cho et al., 1992). Cross protection has several disadvantages that have been summarized by Fulton (1986), but is not widely used in the United States for controlling viruses.
Genes for resistance to WMV2 have been identified in C. ecuadorenis, C. ficifolia, C. foetidissima, C.pedatifola, and C. moschata (Nigerian squash) and for ZYMV in C. ecuadorenis, and C. moschata. However, introduction of these resistance genes into domestic cultivars via traditional plant breeding has proven difficult because of genetic incompatibility among species (Provvidenti, 1990). A traditionally bred cultivar phenotypically identical to ZW-20 is commercially available from Harris Moran Seed Company. Virus resistant zucchini squash (specific virus not described but possibly CMV) and CMV-resistant marrow squashes are available commercially from Thompson and Morgan, Inc. of Jackson, New Jersey. Also, several traditionally bred virus resistant cultivars developed by Upjohn/Asgrow or Cornell University are, or shortly will be, on the market.
If virus resistant squashes, developed by genetic engineering or by traditional breeding techniques, become widely accepted, the use of certain agricultural chemicals may be reduced. Whether there is or is not a reduction at any specific production site probably depends on whether whiteflies are a major problem at that site. Because precise data on the amount of insecticide used exclusively on yellow crookneck squash is not available, APHIS cannot hypothesize on the absolute amount of any reduction in use but we do not think that speculation on the possible reduction in pesticide use will have any bearing on our determination or FONSI. We would hypothesize that a reduction, if any, in insecticide use would be minor relative to total insecticide use on U.S. crops. A reduction in usage would be most likely in States where whiteflies are only a minor pest. In States where whiteflies are a major problem we might predict that insecticide use would be unlikely to change. Whiteflies are a major problem in the Southern tier of States from North Carolina to California. Cucurbit viruses are problems in many of these States (see Appendix, Table 1). If whiteflies are a major problem at the site, chemicals will probably still be applied. Without whiteflies, genetic resistance will probably be sufficient since aphids alone usually do not cause sufficient damage to warrant chemical application.
IV. Analysis of the Properties of ZW-20 Squash
To reach its determination that ZW-20 squash does not present a plant pest risk, APHIS has addressed not only issues raised in public comments, but also considered basic information on the biology of squash and data presented by Upjohn/Asgrow or otherwise available to APHIS that are relevant to consideration of plant pest risk. Based on the data described, APHIS has arrived at a series of additional conclusions regarding the properties of ZW-20 squash.
The Introduced Genes, Their Products, and the Added Regulatory Sequences Controlling Their Expression Do Not Present a Plant Pest Risk in ZW-20
The ZW-20 squash plants were derived by transforming yellow crookneck squash via a well-characterized technique that uses DNA sequences from A. tumefaciens to introduce genes into the chromosome of the recipient plant (see reviews by Klee and Rogers, 1989; and Zambryski, 1988). Although some DNA sequences used in the transformation process were derived from the plant pathogen A. tumefaciens (the causal agent of crown gall disease), the genes that cause crown gall disease were removed, and therefore the squash plant does not develop crown gall disease. Once inserted into the chromosome of the squash plant, the introduced genes are maintained and transmitted in the same manner as any other genes. Squash plants pass their genes to their progeny by sexual reproduction that involves self pollination, or pollination of other squash plants or sexually compatible relatives.
The ZW-20 squash line was produced using an Agrobacterium-meditated transformation protocol to transform yellow crookneck squash with genes designed to confer resistance to ZYMV and WMV2, two plant viruses that frequently infect squash. The genes that confer this resistance are derived from virus genes that encode the CP of ZYMV and WMV2. Expression of these CP genes in the squash does not cause plant disease, but rather confers resistance to infection by ZYMV and WMV2.
The introduced DNA that encodes the CP genes also has accompanying DNA regulatory sequences that modulate the expression of the CP genes. The DNA regulatory sequences were derived from three plant pathogenic organisms: the bacterium Agrobacterium tumefaciens, CaMV, and CMV. Specifically, the DNA regulatory sequences associated with the viral CP coding regions comprise promoter and transcriptional termination sequences derived from the 35S gene of CaMV and translational initiation sequences from CMV. In addition, the amino-terminus of the WMV2 coding region is fused to the 5' intergenic region and the first 48 nucleotides (N-terminus) of the CMV CP gene. Although these regulatory sequences were derived from plant pathogens, the regulatory sequences cannot cause plant disease by themselves or with the genes that they regulate. Because of the physical and biological properties of ZYMV and WMV2, the likelihood of creating new plant viruses with novel biological properties through field cultivation of ZW-20 plants is no greater than in naturally occurring potyvirus-infected squash.
During characterization of the performance of ZW-20 squash in laboratory, greenhouse, and field experiments, the plants exhibited the typical agronomic characteristics of the parent crookneck squash, with the addition of resistance to ZYMV and WMV2 infection. In APHIS' opinion, the components and processing characteristics of ZW-20 squashes reveal no differences in any component that could have an indirect plant pest effect on any processed plant commodity. The ZW-20 plants have no plant pest characteristics.
The ZW 20 Squash is No More Likely to Become a Weed Than a Virus-Resistant Plant Developed by Traditional Breeding Techniques
APHIS' analysis of this issue can been found in Section III, Response to Comments, Issue 2. Briefly, yellow crookneck squash is not listed as a weed in the Federal Noxious Weed Act (7 U.S.C. 2801-2813) and is not reported by the Weed Society of America to be a common or troublesome weed anywhere in the United States (Bridges and Baumman, 1992). Upjohn/Asgrow has reported that there are no major changes in seed germination, cucurbitin levels, seed set viability, susceptibility or resistance to pathogens or insects (except ZYMV and WMV2), and there are no differences in overwintering survivability between ZW-20 squash and nontransgenic squash. APHIS concludes that ZW 20 is unlikely to increase the weediness of yellow crookneck squash and is no more likely to become a weed than virus-resistant plants
developed by traditional breeding techniques.
The ZW 20 Squash is Unlikely to Increase the Weediness Potential for Any Other Cultivated Plant or Native Wild Species With Which the Organism Can Interbreed
APHIS' analysis of this issue can been found in Section III, Response to Comments, Issue 3, 4, and 5. Based on review of current data, FLCP plants are not serious weeds in unmanged or agricultural ecosystems. The virus resistance gene from ZW-20 plants will move via pollen to the FLCP plants. Since all evidence supports the conclusion that FLCP populations are not under significant environmental stress from viral infection, the selective pressure to maintain the virus resistance genes in natural populations of FLCP plants should be minimal. APHIS concludes that widespread cultivation of ZW 20 squash is unlikely to increase the weediness potential for any other squash or native wild species with which ZW 20 can interbreed.
The ZW-20 Squash Should Not Cause Damage to Processed Agricultural Commodities
There is no reason to believe that the development of virus-resistant squash plants would result in a change in fresh marketing or processing procedures. Most yellow crookneck squash is consumed as a raw table vegetable or processed for the frozen food market.
The ZW 20 Squash Should Not Increase the Likelihood of the Emergence of New Plant Viruses
APHIS' analysis of this issue can been found in Section III, Response to Comments, Issue 1. APHIS concludes that based on the known physical and biological properties of ZYMV and WMV2 and data provided by Upjohn/Asgrow, the likelihood of the appearance of new plant viruses with novel biological properties through field cultivation of ZW-20 plants is no greater than in naturally occurring potyvirus-infected squash.
The ZW-20 Squash Should Not Be Harmful to Beneficial Organisms, Including Bees
There is no reason to believe deleterious effects on beneficial organisms could result specifically from the cultivation of ZW-20 squashes, based on two lines of reasoning:
(1) No direct pathogenic properties, nor any hypothetical mechanisms for pathogenesis toward beneficial organisms, such as bees and earthworms, were identified for ZW-20 squash. APHIS also cannot envision any plausible mechanisms for any hypothetical pathogenetic effect since the two proteins engineered in ZW-20 are already present in high concentration in naturally infected squash plants.
(2) Cucurbitin, a naturally occurring toxicant in squash plants, levels are likely to be unchanged and can be easily identified in cucurbit fruits by its bitter taste. Cucurbitin was not detected by taste-testing of fruits from ZW-20 and its descendants (data report for permit No. 92-027-01).
The definition of ZW-20 squash encompasses not only squash lines that already have been field tested, but also new squash lines that may be produced through conventional breeding using ZW-20 squash as one or both parents. APHIS believes that the analysis applied to ZW-20 squash already field tested will apply equally well to these new squash lines, and that the data provided by Upjohn/Asgrow justifies the conclusion that such new ZW-20 squash will not present a plant pest risk. The variation in agronomic characteristics among the ZW-20 squash lines that have been field tested does not differ significantly from that seen in commercial cultivars of squash that have never been considered regulated articles. While it is impossible to predict the exact agronomic characteristics of the progeny of a cross between a ZW-20 squash and another squash cultivar, cross-breeding between well-characterized squash varieties is the traditional means by which new and improved squash varieties are created. These crosses have often used as one-parent squash cultivars that are considerably more genetically different from standard commercial cultivars than are ZW-20 squashes, i.e., other members of the genus Cucurbita.
V. Conclusion
In response to a petition from the Asgrow Seed Company, APHIS has evaluated information regarding the potential plant pest risks presented by the transgenic squash line designated as ZW-20. ZW-20 squashes have been transformed via an Agrobacterium-mediated protocol with the viral CP genes of zucchini yellow mosaic virus (ZYMV) and watermelon mosaic virus, type 2 (WMV2). Expression of the ZYMV and WMV2 CP genes in ZW-20 squash does not cause plant disease, but rather confers resistance to infection by ZYMV and WMV2. APHIS has determined that the ZW-20 squash does not pose a direct or indirect plant pest risk and therefore will no longer be considered a regulated article under APHIS regulations at 7 CFR Part 340. Permits under those regulations will no longer be required from APHIS for field testing, importation, or interstate movement of those squashes or their progeny. (Importation of ZW-20 squashes [and nursery stock or seeds capable of propagation] is still, however, subject to the restrictions found in the Foreign Quarantine Notice regulations at 7 CFR Part 319.) This determination has been made based on an analysis that revealed that ZW-20 squash: (1) exhibits no plant pathogenic properties; (2) is no more likely to become a weed than a virus-resistant plant developed by traditional breeding techniques; (3) is unlikely to increase the weediness potential for any other cultivated plant or native wild species with which the organisms can interbreed; (4) should not cause damage to processed agricultural commodities; (5) should not increase the likelihood of the emergence of new plant viruses; and (6) is unlikely to harm other organisms, such as bees, which are beneficial to agriculture. APHIS has also concluded that there is a reasonable certainty that new progeny ZW-20 squash varieties bred from these lines should not exhibit new plant pest properties, i.e., properties substantially different from any observed for the ZW-20 squash lines already field tested, or those observed for squashes in traditional breeding programs.
Terry L. Medley, J. D. Director Biotechnology, Biologics, and Environmental Protection Animal and Plant Health Inspection Service U.S. Department of Agriculture Date
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VII. Appendices
Appendix I. Distribution of WMV2 and ZYMV, Their Plant Hosts and Insect and Leading Cucurbit Production States
Table 1. Prevalence of WMV2, ZYMV, CMV and PRSV by State
Table 2. Host Plants of WMV2
Table 3. Host Plants of ZYMV
Table 4. Select list of aphids that transmit ZYMV, WMV2, PRSV, and CMV
Table 5. Acreage of cucurbit crops in States containing FLCP plants
Table 6. Results from survey performed by Upjohn/Asgrow of FLCP plants for plant viruses
Appendix II. Free-Living Cucurbita pepo in the United States. Viral Resistance, Gene Flow, and Risk Assessment. Report to USDA, Biotechnology, Biologics, and Environmental Protection by Dr. Hugh Wilson