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University of Wisconsin-Madison; Availability of Environmental Assessment for Field Tests of Genetically Engineered Erwinia carotovora

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Animal and Plant Health Inspection Service, USDA.




We are advising the public that the Animal and Plant Health Inspection Service has prepared an environmental assessment for a field trial of genetically engineered strains of a bacterium, Erwinia carotovora, the causal agent of tuber soft rot disease in potato. The bacteria have been genetically engineered to disrupt the disease causing pathway. This field trial will allow researchers to better understand the function of each mutated gene under field conditions. This environmental assessment is available for public review and comment.


We will consider all comments we receive on or before September 12, 2005.


You may submit comments by either of the following methods:

  • Postal Mail/Commercial Delivery: Please send four copies of your comment (an original and three copies) to Docket No. 05-053-1, Regulatory Analysis and Development, PPD, APHIS, Station 3C71, 4700 River Road Unit 118, Riverdale, MD 20737-1238. Please state that your comment refers to Docket No. 05-053-1.
  • EDOCKET: Go to​feddocket to submit or view public comments. Once you have entered EDOCKET, click on the “View Open APHIS Dockets” link to locate Docket No. 05-053-1.

Reading Room: You may read the environmental assessment and any comments that we receive in our reading room. The reading room is located in room 1141 of the USDA Start Printed Page 47171South Building, 14th Street and Independence Avenue, SW., Washington, DC. Normal reading room hours are 8 a.m. to 4:30 p.m., Monday through Friday, except holidays. To be sure someone is there to help you, please call (202) 690-2817 before coming.

Other Information: You may view APHIS documents published in the Federal Register and related information on the Internet at​ppd/​rad/​webrepor.html.

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Dr. Rudaina Alrefai, Biotechnology Regulatory Services, APHIS, 4700 River Road Unit 147, Riverdale, MD 20737-1236; (301) 734-4866. To obtain copies of the petition or the environmental assessment (EA), contact Ms. Ingrid Berlanger at (301) 734-4885; e-mail: The EA is also available on the Internet at​brs/​aphisdocs/​05_​09701r_​ea.pdf.

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The regulations in 7 CFR part 340, “Introduction of Organisms and Products Altered or Produced Through Genetic Engineering Which Are Plant Pests or Which There Is Reason to Believe Are Plant Pests,” regulate, among other things, the introduction (importation, interstate movement, or release into the environment) of organisms and products altered or produced through genetic engineering that are plant pests or that there is reason to believe are plant pests. Such genetically engineered organisms and products are considered “regulated articles.” A permit must be obtained or a notification acknowledged before a regulated article may be introduced. The regulations set forth the permit application requirements and the notification procedures for the importation, interstate movement, or release into the environment of a regulated article.

On April 7, 2005, the Animal and Plant Health Inspection Service (APHIS) received a permit application (APHIS permit number 05-097-01r) from the University of Wisconsin-Madison, Department of Plant Pathology, Madison, WI, for a permit for a field trial of Erwinia carotovora. These bacteria have been genetically engineered not to express specific hrp/hrc (hypersensitive reaction on non-host plants and pathogenesis on host plants or conserved among plant and animal pathogens) genes resulting in the disruption of the disease-causing mechanism. These mutations are expected to make the bacterial strains avirulent or non-pathogenic. The application describes four genetically engineered strains to be used in this field trial.

The E. carotovora ssp. carotovora WPP14 strain was initially isolated from a diseased potato plant obtained from a commercial farm in Waushara County, WI. This strain was used to create four new genetically engineered strains by inserting a marker gene into genes that may be necessary for E. carotovora infection of potatoes. The four strains resulting from this mutatgenesis that are proposed for use in this field trial are described below.

  • Strain WPP40 contains an insertion of a kanamycin resistance gene (aph) cassette into out D. The out D gene encodes for an outer membrane porin that is required for a functional type II secretion system. This mutant is unable to secrete plant cell wall degrading enzymes and is avirulent. The kanamycin resistance gene cassette contains aph, which encodes neomycin phosphotransferase which was originally isolated from Tn5, and two FRT sites derived from Saccharomyces cerevisiae.
  • Strain WPP60 has an insertion of a spectromycin resistance gene (aad A cassette into hrc C, an outer membrane porin which is required for a functional type III secretion system. This mutant is unable to secrete harpins or effector (Avr) proteins. It is hypersensitive response minus and reduced in virulence. The spectinomycin resistance gene cassette is constructed from the aad D gene which encodes aminoglycoside-3 adenyltransferase, originally derived from Shigella flexneri, with termination sequences derived from bacterophage T4.
  • Strain Wpp195 has a deletion of hrp N and an insertion of a chloramphenicol resistance gene (cat) cassette and a modified green fluorescent protein (gfpmut 2) into this locus. This mutant is unable to produce or secrete the harpin, HrpN. The gfpmut 2 gene was originally cloned from Aequorea victoria and was modified to be brighter. Its expression is driven by the nptII kan promoter from Tn5. The cat gene encodes cholramphenicol acetyltransferase, which was originally isolated from Escherichia coli. This construct also contains FRT sites.
  • Strain Wpp198 is an insertion of a similar chloramphenicol resistance cassette into hrpL, which is a sigma factor required for expression of the type III secretion system and its secreted substrates. The mutant is unable to produce or secrete harpins or effector (Avr) proteins. It is hypersensitive response minus and reduced in virulence.

The genetically engineered E. carotovora are considered regulated articles under the regulations in 7 CFR part 340 because they may be plant pests. The purpose of the field trial is to use genetically engineered E. carotovora strains with mutations in homologs of the well-characterized Pseudomonas syringae hrp genes as tools to:

  • Understand the effects of specific genes on the fitness of E. carotovora,
  • Use the results from these experiments to better understand the function of these genes in plant-bacterial interactions, and
  • Compare the results obtained with E. carotovora mutants with those found for P. syringae to determine if homologous genes play similar roles in fitness in different environments.

To provide the public with documentation of APHIS' review and analysis of any potential environmental impacts and plant pest risk associated with the proposed field trial of theses strains of genetically engineered E. carotovora, an environmental assessment (EA) has been prepared. The EA was prepared in accordance with (1) The National Environmental Policy Act of 1969 (NEPA), as amended (42 U.S.C. 4321 et seq.), (2) regulations of the Council on Environmental Quality for implementing the procedural provisions of NEPA (40 CFR parts 1500-1508), (3) USDA regulations implementing NEPA (7 CFR part 1b), and (4) APHIS' NEPA Implementing Procedures (7 CFR part 372). Copies of the EA are available as indicated in the FOR FURTHER INFORMATION CONTACT section of this notice.

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Done in Washington, DC, this 8th day of August 2005.

Kevin Shea,

Acting Administrator, Animal and Plant Health Inspection Service.

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[FR Doc. E5-4382 Filed 8-11-05; 8:45 am]