West Virginia University College of Agriculture, Forestry and Consumer Sciences April 16, 2004 Dr. William R. Schneider Biopesticides and Pollution Prevention Division Office of Pesticide Programs US Environmental Protection Agency 1200 Pennsylvania Avenue Washington, DC 20460 Dear Dr. Schneider:

This letter is in response to your email of 15 April 2004 regarding the submission of a Biotechnology Notification letter about our proposed release of genetically modified ( transgenic) Cryphonectria parasitica strains. The materials and methods of the proposed research are detailed in the attached APHIS permit ( 04 077 02r, granted 13 April 2004). We propose to compare the dissemination of two hypovirulent ( less virulent) treatments ( genetically modified [ transgenic] and non genetically modified [ cytoplasmic]) with a treatment of virulent C. parasitica. Treatment isolates will be grown in the laboratory at West Virginia University, mixed with water agar to the consistency of applesauce and applied to cankers on American chestnut with a paintbrush. The experiment will be conducted in the Monongahela National Forest, near Circleville, WV ( Pendleton County) on nine 0.1 acre plots ( 0.9 acres or 0.36 hectares). The transgenic strains will be released in only three plots; the remaining six plots will test the cytoplasmic hypovirulent and virulent strains. In the transgenic plots, only a percentage of trees will be treated with transgenic inoculum; the remaining trees will be untreated and serve as trap trees. Therefore, the total area proposed for treatment with trangenic strains is less than 0.3 acres or 0.13 hectares. The research is proposed to be conducted from May 2004   August 2006.

Referencing 40 CFR 172.4 ( b) Name and address of applicant: Dr. William L. MacDonald, 401 Brooks Hall, West Virginia University, Morgantown, WV 26506 6058 telephone 304 293 3911 ext 2236. ( b) ( iii) Purpose of objective of proposed testing: The goal of this project is to examine the dissemination and subsequent establishment of transgenic hypovirulent strains of Cryphonectria parasitica. The objectives are three fold: ( 1) comparison of growth and sporulation of artificially established cankers initiated with either transgenic or cytoplasmic hypovirulent strains or virulent strains of C. parasitica; ( 2) the spermatizing potential of transgenic hypovirulent conidia; and, ( 3) the ability of transgenic strains to disseminate. ( b) ( iv) Name and address of all participants: William Rittneour, 401 Brooks Hall, West Virginia University, Morganwtown, WV 26506 6058, 304 293 3911 ext 2150; Mark Double, 401 Brooks Hall, West Virginia University, Morgantown, WV 26506 6058, 304 29303911 ext 2239. ( b) ( v) Name and address of all cooperators: Dr. Donald L. Nuss, Center for Biosystems Research, University of Maryland Biotechnology Institute, 5115 Plant Sciences Building, College Park, MD 20742, phone 301 405 0334. ( b) ( vi) Description of specific results of prior testing: A greenhouse test, utilizing a similar transgenic strain ( CHV1 EP713/ pXH9) was conducted on twelve woody stems. The results are listed below. CHV1 EP713 and CHV1 Euro7, the hypovirus proposed for the 2004 release, show extensive sequence identities: 87 to 93% and 90 to 98% at the nucleotide and amino acid levels, respectively. Lesion length ( mm) following 49 day inoculation of Cryphonectria parasitica virulent strain Ep155 and hypovirulent strain CN2 ( CHV1 Ep713/ pXH9) into woody stems. Plant Lesion Length ( mm)* of Virulent Ep155* Lesion Length ( mm)* of Hypovirulent CN2** Blueberry 0, 0 0, 0, 0, 0 Apple 3, 3 1, 1, 3, 3 Red Maple 28, 14 4, 3, 6, 11 Sugar Maple 2, 8 6, 1, 2, 1 Sweet Birch 9, 1 8, 1, 4, 3 American Beech 6 1, 1, 1 American Chestnut 59, 34 4, 1, 3, 4 Pin Oak 6, 1 1, 1, 3, 1 White Oak 1 3, 3, 1 Red Oak 6, 9 3, 3, 6, 4 Black Walnut 6, 9 3, 3, 3, 3 White Pine 3, 3 3, 1, 1, 0 * Lesion length includes only visible growth beyond the 4 mm diamater inoculation hole. * Two lesions were made to each woody stem with the virulent isolate. ** Four lesions were made to each woody stem with the hypovirulent isolate. ( b) ( vii) Proposed method of storage and disposal: Manipulation of fungal isolates in the laboratory will be conducted in a Labconco Class II biosafety cabinet. Transgenic isolates will be grown in temperature controlled growth chamber, secured from the general public. Slurry inoculum for field deployment will be prepared in the laboratory using " good microbiological practices". The West Virginia University Institutional Biosafety Committee has approved all practices described in this proposal. All fungi will be transported from West Virginia University to the field test sites in an automobile under the supervision of West Virginia University personnel who are directly responsible for supervising the field trial. Fungi will be transported in containers that meet the requirements of § 340.7 ( container requirements for movement of regulated articles). Bark samples ( 2 mm) from cankers will be collected in plastic microtiter or tissue culture plates ( 13 x 8.5 cm), secured with tape to prevent mixing or escape, and transported in a cooler to the laboratory at West Virginia University. Bark samples will be stored in a low temperature freezer until processed. Isolates will be maintained in incubators and temperature controlled growth chambers, secured from the general public. Following cultural assessment and molecular analyses, all fungal cultures will be autoclaved prior to disposal.

I trust this brief narrative is sufficient to address the notification issues. If further information is required, please feel free to contact me.

Sincerely,

William L. MacDonald Professor, Plant Pathology Division of Plant and Soil Sciences 401 Brooks Hall P. O. Box 6058 Morgantown, WV 26506 6058 Phone: 304 293 3911 Fax: 304 293 2872