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TSCA Environmental Release Application (TERA) for Alcaligenes xylosoxidans subspecies denitrificans strain AL6.1, R04-03

On April 21, 2004, the Office of Pollution Prevention and Toxics approved the TSCA Environmental Release Application (TERA) under the biotechnology regulations promulgated under the Toxic Substances Control Act (TSCA). The TERA, submitted by the University of California, Riverside, involves field trials of a modified strains of Alcaligenes xylosoxidans subspecies denitrificans (Axd) strain AL6.1. The TERA was given the tracking designation of R04-0003 (previously reviewed under tracking designation R03-0001). The microorganism will be tested at three of the same four test sites as in TERA R03-0001 in California to determine its biology and behavior in vineyard ecosystems. The eventual application of such technology would be the development of a paratransgenesis system to control Pierce’s disease of grapes, a widespread devastating disease caused by the bacterium Xylella fastidiosa (X. fastidiosa). The field trials were set to run from the week of May 1, 2004 until the end of the growing and harvest season, approximately December 15, 2005.

Proposed Use and Field Study

The bacterium will be used for small-scale research and development field trails to continue studying its biology and behavior in vineyard ecosystems at 3 of the 4 sites used in the previous submission, R03-0001. The X. fastidiosa pathogen is spread throughout grape growing regions by the glassy-winged sharpshooter (GWSS), a large leafhopper insect. Since the parental strain of Axd AL6.1 was originally isolated from the GWSS, the researchers ultimately plan to use the GWSS as a delivery vector of Axd carrying a pesticidal gene antagonist to X. fastidiosa to the xylem of grapevines where the pathogenic bacterium resides.

The current proposed field trials, as well as the previous field trials, are preliminary investigations whereby the AL6.1 stain contains only the DsRed marker gene to evaluate the survival of Axd in and on the vines and fruit, and to discover if the bacterial inoculant will persist long enough in the xylem to serve as a delivery vehicle for a pesticidal gene product antagonistic to X. fastidiosa. This is a TSCA application because there is a field release of an intergeneric microorganism, AL6.1, without a pesticidal gene insert. If positive results are obtained from these field tests and the researchers subsequently insert a pesticidal gene into the AL6.1 strain of Axd, any future testing will fall under the Office of Pesticide Programs.

Regulatory Background

The EPA requirements concerning microbial products subject to TSCA (15 U.S.C. Section 2601, et seq.) are set forth in “Microbial Products of Biotechnology; Final Regulation under the Toxic Substances Control Act” (62 FR 17910 (April 11, 1997)) and codified at 40 C.F.R. Part 725. Microorganisms resulting from the deliberate combination of genetic material originally isolated from organisms of different taxonomic genera (intergeneric microorganisms) constitute “new” microorganisms subject to TSCA Section 5 notification requirements. Persons who manufacture, import, or process intergeneric microorganisms for commercial purposes subject to EPA jurisdiction under TSCA, are required to submit a Microbial Commercial Activity Notice (MCAN). Persons conducting commercial research and development activities may submit a TERA, instead of an MCAN, before initiation of such testing. EPA conducts a review of these submissions to determine whether the intergeneric microorganisms present an unreasonable risk to health or the environment. The Agency can impose regulatory controls under Section 5 of TSCA.

Genetic Characteristics of Microorganism

As discussed in the risk assessment for R03-0001, there were proposed changes in the taxonomy of the submission strain, Alcaligenes xylosoxidans subsp. denitrificans. In 1998, it was proposed that Alcaligenes xylosoxidans be transferred to the genus Achromobacter to become Achromobacter xylosoxidans subsp. denitrificans. More recently, this proposal was refuted and a new name, Achromobacter denitrificans, was proposed.

Summary of the Risk Assessment

Based on available data and the R03-01 submission, there is low concern for potential toxicity of the recipient microorganism and the introduced genetic material. Although there are some uncertainties associated with the genetic construct, the risk to humans or the environment is low due to the low exposure conditions of the contained field tests. Additionally, the recipient species, Axd, is not a frank pathogen, although it has been associated with some opportunistic infections. No data is available regarding the toxicity associated with exposure to the DsRed fluorescent protein. However, the DsRed fluorescent protein may present some cytotoxicity when expressed in certain cells.


EPA has determined that the proposed small-scale field trials of these intergeneric microorganisms will not present an unreasonable risk of injury to health or the environment. In the TERA approval letter, EPA advised the submitter that each grape plant should be destroyed at the end of the experiment, by incinerating the vines, as well as digging up and autoclaving the root balls, as there is potential for widespread exposure of the bacterium once the Reemay fabric covers are removed and insect vectors can transport the bacterium to other crops. In addition, the submitter is informed that the issue of gene transfer must be addressed in greater detail if the microorganisms are ever proposed for large scale testing or submitted as MCANs.

For a copy of the original nonconfidential TERAs and the nonconfidential approval letter, please contact the TSCA Non-Confidential Information Center (NCIC) by phone at (202) 566-0280, or by fax at (202) 566-9744.