USDA APHIS Environmental Assessment

In response to a permit application (06-111-01r) received from

Dr. Milton Rush of Louisiana State University for a field test of

two non-pathogenic, genetically engineered mutant strains of

*Burkholderia glumae*

U.S. Department of Agriculture

Animal and Plant Health Inspection Service

Biotechnology Regulatory Services

TABLE OF CONTENTS

[I. INTRODUCTION 4](#i.-introduction)

[A. Summary 4](#a.-summary)

[B. Regulatory Authority 4](#b.-regulatory-authority)

[II. NEED FOR THE PROPOSED ACTION 5](#ii.-need-for-the-proposed-action)

[III. ALTERNATIVES 5](#iii.-alternatives)

[A. No Action 5](#a.-no-action)

[B. Issue the Permit as Received 5](#b.-issue-the-permit-as-received)

[C. Issue the Permit with Supplemental Conditions
6](#c.-issue-the-permit-with-supplemental-conditions)

[IV. Research Design for *Burkholderia glumae* Release
7](#iv.-research-design-for-burkholderia-glumae-release)

[A. Purpose of the Research 7](#a.-purpose-of-the-research)

[B. Description of the Research 7](#b.-description-of-the-research)

[V. ENVIRONMENTAL CONSEQUENCES OF THE PROPOSED ACTION AND ALTERNATIVES
8](#__RefHeading___Toc163361718)

[A. Analysis of Issues, Consequences, and Theoretical Risks of Field
Research using Transgenic Burkholderia glumae
8](#a.-analysis-of-issues-consequences-and-theoretical-risks-of-field-research-using-transgenic-burkholderia-glumae)

[1. Risk of Transgenic, Non-Pathogenic *Burkholderia glumae* to the
Environment
8](#risk-of-transgenic-non-pathogenic-burkholderia-glumae-to-the-environment)

[2. Persistence of Transgenic *Burkholderia glumae*
9](#persistence-of-transgenic-burkholderia-glumae)

[3. Alteration in Susceptibility to Disease or Insects
9](#alteration-in-susceptibility-to-disease-or-insects)

[4. Horizontal Gene Transfer to Other Organisms
9](#__RefHeading___Toc163361723)

[5. Fate of Transgenic DNA 10](#__RefHeading___Toc163361724)

[6. Potential Impacts on Humans, Including Minorities, Low Income
Populations, and Children
10](#potential-impacts-on-humans-including-minorities-low-income-populations-and-children)

[7. Risks to Threatened and Endangered Species and Designated Critical
Habitat
11](#risks-to-threatened-and-endangered-species-and-designated-critical-habitat)

[8. Effects on Native Floral and Faunal Communities
11](#effects-on-native-floral-and-faunal-communities)

[9. Impact on Existing Agricultural Practices
12](#impact-on-existing-agricultural-practices)

[10. Cumulative Environmental Effects
12](#cumulative-environmental-effects)

[11. Consistency of proposal with other environmental requirements
13](#consistency-of-proposal-with-other-environmental-requirements)

[B. Analysis of the Alternatives 13](#b.-analysis-of-the-alternatives)

[1. Deny the Permit 13](#deny-the-permit)

[2. Issuance of the Permit as Received
13](#issuance-of-the-permit-as-received)

[3. Issuance of the Permit with Additional Conditions
13](#issuance-of-the-permit-with-additional-conditions)

[4. Preferred Alternative 14](#preferred-alternative)

[REFERENCES 16](#references)

[Table 1. 18](#table-1.)

[APPENDIX 1: Biology of *Burkholderia glumae*
19](#appendix-1-biology-of-burkholderia-glumae)

[APPENDIX 2: Description of the Regulated Bacteria
20](#appendix-2-description-of-the-regulated-bacteria)

[APPENDIX 3: Threatened and Endangered Species Analysis
21](#appendix-3-threatened-and-endangered-species-analysis)

[APPENDIX 4: Supplemental Permit Conditions
23](#appendix-4-supplemental-permit-conditions)

# I. INTRODUCTION

## A. Summary

*Burkholderia glumae* Kurita et Tabei is a bacterial plant pathogen that
causes bacterial panicle blight in rice (*Oryza sativa*), and is
transmitted by infected seed (Sayler et al. 2006). This bacterium was
first described in Japan as the cause of grain rotting and seedling
blight (Goto and Ohata 1956, Uematsu et al. 1976) and is considered one
of the most important rice pathogens in Japan (Azegami et al. 1987).
Epidemics of panicle blight occurred in the southern rice producing area
of the United States during the 1995 and 1998 growing seasons, with
yield losses in some fields estimated to be as high as 40 percent (Rush,
M.C., *pers. communication*).

Currently, there is no control method for panicle blight in the United
States (Sayler et al. 2006), and most commercially grown rice varieties
in the United States are susceptible to the disease (Shahjahan et al.
2000, Sayler et al. 2006). By field testing non-pathogenic, transgenic
strains of *B. glumae*, the proposed field release will provide
information on bacterial panicle blight infection of rice, and potential
routes of pathogen control. The transgenic *B. glumae* strains have been
modified to disrupt the disease-causing gene (gene that produces
toxoflavin), producing avirulent, non-pathogenic strains. The transgenic
*B. glumae* strains also express the genes for kanamycin and ampicillin
resistance as selectable markers. The proposed field release will also
involve the challenge of rice plants with a wild-type, pathogenic *B.
glumae* strain and a naturally-occurring, non-pathogenic *B. glumae*
strain, both of which were isolated from and are widely prevalent in
Louisiana, the site of the proposed release.

## 

## B. Regulatory Authority

The authorities for regulation of genetically engineered (GE)
*Burkholderia glumae* are the Plant Protection Act of 2000, 7 U.S.C.
7701-7772, and the United States Department of Agriculture's (USDA)
Animal and Plant Health Inspection Service (APHIS) regulations under 7
CFR § 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." A GE organism is considered a
regulated article if the donor organism, recipient organism, vector, or
vector agent used in engineering the organism belongs to one of the
taxonomic groups listed in the regulation and is also a plant pest, or
if there is a reason to believe it is a plant pest. The disease-causing
wild-type *B. glumae* is considered the causal pathological agent of
panicle blight in rice.

This environmental assessment (EA) was conducted under the authority of
the National Environmental Policy Act (NEPA), 42 U.S.C. 4321 and 7 CFR §
372, NEPA Implementing Procedures. Except for actions that are
categorically excluded, approvals and issuance of permits for proposals
involving GE or non-indigenous species normally require EAs, but not
necessarily environmental impact statements (7 CFR § 372.5(b)(4)). The
actions described in the application for permit 06-111-01r involve the
release of a transgenic, endemic plant pathogen, *B. glumae*. APHIS
analysis of the conditions proposed in the permit applications suggests
that these actions constitute a confined field release and thus are
categorically excluded actions under 7 CFR 372. However, the species
being considered has not been previously considered for a release
permit, and in its untransformed state is a plant pest of rice. Thus,
following 7 CFR 372.5(d)(4) APHIS is preparing an EA to address the
confined release of this new species that raises new issues.

# 

# II. NEED FOR THE PROPOSED ACTION

The purpose of this EA is to assess any potential adverse environmental
effects of a field study involving transgenic *Burkholderia glumae* in
East Baton Rouge Parish, Louisiana. The permit application was received
by APHIS BRS on April 21, 2006, submitted by Dr. Milton C. Rush,
Department of Plant Pathology and Crop Physiology, Louisiana State
University, Baton Rouge, East Baton Rouge Parish, Louisiana. The
application number is 06-111-01r. Under APHIS regulations, the receipt
of a permit application to introduce a GE organism requires a response
from the Administrator:

*Administrative action on applications*. After receipt and

review by APHIS of the application and the data submitted pursuant

to paragraph (a) of this section, including any additional information

requested by APHIS, a permit shall be granted or denied. 7 CFR §
340.4(e)

# III. ALTERNATIVES

## A. No Action

Under APHIS BRS regulations, the Administrator must either grant or deny
permits properly submitted under 7 CFR part 340. For the purposes of
this EA, the No Action alternative would be the denial of permit
application 06-111-01r, which would prevent any environmental release of
the two transgenic *B. glumae* strains.

## 

## B. Issue the Permit as Received

Issuing this permit as received would allow the field release to proceed
at the Louisiana State University AgCenter Central Research Station in
East Baton Rouge Parish, LA, under the conditions provided by the
applicant (see below, conditions a-p) and the standard permit conditions
under 7 CFR §340.4. Under this alternative, APHIS BRS would authorize
the field release of the GE *B. glumae* at the specified location with
no supplemental conditions implemented.

The following redundant mitigation measures are incorporated into the
experimental procedures by the applicant to promote a confined field
release, contain seed infected with transgenic *B. glumae*, and ensure
the least amount of harm to the environment:

a.  The field site is on land owned by Louisiana State University (LSU)
    and is expected to provide adequate physical security.
    Non-university visitors are monitored.

b.  The 0.4 acre field site containing inoculated rice plants and
    non-inoculated border plants will be located in an isolated levied
    area and irrigated separately to prevent accidental contamination
    and release of inoculated seed at harvest.

c.  The field site receiving the transgenic *B. glumae* is separated
    from other research areas by a 25 ft barren zone.

d.  Plants will be inoculated with the bacteria by spraying under
    conditions that do not favor drift. Spraying will be at 4 inches
    above the plants, to further minimize accidental drift.

e.  A tall rice variety, Wells, will be planted around each experimental
    treatment plot at the field site to further minimize drift from the
    bacterial inoculations (see Table 1).

f.  Plants outside the field site will be monitored for disease during
    and after the field trial, and if symptoms are observed, samples
    will be brought back to the lab for testing.

g.  Seed heads will be monitored daily for maturity during the seed
    ripening period.

h.  Rice seeds will be mechanically harvested by small-plot combine.
    After harvest, the combine will be moved by trailer from the field
    site to the steam cleaning station at the LSU AgCenter Central
    Research Station.

i.  The field site will be left fallow for next cropping season and will
    continue to be monitored daily until the next growing season begins
    (approximately March 15th).

j.  Harvested seeds will be transferred to the laboratory following
    container requirements in 7 CFR § 340.8.

k.  After harvest, the field site will be plowed to bury any unharvested
    seeds.

l.  Volunteer seedlings that emerge after the field trial will be
    sprayed with herbicide before flowering.

m.  Seed harvested for yield analysis will be burned at the LSU AgCenter
    Central Research Station burning site.

n.  Seed harvested for laboratory analysis will be destroyed by
    autoclaving.

o.  Containers of bacterial cultures will be autoclaved after use in the
    field. Sprayers will be disinfected with ammonia solution and washed
    several times for decontamination. Tyvek suits and masks used for
    bacterial inoculations will also be autoclaved and discarded.

p.  Volunteer seedlings that emerge after the field trial will be
    sprayed with herbicide.

q.  No commercial rice is found in East Baton Rouge Parish.

## C. Issue the Permit with Supplemental Conditions

Issuing this permit with supplemental conditions would allow the release
to proceed at the Louisiana State University AgCenter Central Research
Station in East Baton Rouge Parish, LA where supplemental permit
conditions would be established based on APHIS' scientific analysis of
the permit application, input from the State of Louisiana, and public
comment from this EA. If warranted, based on environmental risk of
escape of the engineered organism, APHIS will require mitigating
measures to prevent spread of the organism outside the field release
area.

Under APHIS regulations, compliance with all mitigating measures is
required:

> *Permit conditions.* A person who is issued a permit and his/her
> employees or agents shall comply with the following conditions
> (Standard Permit Conditions, Appendix VI), and any supplemental
> conditions (Supplemental Permit Conditions, Appendix VII) which shall
> be listed on the permit, as deemed by the Deputy Administrator to be
> necessary to prevent the dissemination and establishment of plant
> pests 7 CFR 340.4(f)

Currently APHIS proposes to include duplicative safety measures to
promote a confined field release, contain seed infected with transgenic
*B. glumae*, and to ensure no significant harm to the environment. The
proposed supplemental conditions are listed at Section V., C. Issuance
of the Permit with Additional Conditions, below.

APHIS also proposes to include reporting requirements for the field
trial:

a.  Activity reports will be submitted 28 days after the release of
    transgenic *B. glumae* and the termination of the field test. The
    activity report will include approximate acreage of rice plants
    inoculated, total acreage of the field test and the actual release
    and termination date.

b.  Disposition of harvested material is to be included in the 6 month
    Field Test Report.

c.  Monitoring reports will be submitted within 3 months after the end
    of the monitoring period. The monitoring report will include dates
    of volunteer monitoring, number of volunteers observed, and any
    action taken to remove, destroy, or test volunteers for *B. glumae*
    infection.

# IV. Research Design for *Burkholderia glumae* Release

## 

## A. Purpose of the Research

The field experiment proposed by the applicant will provide information
on the pathogenicity of *B. glumae* and work toward developing control
methods to reduce yield loss caused by panicle blight. The experiment
will test the role of toxoflavin, a potential virulence factor for *B.
glumae* (Iiyama et al. 1995). The phytotoxicity of toxoflavin has been
demonstrated under greenhouse conditions, but has not yet been studied
under field conditions. The transgenic strains of *B. glumae* are
engineered to suppress toxoflavin production, causing the transgenic
strains to become avirulent (non-pathogenic), and not cause plant
disease. By comparing rice infected by transgenic, non-disease causing
strains of *B. glumae* to infection by both a native, wild-type strain
and an endemic avirulent strain of *B. glumae*, the data gathered during
the proposed field trial will help determine if toxoflavin production is
required for panicle blight disease.

## 

## B. Description of the Research

The experiment will be conducted on a single field site located at the
LSU AgCenter Central Research Station in East Baton Rouge Parish,
Louisiana. This experiment will examine the pathogenicity associated
with the wild-type bacterial pathogen, as compared to two transgenic,
avirulent strains and one naturally-occurring, avirulent strain of *B.
glumae*. Rice plants will be planted and subjected to one of five
experimental treatments: spraying with native, non-transgenic *B.
glumae* (strain 336gr-1); spraying with native, non-transgenic,
non-pathogenic *B. glumae* (strain AV); spraying with transgenic,
non-pathogenic *B. gluma*e strain BGM15, spraying with transgenic,
non-pathogenic *B. glumae* strain BGM16, and an unsprayed control plot.
Each treatment will be tested on two commercial rice varieties, Trenasse
and Cocodrie, for a total of 10 treatments (5 inoculation treatments x 2
cultivars). The experimental design will consist of a randomized
complete block design with 4 block replications, for a total of 40 plots
(10 treatments x 4 replicates of each treatment). Although the total
acreage of rice planted for this field trial is 0.4 acres, the total
acreage of rice that will receive inoculation of transgenic *B. glumae*
strains is 0.02 acres.

The standard treatment plot size for this experiment is 4 x 12 ft,
consisting of 7 rows, each with 18 cm spacing. Surrounding each
treatment plot will be a 4 x 12 ft border plot of the rice variety,
Wells, which is a tall variety planted to minimize drift during the
experimental inoculations (see Table 1). The disease intensity will be
recorded at 15 and 30 days after spraying (at panicle emergence). At
maturity, grains from the center 4 rows of each plot will be harvested
by small-plot combine and grain weight at 12 percent moisture will be
recorded.

# V. ENVIRONMENTAL CONSEQUENCES OF THE PROPOSED ACTION AND ALTERNATIVES 

## 

## A. Analysis of Issues, Consequences, and Theoretical Risks of Field Research using Transgenic Burkholderia glumae

### 

### Risk of Transgenic, Non-Pathogenic *Burkholderia glumae* to the Environment

The *Burkholderia glumae* strains (transgenic strains BGM15 and BGM16,
and wild-type strains 336gr-1 and AV) proposed for this release were
either derived from or were isolated from blighted rice panicles
collected in Louisiana. Thus, the strains to be released are native to
Louisiana and do not constitute bacteria novel to the proposed area of
release.

*Burkholderia glumae* is a plant pathogen and causal agent of panicle
blight in rice. Most members of the genus *Burkholderia* are soil
inhabiting bacteria. Only a few *Burkholderia* species (e.g.
*Burkholderia cepacia*) are considered opportunistic human pathogens in
immuno-compromised individuals. Recently, a report cited *B. glumae* as
the likely disease-causing agent in a single immuno-compromised
individual (Weinberg et al. 2006). However, *B. glumae* is a
naturally-occurring, ubiquitous organism endemic to and widely prevalent
in Louisiana that, prior to the Weinberg et al. (2006) study, had not
been implicated as a potential causal agent of adverse effects in
humans. It is highly unlikely that releasing both transgenic and
non-transgenic *B. glumae* over 0.04 acres during this field trial will
significantly increase inoculum levels as these bacterial strains are
already present in the environment, Further, the field site proposed for
this release is under control of LSU and is not open to the general
public. Therefore, it is highly unlikely that this field trial will
significantly increase the risk of *B. glumae* infection in susceptible
individuals.

The addition of 216 base pairs of the methyltransferase gene inserted
into the transgenic strains of *B. glumae* results in the disruption of
the gene sequences that produce disease-causing toxoflavin (see Appendix
1: Biology of *Burkholderia glumae* and Appendix 2: Description of the
Regulated Article for more information) as shown in greenhouse studies
(see 3. Alteration in Susceptibility to Disease and Insects, below). As
the addition of this DNA to *B. glumae* will result in the suppression
of toxoflavin production, the transgenic *B. glumae* strains proposed
for release are not expected to cause plant disease. Consequently, the
engineered strains are expected to have no adverse effect on the
environment as these strains are non-pathogenic. In the unlikely event
that individual transgenic *B. glumae* bacteria revert and begin
producing toxoflavin, the resulting *B. glumae* will be no more
infectious than the wild-type, native *B. glumae* endemic to Louisiana.

Because the strains to be released are native to and widely prevalent in
Louisiana, the inoculum load will not be significantly increased
compared to the environmental baseline, and the transgenic modification
to the *B. glumae* strains will not result in strains that are more
virulent than the endemic *B. glumae* strains already present in the
environment, APHIS concludes that the introduction of the transgenic,
non-pathogenic *Burkholderia glumae* strains will not significantly
affect the environment.

### 2. Persistence of Transgenic *Burkholderia glumae*

*Burkholderia glumae* is transmitted by infected seed (Sayler et al.
2006). Infected rice seeds collected at the termination of the
experiment are unlikely to be sources of inoculum for future rice
infections because of the numerous confinement measures described in
above text and APHIS inspections. During the inoculation of the test
plants, the applicant proposes an infection strategy (see III. C. 2.
Description of Research, above, and Table 1) that minimizes drift and
significantly reduces the potential of *B. glumae* persistence outside
the release site. The applicant will also monitor plants that border the
release site. If any symptoms are observed, a sample will be brought to
the lab and tested for the presence of *B. glumae*.

In Korea, *B. glumae* infection was recently attributed to bacterial
wilt symptoms often seen in Korean plants \[peppers, tomato, potato,
eggplant, sesame, perilla (*Perilla frutescens*) and sunflower\] and
usually ascribed to *Ralstonia* infection (Jeong et al. 2003). In the
United States , *B. glumae* has no known hosts other than rice (Rush,
M.C., *pers. communication*). Because East Baton Rouge Parish is not a
commercial rice producing Parish, and *B. glumae* is not known to
survive in hosts other than rice in the United States, there will be no
host available for *B. glumae* infection after the field test. The
applicant will also monitor for and remove volunteer rice plants after
the release to ensure that transgenic *B. glumae* does not persist in
the local area after the field test is terminated.

Thus, APHIS concludes that it is highly unlikely for transgenic strains
of *B. glumae* to persist in the environment. Additionally, in the
unlikely event that transgenic *B. glumae* strains remain in the
environment after the field trial, the transgenic, avirulent *B. glumae*
strains are not likely to have a selective advantage over virulent,
native strains and, therefore, are unlikely to persist.

### 3. Alteration in Susceptibility to Disease or Insects

The transgenic strains of *B. glumae* were assessed under greenhouse
conditions by artificial inoculation to test their ability to cause
panicle blight. The transgenic strains did not show any sheath lesions
or panicle blight symptoms, whereas inoculation with the wild-type
strain produced typical panicle blight symptoms (Rush, M.C., *pers.
communication*). Thus, the transgenic *B. glumae* strains are less
virulent than the wild-type, native strain and are unlikely to cause an
increase in disease susceptibility to rice plants in the field. There
have been no reports of increased insect susceptibility in rice plants
during times of increased panicle blight disease pressure.

Execution of the prescribed periodic monitoring of the field site will
allow the detection of any unexpected infestation by plant disease
organisms or animal pests. The applicant is required to report any such
unanticipated effects to APHIS under the terms of the permit. See 7 CFR
§ 340.4(f)(10)(ii).

### 4. Horizontal Gene Transfer to Other Organisms 

Besides the gene segment that renders the transgenic *Burkholderia
glumae* strains avirulent, these non-pathogenic strains also contain two
antibiotic resistance genes as selectable markers. Expression of
neomycin phosphotransferase *(nptII)* confers tolerance to the
antibiotic kanamycin, and expression of beta-lactamase (*bla)* confers
tolerance to the antibiotic ampicillin. The *nptII* gene is devoid of
inherent plant pest characteristics (Fuchs et al. 1993), as is the *bla*
gene. The Environmental Protection Agency has granted an exemption from
the requirement of a tolerance for NPTII and the nucleic acids necessary
for its production in plants (FR 59 49353).

Horizontal gene transfer and expression of DNA from the transgenic *B.
glumae* strains to other bacteria species are unlikely to cause an
increase in antibiotic resistance as antibiotic resistance genes are
derived from and/or widely prevalent in enteric and soil-borne bacteria
(Van Dijck and van de Voorde 1976, DeBoy et al. 1980, Jensen et al.
2001, Sengelov et al. 2003). Under natural conditions, these antibiotic
resistance genes can be transferred to related microorganisms (Wilson
and Salyers 2003). Gene transfer from transgenic *B. glumae,* should not
be any different than what occurs naturally. While horizontal gene
transfer is widespread between microorganisms, such transfer from
microorganisms to animals and plants is highly unlikely under the
conditions of this field test (Syvanen 1999).

Therefore, in the unlikely event that the genes for kanamycin resistance
and ampicillin resistance would migrate to other bacteria outside the
field trial, APHIS concludes there would be no significant impact to the
environment.

### 5. Fate of Transgenic DNA

As rice plants infected with transgenic *Burkholderia glumae* will not
be used for food or feed, the information presented in this section is
for the unlikely event of accidental consumption.

Transgenic DNA is no different from other DNA consumed as part of the
normal diet. GE organisms have been used in drug production and
microbial fermentation (cheese and yogurt) since the late 1970\'s. More
than 1.4 billion cumulative acres of engineered food and feed crops have
been grown and consumed world wide in the past 7 years (International
Service for the Acquisition of Agri-biotech Applications, (ISAAA) at:
<http://www.isaaa.org/Resources/Publications/briefs/35/executivesummary/default.html>[)]{.underline}.
The FDA has not reported any significant concerns with bioengineered
food and feed currently on the market. Because of a lack of toxicity,
EPA has exempted from the requirement of a tolerance DNA that encodes
currently registered plant incorporated protectants (FR 66 37817-37830).

There have been several studies in humans and animals following the fate
of DNA once consumed (Mercer et al. 1999, Beever and Kemp 2000, Duggan
et al. 2000, Einspanier et al. 2001, Chambers et al. 2002, Netherwood et
al. 2002, Duggan et al. 2003). The majority of DNA consumed is degraded
in the gastro-intestinal tract although the degradation is not 100
percent efficient. There is evidence that DNA from consumed food can
move from the GI tract lumen to other areas of the body and that this is
a normal occurrence. No risks have been identified as a result of this
movement.

### 6. Potential Impacts on Humans, Including Minorities, Low Income Populations, and Children

The *B. glumae* strains used in this experiment are endemic to and
widely prevalent in Louisiana. Because the field site is on property
owned by Louisiana State University and is a confined field test, the
general public will not be exposed to the transgenic bacteria. Due to
the small size of the field trial (0.4 acres, of which only 0.02 acres
will be inoculated with transgenic *B. glumae*, see Table 1), and the
fact that *B. glumae* is endemic to and prevalent in Louisiana, the
amount of *B. glumae* proposed to be released will not result in a
significant increase in inoculum load. Infected rice seeds collected at
the termination of the experiment are unlikely to be mixed with any
seeds intended for human or animal consumption because of the numerous
measures described in above text and APHIS inspections during
harvesting. The applicant will store all the harvested seeds in
dedicated storage bags on site and will transfer seeds to a laboratory
setting for seed cleaning and analysis.

Consideration of these potential impacts are specified in Executive
Orders 13045 and 12898 and address the identification of health or
safety risks that might disproportionately affect children or have
adverse impacts on minorities and low-income populations. The proposed
actions are not expected to adversely affect any of these groups.

### 7. Risks to Threatened and Endangered Species and Designated Critical Habitat

APHIS has reached a determination that the proposed environmental
release under permit 06-111-01r would have no effect on federally listed
threatened or endangered species (TES), or species proposed for listing,
and no effect on designated critical habitat or habitat proposed for
designation in the action area. Consequently, consultation under Section
7 of the Endangered Species Act with U.S. Fish and Wildlife Service is
not required for the action described in permit 06-111-01r and
supplemental conditions proposed by APHIS. Appendix 3 includes the APHIS
analysis of TES and critical habitat in the area of the field release.

### 8. Effects on Native Floral and Faunal Communities

[a. Native Floral Communities]{.underline}

The field site proposed in the permit application and the surrounding
fields have a history of use as an agricultural research experiment
station, in association with Louisiana State University. As the
conditions imposed by the applicant and APHIS are expected to confine
transgenic *B. glumae* and infected rice plants to the field site, APHIS
concludes there would be no significant effect on any native floral
species. Additionally, rice is the only known host of *B. glumae* in the
United States. In the highly unlikely event that *B. glumae* would move
outside the field site, non-transgenic, virulent *B. glumae* is already
present in the environment. The addition of non-pathogenic *B. glumae*
would not significantly increase the inoculum load already present in
the environment, and does not present a plant disease risk.

[b. Terrestrial Vertebrate Animals]{.underline}

Vertebrate animals are not affected by the plant pathogen *B. glumae*.
Thus, any accidental consumption of rice leaves or seeds infected with
transgenic *B. glumae* during the field trial will not result in animal
disease. Additionally, as the bacteria strains proposed for the field
release were collected from Louisiana, typical vertebrate residents have
been previously exposed to *B. glumae*. Thus, APHIS concludes there
would be no significant effect on any terrestrial vertebrate animal
species.

[c. Terrestrial Invertebrate Animals]{.underline}

The most likely invertebrate animals exposed to the transgenic *B.
glumae* would be seed-eating or plant-eating invertebrates. However, *B.
glumae* is not an invertebrate pathogen. Additionally, as the bacteria
strains proposed for the field release were collected from Louisiana,
typical invertebrate residents have been previously exposed to *B.
glumae*. Thus, APHIS concludes there would be no significant effect on
any invertebrate species.

[d. Aquatic Organisms]{.underline}

The field site, as commonly practiced in rice growing regions, includes
a levy irrigation system. However, *B. glumae* is not a
water-transmissible pathogen, nor a pathogen of aquatic organisms, and
is thus highly unlikely to affect aquatic organisms that inhabit the
levies within the proposed release site. APHIS therefore concludes there
would be no significant effect on any aquatic species.

### 9. Impact on Existing Agricultural Practices

The strains of *B. glumae* used for this field trial were collected in
Louisiana, where it is widely prevalent. *B. glumae* has also been
collected from Texas and Arkansas. The proposal field site is on land
owned by LSU, and rice is not commercially produced in East Baton Rouge
Parish. The confinement measures as given by the applicant and the
supplemental permit conditions proposed by APHIS should confine the
transgenic *B. glumae* to the field site. Therefore, APHIS concludes
that this small field test will not increase the incidence of plant
disease and will not have any significant impact on existing
agricultural practices.

### 10. Cumulative Environmental Effects

The only past, present, and reasonably foreseeable actions associated
with the location for the proposed release are those related to the
release of avirulent, transgenic *B. glumae*; release of native,
non-transgenic *B. glumae*; and agricultural production of rice. As
stated previously, APHIS has determined that there is no significant
impact to the environment due to the release of transgenic *B. glumae*
strains because: (1) the non-transgenic versions of these strains were
originally collected from, and are native to, Louisiana; (2) the
inoculum load will not be significantly increased compared to the
environmental baseline; (3) the transgenic bacteria will not persist in
the environment; (4) and the transgenic modification to the *B. glumae*
strains will not result in strains that are more virulent than the
endemic *B. glumae* strains already present. Additionally, there is no
significant impact on disease or insect susceptibility for rice plants
inoculated with transgenic *B. glumae*, no effect of the release of
transgenic *B. glumae* on federally listed threatened or endangered
species, or species proposed for listing, and no effect on designated
critical habitat or habitat proposed for designation in the action area,
or any animal or plant species in the proposed area of release. The
proposed field site is the LSU Ag Center Central Research Station, which
has been in agricultural production for the past 20 years, and will
likely remain in agricultural cultivation for the foreseeable future.
Therefore, APHIS has determined that there are no past, present, or
reasonably foreseeable actions that would aggregate with effects of the
proposed action to create cumulative impacts or in any way reduce the
long-term productivity or sustainability of any of the resources (soil,
water, ecosystem quality, biodiversity, etc.) associated with the
release site or the ecosystem in which it is situated.

### 11. Consistency of proposal with other environmental requirements

The proposal is believed to be consistent with other environmental
requirements. This environmental assessment was prepared in accordance
with: (1) The National Environmental Policy Act of 1969 (NEPA), as
amended (42 U.C § 4321 et seq.); (2) regulations of the Council on
Environmental Quality for implementing the procedural provisions of NEPA
(40 CFR §§ 1500-1508); (3) USDA regulations and implementing NEPA (7 CFR
§ 1b); and (4) APHIS NEPA Implementing Procedures (7 CFR § 372).

## 

## B. Analysis of the Alternatives

### 1. Deny the Permit

Denying the permit application would have no expected potential adverse
environmental impacts, would prevent the field research from proceeding,
and prevent any benefits associated with the knowledge gained from this
research study.

### 2. Issuance of the Permit as Received

The proposed action is not expected to have any adverse environmental
impacts for the following biological and physical reasons:

> a\. No adverse consequences to non-target organisms or environmental
> quality are expected from the field release of the transgenic bacteria
> strains.
>
> b\. Neither the genes introduced, nor the proteins produced by these
> bacteria are expected to have toxicological or allergenic properties.
>
> c\. None of the genes introduced, nor the protein produced in these
> bacteria provide the engineered, non-pathogenic bacteria with any
> selective advantage over non-engineered bacteria in the ability to be
> disseminated or to become established in the environment.

### 3. Issuance of the Permit with Additional Conditions

The proposed action is not expected to have any adverse environmental
impacts for the following biological and physical reasons:

> a\. No adverse consequences to non-target organisms or environmental
> quality are expected from the field release of these transgenic
> bacteria.
>
> b\. Neither the genes introduced, nor the proteins produced by these
> bacteria are expected to have toxicological or allergenic properties.
>
> c\. None of the genes introduced, nor the protein produced in these
> bacteria provide the engineered, non-pathogenic bacteria with any
> selective advantage over non-engineered bacteria in the ability to be
> disseminated or to become established in the environment.

Under this alternative, APHIS proposes to include the following
duplicative safety measures to promote a confined field release and
ensure no significant harm to the environment:

a.  Bacterial liquid inoculum will be doubly contained in water tight
    containers for transport to and from the field site to ensure that
    wild-type and transgenic strains of *B. glumae* will remain
    contained during transport.

b.  After inoculation in the field, Tyvek suits and sprayers used during
    inoculation will be doubly contained in plastic bags before leaving
    the field site to ensure that wild-type and transgenic strains of
    *B. glumae,* that may be present on applicator clothing and in
    sprayers, remain contained after spraying and during transport to
    and from the field site.

c.  Combines used to harvest seed will be brushed down at the field site
    before transport to steam-cleaning site to limit the accidental
    release of seeds potentially infected with transgenic *B. glumae*
    during transport of the combine from the field site to the
    steam-cleaning site at the LSU AgCenter Central Research Station.

d.  Harvested seed will be contained via 7 CFR § 340.8 during transport
    to the weighing facility at the LSU AgCenter Central Research
    Station to limit the accidental release of seeds potentially
    infected with transgenic *B. glumae* during transport of harvested
    seeds to the weighing facility at the LSU AgCenter Central Research
    Station.

e.  All plants within the field site will be treated as regulated
    articles to ensure the appropriate disposition of rice infected with
    transgenic *B. glumae*.

### 4. Preferred Alternative

APHIS has chosen Alterative C, issue the permit with supplemental
conditions, as the preferred alternative, in order to implement
supplemental conditions and reporting requirements.

Preparer and Agency Contact

Preparer:

Andrea F. Huberty, Ph.D.

Biotechnologist

USDA, APHIS

Biotechnology Regulatory Services

4700 River Road, Unit 147

Riverdale, MD 20737

Phone: 301-734-0659

Fax: 301-734-0767

Email: <Andrea.F.Huberty@aphis.usda.gov>

Agency Contact

Cynthia Eck

USDA, APHIS

Biotechnology Regulatory Services

4700 River Road, Unit 147

Riverdale, MD 20737

Phone: 301-734-0667

FAX: 301-734-0767

Email: <Cynthia.A.Eck@aphis.usda.gov>

# REFERENCES

# Table 1. 

# 

The table below is a description of the plot layout for the proposed
field site described in 06-111-01r. The rice variety Wells \[W\] will be
planted to surround each of the plots that receive an experimental
treatment. No plants will be inoculated in these plots. Two rice
varieties are proposed to receive experimental treatments, Tenasse
(columns T-I to T-IV) and Cocodrie (columns C-I to C-IV). There are 5
proposed experimental treatments: a non-inoculated control \[Cont
(T-1)\]; inoculation with wild-type, pathogenic *B. glumae* (strain
336gr-1) \[Wild (T-2\]; inoculation with transgenic, avirulent *B.
glumae* strain BGM15 \[M-1 (T-3)\]; inoculation with transgenic,
avirulent *B. glumae* strain BGM16 \[M-2 (T-4)\]; or inoculation with a
naturally-occurring avirulent *B. glumae* strain (AV) \[AV (T-5)\]. The
total acreage of the field test is 0.4 acres, only 0.02 acres of which
will be inoculated with transgenic *B. glumae* strains.

<table>
<colgroup>
<col style="width: 4%" />
<col style="width: 6%" />
<col style="width: 5%" />
<col style="width: 6%" />
<col style="width: 5%" />
<col style="width: 6%" />
<col style="width: 5%" />
<col style="width: 6%" />
<col style="width: 5%" />
<col style="width: 6%" />
<col style="width: 5%" />
<col style="width: 6%" />
<col style="width: 5%" />
<col style="width: 6%" />
<col style="width: 5%" />
<col style="width: 7%" />
<col style="width: 5%" />
</colgroup>
<tbody>
<tr class="odd">
<td></td>
<td><blockquote>
<p>T-I</p>
</blockquote></td>
<td></td>
<td><blockquote>
<p>T-II</p>
</blockquote></td>
<td></td>
<td><blockquote>
<p>T-III</p>
</blockquote></td>
<td></td>
<td><blockquote>
<p>T-IV</p>
</blockquote></td>
<td></td>
<td><blockquote>
<p>C-I</p>
</blockquote></td>
<td></td>
<td><blockquote>
<p>C-II</p>
</blockquote></td>
<td></td>
<td><blockquote>
<p>C-III</p>
</blockquote></td>
<td></td>
<td><blockquote>
<p>C-IV</p>
</blockquote></td>
<td></td>
</tr>
<tr class="even">
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
</tr>
<tr class="odd">
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>Cont</strong></p>
<p><strong>(T-1)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>M-1</strong></p>
<p><strong>(T-3)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>AV</strong></p>
<p><strong>(T-5)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>M-1</strong></p>
<p><strong>(T-3)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>Cont</strong></p>
<p><strong>(T-1)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>M-1</strong></p>
<p><strong>(T-3)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>AV</strong></p>
<p><strong>(T-5)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>M-1</strong></p>
<p><strong>(T-3)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
</tr>
<tr class="even">
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
</tr>
<tr class="odd">
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>Wild</strong></p>
<p><strong>(T-2)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>AV</strong></p>
<p><strong>T-5</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>Wild</strong></p>
<p><strong>(T-2)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>Cont</strong></p>
<p><strong>(T-1)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>Wild</strong></p>
<p><strong>(T-2)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>AV</strong></p>
<p><strong>T-5</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>Wild</strong></p>
<p><strong>(T-2)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>Cont</strong></p>
<p><strong>(T-1)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
</tr>
<tr class="even">
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
</tr>
<tr class="odd">
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>M-1</strong></p>
<p><strong>(T-3)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>M-2</strong></p>
<p><strong>(T-4)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>Cont</strong></p>
<p><strong>(T-1)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>M-2</strong></p>
<p><strong>(T-4)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>M-1</strong></p>
<p><strong>(T-3)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>M-2</strong></p>
<p><strong>(T-4)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>Cont</strong></p>
<p><strong>(T-1)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>M-2</strong></p>
<p><strong>(T-4)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
</tr>
<tr class="even">
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
</tr>
<tr class="odd">
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>M-2</strong></p>
<p><strong>(T-4)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>Wild</strong></p>
<p><strong>(T-2)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>M-1</strong></p>
<p><strong>(T-2)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>AV</strong></p>
<p><strong>(T-5)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>M-2</strong></p>
<p><strong>(T-4)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>Wild</strong></p>
<p><strong>(T-2)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>M-1</strong></p>
<p><strong>(T-2)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>AV</strong></p>
<p><strong>(T-5)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
</tr>
<tr class="even">
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
</tr>
<tr class="odd">
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>AV</strong></p>
<p><strong>(T-5)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>Cont</strong></p>
<p><strong>(T-1)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>M-2</strong></p>
<p><strong>(T-4)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>Wild</strong></p>
<p><strong>(T-2)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>AV</strong></p>
<p><strong>(T-5)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>Cont</strong></p>
<p><strong>(T-1)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>M2</strong></p>
<p><strong>(T-4)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>Wild</strong></p>
<p><strong>(T-2)</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
</tr>
<tr class="even">
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
<td><blockquote>
<p><strong>W</strong></p>
</blockquote></td>
</tr>
</tbody>
</table>

# 

# APPENDIX 1: Biology of *Burkholderia glumae*

*Burkholderia* *glumae* are rod-shaped, motile, gram-negative,
obligately aerobic bacteria commonly found in soil worldwide. Originally
placed in the genus *Pseudomonas*, Urakami et al. (1994) presented
morphological, biochemical, and physiological data that moved
*Pseudomonas glumae* into the *Burkholderia* genus (Urakami et al.
1994). Further taxonomic analysis established the similarity between *B.
glumae*, *B. gladioli*, and *B. plantarii* (Coenye et al. 2001, Maeda et
al. 2006). Accordingly, an analysis also established that *B. glumae* is
not within the *B. cepacia* complex of human pathogens (Coenye et al.
2001).

In the United States, *B. glumae* causes panicle blight in rice,
resulting in sterile spikelets (Sayler et al. 2006) with brown streaks
on the paleae and lemmata (Iiyama et al. 1995) and chlorosis of grain on
infected panicles (Suzuki et al. 2004). *B. glumae* invades rice
spikelets by entering the stomata of the epidermis of the glume (Hikichi
et al. 1994) and grows epiphytically through the booting stage (Goto
1992, Sayler et al. 2006). Only plumules in germinating seeds (causing
seedling rot in nursery produced rice plants in Asia (Cottyn et al.
1996)) and spikelets during heading (causing panicle blight) are
susceptible to *B. glumae* infection (Hikichi et al. 1994). Invasion of
one bacterial cell is enough to complete spike infection (Hikichi et al.
1994) and the bacteria also multiplies rapidly on emerging panicles and
infects flowers just after emergence (Goto 1992, Sayler et al. 2006).
Pathogen transmission is through infected seed (Sayler et al. 2006).

*Burkholderia glumae* produces a yellowish substance that has been
identified as the phytotoxin toxoflavin (Azegami et al. 1987, Suzuki et
al. 1998b). Toxoflavin production results in significant rice damage to
inoculated plants (Azegami et al. 1987). When non-toxoflavin producing
mutants were inoculated on rice plants, no disease symptoms developed,
indicating that toxoflavin production by *B. glumae* is a virulence
factor for *Burkholderia glumae* (Iiyama et al. 1995) and plays a
significant role in disease development (Suzuki et al. 1998b).
Toxoflavin production in *B. glumae* is limited to ambient temperatures
of 30º to 40º C (Suzuki et al. 1998b), which is consistent with
infection occurring predominately during high temperatures and high
humidity (Suzuki et al. 2004).

Toxoflavin is produced in *Burkholderia* by an operon consisting of the
*tox* gene cluster (*toxABCDE*) and the *toxR* gene (Shingu and Yoneyama
2004, Suzuki et al. 2004). Disruption of the *toxA* gene
(methyltransferase) results in mutants that do not produce toxoflavin
(Suzuki et al. 1998a) and do not cause chlorosis of the rice grain
(Suzuki et al. 2004).

# APPENDIX 2: Description of the Regulated Bacteria

Transformed *Burkholderia glumae* strains (BGM15 and BGM16, derived from
wild-type strains) were developed in Dr. M. C. Rush's laboratory,
Department of Plant Pathology and Crop Physiology, Louisiana State
University, Baton Rouge, LA. The vector plasmid was purchased from
Qiagen, Inc., in Valencia, CA.

The transgenic *B. glumae* strains were created by placing 216 base
pairs of the methyltransferase (*toxA*) gene from *B. glumae* into the
pDRIVE cloning vector (Qiagen, Inc.). The introduced vector, along with
the methyltransferase gene will integrate into the bacterial chromosome
by homologous recombination. Disruption of the *tox* operon results in a
non-toxoflavin producing strain. The system does not require the use of
the plant pathogen, *Agrobacterium tumefaciens*, or other transformation
vectors. The cloning vector also contains two selectable markers, the
gene (*nptII*) for neomycin phosphotransferase from *Streptomyces
kanamyceticus* and the gene (*bla*) for beta-lactamase from *Escherichia
coli*. The selectable markers provide resistance to kanamycin and
ampicillin, respectively. The promoter for each of the genes is the
Bacteriophage T7 promoter, and the terminator for each of the selectable
markers is a synthetic TAA codon sequence. The donor DNA sequences are
stably and irreversibly integrated into the bacterial genome, where they
are maintained and inherited as any other genes of the bacteria cell.

# APPENDIX 3: Threatened and Endangered Species Analysis

There is no designated critical habitat or proposed designated critical
habitat in the Parish of release (East Baton Rouge Parish, Louisiana)
(<http://crithab.fws.gov/>, accessed 03/30/2007). According to the Fish
and Wildlife Service
(<http://ecos.fws.gov/tess_public/StateListingAndOccurrence.do?state=LA>;
accessed on 03/30/2007) there are 20 federally listed threatened and
endangered animals occurring in Louisiana and 3 threatened and
endangered plant species in the state of Louisiana. Of those listed in
the state of Louisiana, the following are not found in East Baton Rouge
Parish (all websites accessed on 03/30/2007):

> Mammals

-   Louisiana Black Bear (*Ursus americanus luteolus*)
    (<http://www.fws.gov/endangered/i/a/saa9e.html>,
    http://www.natureserve.org/explorer/servlet/NatureServe?searchName=Ursus+americanus+luteolus)

-   Finback Whale (*Balaenoptera physalus*)

> (<http://www.natureserve.org/explorer/servlet/NatureServe?searchName=Balaenoptera+physalus>)

-   Humpback Whale (*Megaptera novaeangliae*)
    (<http://www.natureserve.org/explorer/servlet/NatureServe?searchName=Megaptera+novaeangliae>)

> Birds

-   Interior Least Tern (*Sterna antillarum*)
    (<http://www.natureserve.org/explorer/servlet/NatureServe?searchName=Sterna+antillarum>,
    <http://ecos.fws.gov/speciesProfile/SpeciesReport.do?spcode=B07N>)

-   Black-capped Vireo (*Vireo atricapillus*)
    (<http://www.natureserve.org/explorer/servlet/NatureServe?searchName=Vireo+atricapilla>)

-   Red-cockaded Woodpecker (*Picoides borealis*)
    (<http://www.natureserve.org/explorer/servlet/NatureServe?searchName=Picoides+borealis>)

-   Piping Plover (*Charadrium melodus*)
    (<http://www.natureserve.org/explorer/servlet/NatureServe?searchName=Charadrius+melodus>)

-   Bald Eagle (*Haliaeetus leucocephalus*)

Fish

-   Gulf Sturgeon (*Acipenser oxyrinchus desotoi*)
    (<http://www.natureserve.org/explorer/servlet/NatureServe?searchName=Acipenser+oxyrinchus+desotoi>)

-   Pallid Sturgeon (*Scaphirhynchus albus*)

> Reptiles

-   Green Sea Turtle (*Chelonia mydas*)
    (<http://www.natureserve.org/explorer/servlet/NatureServe?searchName=Chelonia+mydas>)

-   Hawksbill Sea Turtle (*Eretmochelys imbricata*)

> (<http://www.natureserve.org/explorer/servlet/NatureServe?searchName=Eretmochelys+imbricata>)

-   Kemp's Ridley Sea Turtle (*Lepidochelys kempii*)
    (<http://www.natureserve.org/explorer/servlet/NatureServe?searchName=Lepidochelys+kempii>)

-   Leatherback Sea Turtle (*Dermochelys coriacea*)
    (<http://www.natureserve.org/explorer/servlet/NatureServe?searchName=Dermochelys+coriacea>)

-   Loggerhead Sea Turtle (*Caretta caretta*)
    (<http://www.natureserve.org/explorer/servlet/NatureServe?searchName=Caretta+caretta>)

-   Gopher Tortoise (*Gopherus polyphemus*)
    (<http://www.natureserve.org/explorer/servlet/NatureServe?searchName=Gopherus+polyphemus>)

-   Ringed Map Turtle (*Graptemys oculifera*)
    (<http://www.natureserve.org/explorer/servlet/NatureServe?searchName=Graptemys+oculifera>)

> Invertebrates

-   Pink (Pearlymussel) Mucket (*Lampsilis abrupta*)
    (<http://www.natureserve.org/explorer/servlet/NatureServe?searchName=Lampsilis+abrupta>)

-   Louisiana Pearl Shell (*Margaritifera hembeli*)
    (<http://www.natureserve.org/explorer/servlet/NatureServe?searchName=Margaritifera+hembeli>)

> Plants -- None of these plants are sexually compatible with rice and
> *Burkholderia glumae* is not known to infect any species within the 3
> plant families to which these species belong.

-   American Chaffseed (*Schwalbea americana*)
    (<http://www.natureserve.org/explorer/servlet/NatureServe?searchName=Schwalbea+americana>)

```{=html}
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```
-   Louisiana Quillwort (*Isoetes louisianensis*)
    (<http://www.natureserve.org/explorer/servlet/NatureServe?searchName=Isoetes+louisianensis>)

-   *Geocarpon minimum*
    (<http://www.natureserve.org/explorer/servlet/NatureServe?searchName=Geocarpon+minimum>)

Three listed threatened and endangered species are found in East Baton
Rouge Parish:

The Alabama Heelsplitter is a mussel found only in Louisiana in the
Amite River in East Baton Rouge Parish. The Alabama Heelsplitter is not
found in the Mississippi River, which is approximately 2 kilometers from
the field site. This mussel requires riverine habitat and will not
colonize rice levees
(<http://ecos.fws.gov/speciesProfile/SpeciesReport.do?spcode=F01O>). The
Pallid Sturgeon is found in East Baton Rouge Parish, within the
Mississippi River
(<http://www.natureserve.org/explorer/servlet/NatureServe?searchName=Scaphirhynchus+albus>).
This sturgeon is a bottom-dwelling river fish, and will not be affected
by this small field trial in an area that is already under agricultural
production. Additionally, rice levees are not suitable habitat for
pallid sturgeon. The Bald Eagle is found in East Baton Rouge Parish
(<http://www.natureserve.org/explorer/servlet/NatureServe?searchName=Haliaeetus+leucocephalus>),
and is found primarily in habitats neighboring large bodies of water.
However, the Bald Eagle is a carnivore and does not feed on rice plants
or seeds. As transgenic *B. glumae* is not an animal pathogen, there are
no foreseeable indirect effects on the potential food resources for the
Bald Eagle due to this field trial. Thus the field release of
non-pathogenic, avirulent *B. glumae* will have no effect on federally
listed threatened and endangered species, species proposed for listing,
designated critical habitat or habitat proposed for designation.

# APPENDIX 4: Supplemental Permit Conditions

1.  Bacterial liquid inoculum will be doubly contained in water tight
    containers for transport to and from the field site.

2.  After inoculation in the field, Tyvek suits and sprayers used during
    inoculation will be doubly contained in plastic bags before leaving
    the field site.

3.  Combines used to harvest seed will be brushed down at the field site
    before transport to steam-cleaning site.

4.  Harvested seed will be contained via 7 CFR 340.8 during transport to
    the weighing facility at the LSU AgCenter Central Research Station.

5.  All plants within the field site will be treated as regulated
    articles.

6.  BRS should be notified in writing of any proposed changes to the
    permit application (or approved permit) including for example
    confinement protocols, transgenic lines or constructs, release
    sites, acreage, etc. Changes usually require amendments to the
    permit and must be pre-approved by BRS. Requests should be directed
    to Regulatory Permit Specialist, USDA APHIS BRS, Biotechnology
    Permit Services, 4700 River Road, Unit 147, Riverdale, Maryland
    20737.

7.  A BRS Regional Biotechnologist or a Plant Protection and Quarantine
    Officer may conduct an inspection of the test site at the beginning
    of the test. Additional inspections may be conducted throughout the
    permitted release.

8.  Harvested plant material may not be used for food or animal feed
    unless it is first devitalized and approved for such use by the U.S.
    Food and Drug Administration; and for plant-incorporated
    protectants, a tolerance for the pesticide must first be established
    by the U.S. Environmental Protection Agency.

9.  Send notices and all reports (CBI and CBI-deleted or non-CBI copies)
    to BRS by e-mail, mail, or fax.

> BRS E-mail:
>
> <BRSCompliance@aphis.usda.gov>
>
> BRS Mail:
>
> Animal and Plant Health Inspection Service (APHIS)
>
> Biotechnology Regulatory Services (BRS)
>
> Compliance and Inspection Branch
>
> 4700 River Rd. Unit 147
>
> Riverdale, MD 20737
>
> BRS Fax:
>
> Compliance and Inspection Branch
>
> \(301\) 734-8669

A.  **Activity Report**

> Within 28 calendar days after planting, submit a report, in paper
> format or electronically, that includes the following information for
> each field test site:

i.  Permit number;

ii. Regulated article;

iii. Release site \[provide state, county, internal identification
     number (if available), and either a single GPS coordinate as a
     reference point (center of plot or specify corner) or specific
     address\];

iv. Approximate number plants inoculated per transgenic strain;

v.  Total acreage of inoculated plants and border rows;

vi. The actual release date

```{=html}
<!-- -->
```
A.  **Field Test Report**

> Within 6 months after the expiration date of the permit, the permittee
> is required to submit a Field Test Report. Field Test Reports shall
> include:

i.  Constructs and specific transformed lines released;

ii. Inoculation and harvest dates;

iii. Total acreage of the test;

iv. The methods of observation;

v.  The resulting data and analysis regarding all deleterious effects on
    plants, non-target organisms, or the environment. This should
    include, but not be limited to, data on insect damage, disease
    susceptibility, gross morphology and any indications of weediness of
    the host plant.

vi. a table with the following information for each line and gene
    released:

  --------------------- ------------- ----------------- ------------------
  Site name (or GPS)    Crop          Harvest Date      Disposition of
                                                        Harvested Material

  --------------------- ------------- ----------------- ------------------

> Regarding disposition of harvested material, include the quantity of
> material harvested and the final location of the harvested material
> (i.e., destroyed in the field, processed on site or sent to an
> off-site facility, sent to the laboratory for analysis). If materials
> were moved, identify the location, what type of facility and quantity
> transported. Describe methods used to devitalize and dispose of
> regulated plant materials and what happened to residual material at
> the field site.
>
> We encourage the inclusion of other types of data if the applicant
> anticipates submission of a petition for determination of
> non-regulated status for their regulated article. APHIS considers
> these data reports as critical to our assessment of plant pest risk
> and development of regulatory policies based on the best scientific
> evidence. [Failure by an applicant to provide data reports in a timely
> manner for a field trial may result in the withholding of permission
> by APHIS for future field trials.]{.underline}

B.  **Monitoring Report**

> The report must include:

i.  Dates when the field site and perimeter fallow zone were inspected
    for volunteers;

ii. Number of volunteers observed;

iii. Any actions taken to remove or destroy volunteers.

```{=html}
<!-- -->
```
C.  This report may be included as part of the Field Test Report
    submitted within 6 months of the expiration date of the permit. If
    the volunteer monitoring period continues beyond the date of
    submission of the Field Test Report, a separate report should be
    submitted. The final monitoring report is then due no later than 3
    months from the end of the volunteer monitoring period.

```{=html}
<!-- -->
```
10. APHIS shall be notified orally within 24 hours followed by a written
    notification within 5 days upon discovery in the event of any
    accidental or unauthorized release of the regulated article.

    A.  For immediate oral notification, contact APHIS BRS Compliance
        Staff at (301) 734-7324 and ask to speak to Compliance and
        Inspection staff member.

    B.  In the event of an emergency and you are unable to reach the BRS
        Compliance Staff at the above number, you may call:

> The APHIS/BRS Regional Biotechnology Coordinator assigned to the State
> where the field test occurs.
>
> **[For Western Region]{.underline}**, contact Ralph Stoaks by phone at
> (970) 494-7573 or e-mail <Ralph.D.Stoaks@aphis.usda.gov>
>
> **[For Eastern Region]{.underline}**, contact Ashima SenGupta by phone
> at (919) 855-7622 or e-mail
> [Ashima.SenGupta]{.underline}[\@aphis.usda.gov](mailto:Roger.L.Holman@aphis.usda.gov)
>
> Or
>
> The APHIS PPQ Regional Biotechnology Coordinator assigned to the State
> where the field test occurs
>
> **[For Western Region]{.underline}**, contact Stacy E. Scott by phone
> at (970) 494-7577 or e-mail <Stacy.E.Scott@aphis.usda.gov>
>
> **[For Eastern Region]{.underline}**, contact Susan Dublinski by phone
> at (919) 855-7324 or e-mail
> [Susan.G.Dublinski]{.underline}[\@aphis.usda.gov](mailto:?????????@aphis.usda.gov)
>
> Or
>
> The APHIS State Plant Health Director assigned of the State where the
> field test occurs. The list of APHIS State Plant Health Directors is
> available at <http://ceris.purdue.edu/napis/names/sphdXstate.html>
>
> Louisiana: William Spitzer, Baton Rouge
>
> Phone: (225) 298-5410
>
> Fax: (225) 298-5415
>
> Email: <william.e.spitzer@aphis.usda.gov>

C. Written notification should be sent:

> [By e-mail]{.underline}:
>
> <BRSCompliance@aphis.usda.gov>
>
> [By mail]{.underline}:
>
> Animal and Plant Health Inspection Service (APHIS)
>
> Biotechnology Regulatory Services (BRS)
>
> Compliance and Inspection Branch
>
> 4700 River Rd. Unit 147
>
> Riverdale, MD 20737

11. This approved Biotechnology Permit (APHIS Form 2000) does not
    eliminate the permittee's legal responsibility to obtain all
    necessary Federal and State approvals, including: (1) for the use of
    any non-genetically engineered plant pests or pathogens as challenge
    inoculum; (2) plants, plant parts or seeds which are under existing
    Federal or State quarantine or restricted use; (3) experimental use
    of unregistered chemical; and (4) food or feed use of genetically
    engineered crops harvested from the field experiment.