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Finding of No Significant Impact and Decision Notice

Animal and Plant Health Inspection Service
Issuance of Permit to Release Genetically-Engineered Burkholderia glumae

The Animal and Plant Health Inspection Service (APHIS) of the United States Department of Agriculture (USDA) has received a permit application (APHIS number 06-111-01r) from Dr. Martin Rush at the Louisiana State University to conduct a field trial using strains of the bacterium Burkholderia glumae. Permit application 06-111-01r describes four Burkholderia glumae strains: Two wild-type strains endemic to the United States, one of which causes panicle blight on rice and the other naturally non-pathogenic, and two genetically engineered, non-pathogenic strains that share the same avirulent phenotype. A description of the field tests may be found in the attached Environmental Assessment (EA), which was prepared pursuant to APHIS regulations (7 CFR part 372) promulgated under the National Environmental Policy Act. The field tests are scheduled to begin in August 2007 in East Baton Rouge Parish, Louisiana.

An EA was prepared and submitted for public comment for 30 days. One comment that raised 5 issues was received and the issues are addressed in an attachment to this document.

APHIS proposed three different actions to take in response to the permit application:
• the denial of the permit (Alternative A) • the granting of the permit with no Supplemental Permit Conditions
(Alternative B)
• the granting of the permit with Supplemental Permit Conditions containing duplicative safety measures and reporting requirements (Alternative C)
APHIS chose Alternative C as its Preferred Alternative.

Based upon analysis described in the EA, APHIS has determined that the action proposed in Alternative C will not have a significant impact on the quality of the human environment because:

1. 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 and prevalent in Louisiana and do not constitute bacteria novel to the proposed area of release.
2. It is highly unlikely that the small additional input of B. glumae for this field trial in an area of high B. glumae density will provide significantly greater inoculum to the environment, and significantly increase the risk of B. glumae infection in susceptible individuals.
3. Transgenic B. glumae is not likely to persist in the environment. During the inoculation of the test plants, the applicant proposes an infection strategy that significantly reduces the potential of B. glumae leaving the field trial and

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persisting in the environment. The infection design utilizes border rows of a tall rice variety and allows inoculation only during non-windy conditions to minimize drift. 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. 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.
4. 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.
5. There have been no reports of increased insect susceptibility in rice plants during times of increased panicle blight disease pressure.
6. 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. B. glumae is a rice pathogen and may also inhabit soil. B. glumae may naturally transfer the antibiotic resistance genes to other soil bacteria. The antibiotic resistance genes were originally derived from soil-borne bacteria; thus are likely to already be present in Louisiana soils. Therefore, the presence of these genes in B. glumae is unlikely to cause an increase in the prevalence of antibiotic resistance genes in the environment because the soil-dwelling microorganisms likely already carry these antibiotic resistance genes.
7. Horizontal gene transfer from microorganisms to animals and plants is highly unlikely under the conditions of this field test.
8. 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.
9. 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 they will be contained during movement between field and laboratory, and devitalized after analysis by burning or autoclaving.
10. 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.
11. 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.
12. 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

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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.
13. 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.
14. This small field test will not increase the incidence of plant disease and will not have any significant impact on existing agricultural practices.
15. 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.

Because APHIS has reached a finding of no significant impact of this field release of transgenic Burkholderia glumae, no Environmental Impact Statement will be prepared regarding this decision.

Pursuant to its regulations (7 CFR part 340) promulgated under the Plant Protection Act of 2000, APHIS has determined that this field trial, following conditions described in Alternative C, will not pose a risk of the introduction or dissemination of a plant pest for the following reasons:

1. Transgenic B.glumae is highly likely to be confined to the field site because 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. The 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. d. The field site receiving the transgenic B. glumae is separated from other research areas by a 25 ft barren zone. e. 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.

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f. 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. g. 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. h. After harvest, the field site will be plowed to bury any unharvested seeds. i. Infected rice seeds collected at the termination of the experiment are unlikely to be sources of inoculum for future rice infections because they will be contained during movement between field and laboratory, and devitalized after analysis by burning or autoclaving. j. Harvested seed will be contained via the movement conditions specified in
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. k. All plants within the field site will be treated as regulated articles to ensure the appropriate disposition of rice infected with transgenic B. glumae. l. APHIS inspections will ensure compliance with the Standard and
Supplemental Conditions.
2. Transgenic B. glumae strains are non-pathogenic and do not cause plant disease.
The DNA inserted into B. glumae will result in the suppression of toxoflavin production, resulting in non-pathogenic strains of transgenic B. glumae. The transgenic B. glumae strains proposed for release are not expected to cause plant disease, will not result in an increased disease susceptibility in rice plants, and will not result in strains that are more virulent than the endemic B. glumae strains already present in the environment.
3. Transgenic B. glumae will not persist in the environment. During the inoculation of the test plants, the applicant proposes an infection strategy that significantly reduces the potential of B. glumae persistence. The infection design utilizes border rows of a tall rice variety and allows inoculation only during non-windy conditions to minimize drift. 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. 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.
4. Transgenic B. glumae will not increase insect susceptibility in rice plants. There have been no reports of increased insect susceptibility in rice plants during times of increased panicle blight disease pressure.
5. The nptII and bla genes used as selectable markers are devoid of inherent plant pest characteristics.

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Attachment Finding of no significant impact Response to comments APHIS No. 06-111-01r

In response to a notice published in the Federal Register (Docket No. APHIS-2007-0021, 72 FR 33735-33736) on June 19, 2007, APHIS received 1 comment, that raised 5 issues, regarding the Environmental Assessment prepared in response to permit application 06- 111-01r, during the 30-day comment period. The submitted comment was against issuing the permit for a field release. APHIS’ responses to the issues raised in the submitted comment are as follows:

Issue 1: The genes used as selectable markers may have greater consequences for horizontal gene transfer than the conclusion made by APHIS in the environmental assessment for 06-111-01r. Additionally, APHIS did not address the potential of horizontal transfer of antibiotic resistance to known Burkholderia pathogens.

Response: The issue of potential significant environmental consequence(s) of the selectable marker genes in the transgenic Burkholderia glumae posed by the submitters were not specifically described within the comment, but a reference was cited (Ho et al. 2007). APHIS recognizes horizontal gene transfer may occur between B. glumae and other microorganisms and analyzed the potential risks (page 10 of EA). As antibiotic resistance genes are derived from and/or widely prevalent in enteric and soil-borne bacteria, the use of these genes in this transgenic bacteria species is highly unlikely to cause greater levels of antibiotic resistance (page 10 of EA). These resistance genes are currently widespread in the environment and the release of transgenic B. glumae will not increase the risk of antibiotic resistance being passed to other bacteria species (page 10 of EA), including Burkholderia species. The reference provided in the comment (Ho et al. 2007) did not address the prevalence of antibiotic resistance in microbial communities or provide scientific evidence contrary to this conclusion.

Issue 2: This is a questionable field trial as the pathogen proposed for release could be spread from the experimental plots to other rice crops.

Response: The comment fails to recognize, as is repeatedly stated in the EA (pages 1, 8, 9, 10, 11, and 12 of the EA) that the wild type strains and the strains from which the transgenic strains were derived are native to Louisiana and were collected from rice planted in Louisiana. The wild-type, pathogenic bacterial strains are already present in Louisiana, and thus the release of the wild-type strain does not constitute a release of novel bacteria.

Issue 3: An alternative research approach may be more effective than those posed by the applicant.

Response: APHIS BRS does not determine a priori whether an alternative research approach is more effective than the approach proposed. APHIS will leave it to the applicant to determine the viability of the alternative research direction proposed in the

Page 1 of 2 comment. APHIS considers the potential for the proposed study to impact the human environment. After careful consideration, APHIS concluded that the proposed study will not significantly impact the human environment.

Issue 4: There is concern regarding the potential of negative human effects due to the release of B. glumae.

Response: By authorizing the release of transgenic B. glumae, APHIS would be authorizing the release of a species of bacteria that is already present and endemic in the state of Louisiana. As stated in the EA, some members of the genus Burkholderia, particularly members of the B. cepacia complex, are recognized as opportunistic human pathogens in immuno-compromised individuals, (page 8 and Appendix 1 of EA). Also as stated in the EA, B. glumae has been shown not to be directly related to B. cepecia nor is it considered to be included within the B. cepacia complex of known human pathogens (Appendix 1 of EA). APHIS (page 8 of EA) and the commenter both cite the single known human infection by B. glumae. As stated on page 8 of the EA, APHIS concluded that the field trial will not significantly increase the risk of B. glumae infection in susceptible individuals because the bacterial strains are endemic and prevalent in the release area and the size of the field test is small. In other words, the amount of bacteria being released in this experiment is negligible compared to the amount of B. glumae currently resident in the area.

Issue 5: APHIS did not recommend precautions for those working with the pathogen, and may ‘take the pathogen to their homes, families and neighbours.’

Response: The comment fails to acknowledge the precautionary measures required in the permit conditions. As stated on page 6 of the EA, personnel applying bacteria to rice plants are required to wear Tyvex suits and masks during inoculation. To minimize the movement of B. glumae outside the field site, APHIS recommends a supplemental condition to prevent B. glumae transport away from the field due to accidental clothing contamination (page 14 and supplemental condition 2). After inoculation, clothing must be removed within the field test area and double bagged for transport out of the field and destruction in an autoclave (page 14 of the EA and supplemental condition 2). Although the comment acknowledges that epidemics of panicle blight occurred in southern rice states in 1995 and 1998, the commenter fails to recognize that an epidemic results in the occurrence of enormous quantities of B. glumae. That this bacteria species is so widely prevalent in the area means that anyone working in rice fields has had extensive exposure to the bacteria. Based on the precautions mandated in the permit and the supplemental conditions, no incremental exposure from the execution of the permit is expected.

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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 A. SUMMARY ................................................................................................................................................... 4 B. REGULATORY AUTHORITY ........................................................................................................................ 4

II. NEED FOR THE PROPOSED ACTION............................................................................................... 5 III. ALTERNATIVES.................................................................................................................................... 5 A. NO ACTION ................................................................................................................................................. 5 B. ISSUE THE PERMIT AS RECEIVED .............................................................................................................. 5 C. ISSUE THE PERMIT WITH SUPPLEMENTAL CONDITIONS......................................................................... 6

IV. RESEARCH DESIGN FOR BURKHOLDERIA GLUMAE RELEASE............................................. 7 A. PURPOSE OF THE RESEARCH..................................................................................................................... 7 B. DESCRIPTION OF THE RESEARCH.............................................................................................................. 7

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  • Burkholderia Bacteria Produce Multiple Potentially Novel Molecules That Inhibit Carbapenem-Resistant Gram-Negative Bacterial Pathogens

    Burkholderia Bacteria Produce Multiple Potentially Novel Molecules That Inhibit Carbapenem-Resistant Gram-Negative Bacterial Pathogens

    antibiotics Article Burkholderia Bacteria Produce Multiple Potentially Novel Molecules that Inhibit Carbapenem-Resistant Gram-Negative Bacterial Pathogens Eliza Depoorter 1 , Evelien De Canck 1, Tom Coenye 2 and Peter Vandamme 1,* 1 Laboratory of Microbiology, Department of Biochemistry and Microbiology, Ghent University, 9000 Ghent, Belgium; [email protected] (E.D.); [email protected] (E.D.C.) 2 Laboratory of Pharmaceutical Microbiology, Department of Pharmaceutical Analysis, Ghent University, 9000 Ghent, Belgium; [email protected] * Correspondence: [email protected]; Tel.: +32-9264-5113 Abstract: Antimicrobial resistance in Gram-negative pathogens represents a global threat to human health. This study determines the antimicrobial potential of a taxonomically and geographically diverse collection of 263 Burkholderia (sensu lato) isolates and applies natural product dereplication strategies to identify potentially novel molecules. Antimicrobial activity is almost exclusively present in Burkholderia sensu stricto bacteria and rarely observed in the novel genera Paraburkholderia, Caballero- nia, Robbsia, Trinickia, and Mycetohabitans. Fourteen isolates show a unique spectrum of antimicrobial activity and inhibited carbapenem-resistant Gram-negative bacterial pathogens. Dereplication of the molecules present in crude spent agar extracts identifies 42 specialized metabolites, 19 of which repre- sented potentially novel molecules. The known identified Burkholderia metabolites include toxoflavin, reumycin, pyrrolnitrin,
  • Genetic and Transcriptomic Analyses of the Rice Pathogenic Bacterium, Burkholderia Glumae, Reveal the Important Roles of The

    Genetic and Transcriptomic Analyses of the Rice Pathogenic Bacterium, Burkholderia Glumae, Reveal the Important Roles of The

    Louisiana State University LSU Digital Commons LSU Master's Theses Graduate School 2015 Genetic and Transcriptomic Analyses of the Rice Pathogenic Bacterium, Burkholderia glumae, Reveal the Important Roles of the Regulatory Gene, tepR, for Bacterial Survival in Environmental Stresses Jingyu Peng Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_theses Part of the Plant Sciences Commons Recommended Citation Peng, Jingyu, "Genetic and Transcriptomic Analyses of the Rice Pathogenic Bacterium, Burkholderia glumae, Reveal the Important Roles of the Regulatory Gene, tepR, for Bacterial Survival in Environmental Stresses" (2015). LSU Master's Theses. 2722. https://digitalcommons.lsu.edu/gradschool_theses/2722 This Thesis is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Master's Theses by an authorized graduate school editor of LSU Digital Commons. For more information, please contact [email protected]. GENETIC AND TRANSCRIPTOMIC ANALYSES OF THE RICE PATHOGENIC BACTERIUM, BURKHOLDERIA GLUMAE, REVEAL THE IMPORTANT ROLES OF THE REGULATORY GENE, TEPR, FOR BACTERIAL SURVIVAL IN ENVIRONMENTAL STRESSES A Thesis Submitted to the Graduate Faculty of the Louisiana State University and Agriculture and Mechanical College in partial fulfillment of the requirements for the degree of Master of Science in The Department of Plant Pathology and Crop Physiology by Jingyu Peng B.S., Shandong Agricultural University, 2013 December 2015 ACKNOWLEDGEMENTS It has never been easy for me to gain such a great opportunity to study in Louisiana State University, one of the best public university in America.
  • Pan-Genome Analysis Reveals Host-Specific Functional

    Pan-Genome Analysis Reveals Host-Specific Functional

    microorganisms Article Pan-Genome Analysis Reveals Host-Specific Functional Divergences in Burkholderia gladioli Hyun-Hee Lee 1,†, Jungwook Park 1,2,†, Hyejung Jung 1 and Young-Su Seo 1,* 1 Department of Integrated Biological Science, Pusan National University, Busan 46241, Korea; [email protected] (H.-H.L.); [email protected] (J.P.); [email protected] (H.J.) 2 Environmental Microbiology Research Team, Nakdonggang National Institute of Biological Resources (NNIBR), Sangju 37242, Korea * Correspondence: [email protected] † These authors contributed equally to this work. Abstract: Burkholderia gladioli has high versatility and adaptability to various ecological niches. Here, we constructed a pan-genome using 14 genome sequences of B. gladioli, which originate from different niches, including gladiolus, rice, humans, and nature. Functional roles of core and niche-associated genomes were investigated by pathway enrichment analyses. Consequently, we inferred the uniquely important role of niche-associated genomes in (1) selenium availability during competition with gladiolus host; (2) aromatic compound degradation in seed-borne and crude oil- accumulated environments, and (3) stress-induced DNA repair system/recombination in the cystic fibrosis-niche. We also identified the conservation of the rhizomide biosynthetic gene cluster in all the B. gladioli strains and the concentrated distribution of this cluster in human isolates. It was confirmed the absence of complete CRISPR/Cas system in both plant and human pathogenic B. gladioli and the presence of the system in B. gladioli living in nature, possibly reflecting the inverse relationship Citation: Lee, H.-H.; Park, J.; Jung, between CRISPR/Cas system and virulence. H.; Seo, Y.-S.
  • Common Features of Environmental and Potentially Beneficial Plant-Associated Burkholderia

    Common Features of Environmental and Potentially Beneficial Plant-Associated Burkholderia

    Microb Ecol (2012) 63:249–266 DOI 10.1007/s00248-011-9929-1 MINIREVIEW Common Features of Environmental and Potentially Beneficial Plant-Associated Burkholderia Zulma Rocío Suárez-Moreno & Jesús Caballero-Mellado & Bruna G. Coutinho & Lucia Mendonça-Previato & Euan K. James & Vittorio Venturi Received: 28 March 2011 /Accepted: 1 August 2011 /Published online: 18 August 2011 # Springer Science+Business Media, LLC 2011 Abstract The genus Burkholderia comprises more than 60 highlighted the division of the genus into two main clusters, species isolated from a wide range of niches. Although they as suggested by phylogenetical analyses. The first cluster have been shown to be diverse and ubiquitously distributed, includes human, animal, and plant pathogens, such as most studies have thus far focused on the pathogenic Burkholderia glumae, Burkholderia pseudomallei,and species due to their clinical importance. However, the Burkholderia mallei, as well as the 17 defined species of increasing number of recently described Burkholderia the Burkholderia cepacia complex, while the other, more species associated with plants or with the environment has recently established cluster comprises more than 30 non- pathogenic species, which in most cases have been found to Jesús Caballero-Mellado deceased be associated with plants, and thus might be considered to be potentially beneficial. Several species from the latter Z. R. Suárez-Moreno : B. G. Coutinho : V. Venturi (*) Bacteriology Group, International Centre for group share characteristics that are of use when associating Genetic Engineering & Biotechnology, with plants, such as a quorum sensing system, the presence Padriciano 99, of nitrogen fixation and/or nodulation genes, and the ability 34149 Trieste, Italy to degrade aromatic compounds.