RISK ASSESSMENT OF A PLANT-ASSOCIATED BACTERIUM GENETICALLY ENGINEERED WITH LUX GENES

RISK ASSESSMENT OF A PLANT-ASSOCIATED BACTERIUM GENETICALLY ENGINEERED WITH LUX GENES RISK ASSESSMENT OF A PLANT-ASSOCIATED BACTERIUM GENETICALLY ENGINEERED WITH LUX GENES

ACCESSION NO: 9163838
PROJ NO: ALA-9304456 AGENCY: CSRS ALA
PROJ TYPE: SPECIAL GRANT
START: 15 SEP 93 TERM: 15 APR 96 FY: 1995 GRANT YR: 1924

INVESTIGATOR: Kloepper, J. W.; Shaw, J. J.

PERFORMING INSTITUTION:
PLANT PATHOLOGY
AUBURN UNIVERSITY
AUBURN, ALABAMA 36849

CONTRACT/GRANT/AGREEMENT NO: 93-33120-9472
GRANT YEAR: 1924

OBJECTIVES: Determine the survival and habitat-colonization profiles of a genetically engineered beneficial plant-associated bacterium. Determine how a genetically engineered bacterium affects indigenous soil and root bacterial communities.

APPROACH: The model system for these objectives will be the bacterial strain Pseudomonas fluorescens 89B-27, which has been shown to promote plant growth, induce systemic disease resistance and reduce damping-off on several crops. This strain has been genetic engineered to bioluminesce by a chromosomal insertion of theluciferase operon. Objectives will be addressed using different experimental approaches to elements of a common field experiment on cucumber. Treatments will include a non-treated control, wild-type strain 89B-27, and a lux transformant of 89B-27. A 15 cm diameter soil core centered on the plant stem, 25 cm deep will be taken from each replication of each treatment. Immunofluorescent staining techniques, including immuno-fluorescent colony staining (IFCS), will be employed to monitor the wild-type strain and to detect revertants of the genetically engineered derivative. Fitness will be assessed by comparing survival over time and habitat colonization in soil and internal and external roots. We will focus environmental impact assessment on structural and functional changes of indigenous microbial communities. Structural changes in bacterial communities of three habitats (soil, internal and external root) will be assessed by isolation, identification to species by fatty acid analysis, and calculation of species richness (total number of species present in a community) and evenness (relative distribution of species within a community).

PROGRESS: 9501 TO 9512
The project consists of two field experiments, 1994 and 1995, to determine: a) the survival and habitat colonization profile of a genetically engineered plant growth-promoting rhizobacterium (PGPR); b) how introduction of a genetically modified PGPR strain affects the indigenous soil and root bacterial communities. Treatments included a nontreated control and bacterization with the wild-type PGPR, and a bioluminescent derivative (Lux+). Plant and soil samples were taken 0, 7, 14, 28, 42, and 76 days after planting (DAP) and processed so that populations of the introduced strain and bacterial communities from both the rhizosphere and internal root tissues were examined. Populations of the introduced bacteria were similar for both years and declined over time, until not detectable at 76 DAP, in any soil sample taken during the growing season, or in subsequent sampling of soil during the winter. Over 18,000 bacterial colonies were isolated from soil, rhizosphere, and internal root tissue and identified using GC-FAME analysis and the Sherlock System (Microbial ID, Inc.). Preliminary analysis indicates that the bacterial community structure is altered by the introduction of PGPR strains as seed treatments, but there were no difference between the wild-type and the Lux+ derivative. This indicates that the employed methods are suitable for determining changes in bacterial community structure and can be used to assess possible environmental impacts of introduced rhizobacteria.

PUBLICATIONS: 9501 TO 9512
KOKALIS-BURELLE, N., RODRIGUEZ-KABANA, R., ROBERTSON, D.G., MAHAFFEE, W.F., KLOEPPER, J.W., AND BOWEN, K.L. 1995. Effects of grass rotations on soil microbial ecology and nematode populations. Phytopathology 85:1124.

MAHAFFEE, W.F., BAUSKE, E.M., AND KLOEPPER, J.W. 1995. Structural changes in bacterial communities associated with introduction of plant growth-promoting rhizobacteria. Phytopathology 85:1191. MAHAFFEE, W.F.,

KLOEPPER, J.W., VAN VUURDE, J.W.L., VAN DER WOLF, J.M., VAN DEN BRINK, M., AND BAUSKE, E.M. 1995. Evaluation of marking systems for monitoring environmental releases of rhizobacteria. Phytopathology 85:1153.

PRESS, C.M., MAHAFFEE, W.F., AND KLOEPPER, J.W. 1995. Community-level changes inbacterial endophytes of cucumber caused by plant growth-promoting disease resistance. Phytopathology 85:1154.

SUBFILE: CRIS