PLANTS - PATHOGENIC MICROBES MODULE EXAMPLES test organisms Xanthomonas campestris pv. campestris /end source of donor DNA V. fischeri - luminescent bacteria /end vector(s) The transposon, Tn4331, was used to construct bioluminescent strains of the bacterium. This transposon carries the lux genes in a manner which facilitates transcriptional fusion of the genes into the bacterial chromosomal DNA. Vector or vector agent: Organisms or objects used to transfer genetic material from the donor organism to the recipient organism. Well-characterized and contain only non-coding regulatory regions. (e.g. operators, promoters, origins of replication, terminators and ribosomes binding regions). The genetic material added to a microorganism in which the following can be documented about such genetic material: (a) The exact nucleotide base sequence of the regulatory region and any inserted flanking nucleotides; (b) The regulatory region and any inserted flanking nucleotides do not code for protein or peptide; and (c) The regulatory region solely controls the activity of other sequences that code for protein or peptide molecules or act as recognition sites for the initiation of nucleic acid or protein synthesis. /end other genetic sequences Another gene complex, besides the luciferase genes and the transposon, is incorporated into the chromosomal DNA after transformation. The tetracycline resistance gene complex functions only as a marker in the initial cell selection process following transformation. Neither of the marker genes nor the resultant enzyme products have any inherent plant pest characteristics. /end exact location(s) The experiment will be conducted on a research farm owned by (Institution Name). It is located on a secondary road in (county), (state). This is XX.XX miles from (city), the nearest population center. /end summary statement Pursuant to regulations in 7 CFR Part 340 we propose to field test X. campestris pv. campestris which has been modified to express the luciferase gene complex. The genes were inserted into the bacterial chromosomal DNA using a suicide vector system. The field trial wiil take place on a small plot on agricultural land in (county), (state). The farm will provide adequate physical security. Site monitoring of the field trial and agronomic management practices that create a nonpropogative environment are expected to provide the necessary degree of both biological and physical containment. /end purpose X. campestris pv. campestris has been genetically modified to express the luciferase gene complex. Limited field testing is necessary so that information can be gathered for scientific evaluation of the efficacy of the genetic change. The bacterium has been tested in the greenhouse to obtain initial data relating to the genetic stability of the engineered genes and preliminary data on light production. Field tests are performed after greenhouse testing to confirm the data, which can only be validated in the environment using standard agricultural practices. /end description of the methods A detailed description of the transposon Tn4331 delivery system is described by Shaw et al. (1988). Briefly, Tn4331, a member of the Tn3 family (Sherratt, 1988), was transferred to X. campestris pv. campestris 2D520 in a biparental mating with Escherichia coli containing the suicide transposon delivery plasmid, pUCD623 (a derivative of pSa235). Plasmid pSA325 does not replicate in X. campestris pv. campestris, but can be transferred into X. campestris pv. campestris due to the fertility functions provided. This fact makes it an ideal suicide delivery system. Plasmid pUBD623 (Figure 1) was constructed by allowing Tn4331 to insert into pSA325. Plasmid pUCD623 can be transferred into X. campestris pv. campestris from E. coli but cannot replicate. Upon introduction of pUCD623 into X. campestris pv. campestris, select sequences were introduced into Xanthomonad chromosomal DNA. /end amount and nature Vector/Donor DNA Remaining Mutants containing these sequences (i.e. transposon Tn4331, tetracycline resistance marker, and luciferase) were found (as expected) to be tetracycline resistant and bioluminescent. To verify that only the tetracycline resistance marker and the lux gene complex and no other plasmid sequences retained by the mutants, a limited number of exconjugants were demonstrated to be sensitive to ampicillin and chloramphenicol. A further verification, colony hybridization of a limited number of exconjugants demonstrated that the mutants had acquired DNA homologous to the transposon and had not retained portions of the plasmid vector pSa325. /end containment procedures Information obtained during studies of the modified pathogenic organism in containment (e.g. microcosm, greenhouse, growth chamber) is crucial. Containment conditions that most closely imitate field situations will provide the best idea as to the potential field performance of the test organism. For example, containment studies for soilborne transformed microbes should include tests using soil from the proposed release site. If the methods of monitoring and identifying pathogenic biotypes are well established, provide details (include information on the previous use of these methods). Studies conducted under containment conditions comparing the genetically engineered organism with the non-modified parent organism are recommended prior to field testing. Some of the topics you may wish to address include: 1. Phenotype. 2. Physical characteristics, e.g., colony morphology. 3. Serology. 4. What genes do the recombinants express differently from the parent and what is the manifestation of this expression. 5. Persistence in the environment. 6. Effects on pathogenicity. Provide a brief description. Example - Studies in Containment The recombinant has a slower doubling time than its parental strain. In vegetable, the recombinant is less virulent and its population levels reached is approximately 100-fold less than its parental strain. Based on current knowledge on the genetic basis of host range determinants in Xanthomonads (Daniels et al., 1987; Turner et al., 1985; Keen and Staskawicz, 1988), the recombinant X. campestris pv. campestris could not have a wider host range than the parental bacterium. Host and Alternate Hosts. The natural host range of X. campestris pv. campestris is generally limited to the Brassica family. Many weeds can harbor the bacterium including Brassica campestris, B. nigra, B. geniculata, Lepidium virginicum, and Cardaria pubescens (Sherf and MacNab, 1986). Schaad and Dianese (1981) demonstrated that in Georgia, X. campestris pv. campestris was found on the weeds B. campestris, Lepidium virginicum, Cornopus didymud, and Raphanus sativus. They also demonstrated that X. campestris pv. campestris was disseminated up to 12 meters from infected weeds to cabbage. Laboratory and greenhouse studies in soil survival at different moisture regimes have shown that populations of X. campestris pv. campestris declined very slowly in soils maintained at low moisture levels (-30 bars). In contrast, populations declined more rapidly in soils maintained at higher moisture levels and were quickly eliminated from soils maintained near field capacity (-0.1 bar). The pathogen could no longer be recovered from moist soils 6 days after adding the inoculum, whereas 95 percent of the initial inoculum could be detected in dry soils after infestation (Alvarez et al., 1987). /end transmitted to other species Stability of Vector and/or Other Genetic Sequences. The recombinant bacterium has no detectable plasmids, reducing the likelihood that the lux genes or other introduced genes could transfer to a conjugal plasmid and subsequently vectored to new bacterial hosts. This strain has been refractory to conjugation manipulation under laboratory conditions. The recombinant has been demonstrated to be stable in vegetable. Plants were inoculated with the recombinant, allowed to grow and disease progressed. Bacteria from this infected plant was used as inoculum for another plant and this procedure repeated once again. X. campestris pv. campestris was isolated from the last infected plant and greater than 99 percent of the bacterial colonies were bioluminescent and tetracycline resistant (Shaw, unpublished). /end effects on human health No potential impact on people living in the area of the field test, or any other human population, can be identified. Xanthomonads are not known to be human or animal pathogens (Starr, 1981). /end design of the experiment The main plot will be divided into four subplots. Each subplot will consist of 4 rows of plants, 18 inches apart, with each row about 6 feet in length. A disposal area will be located adjacent to the main plot. An unplanted area will surround the main plot and disposal areas. Surrounding the unplanted area will be a single row of cabbage or cauliflower. These plants will be monitored for the presence of the recombinant X. campestris pv. campestris during the growing season. The total plot size, including the perimeter row, will not exceed 6,000 square feet. The plant varieties are the susceptible hosts: snowball cauliflower (B. oleracea L. boytrytis) and perfect ball cabbage (B. oleracea L. capitata). Plants will be inoculated by: stabbing or injecting bacterial suspensions into the petioles, or spraying a bacterial suspension over the plant with a hand-held spray bottle. /end monitoring plan During the field test, university scientists will monitor the field plots regularly. Plant parts, debris, and soil will be sampled periodically for X. campestris pv. campestris. Samples will be placed in small bags or small containers and transported to the laboratory under contained conditions (7 CFR 340.6). At the laboratory they will be analyzed for light production ability and/or the presence of viable recombinant X. campestris pv. campestris. The recombinant X. campestris pv. campestris under appropriate conditions produces light. This light production ability allows the movement and spread of the bacterium in the environment to be monitored. Plant disease index will be recorded and correlated with the location of the recombinant. Procedures for Sample Processing Soil inoculations with recombinant will involve small amounts of bacteria (less than 100 ml of stationary culture). The bacteria will be mixed with soil and then the mixture will be buried into the soil. Alternately, infected plant parts (from laboratory or field) will be buried in the soil. Areas so inoculated will be marked by a flag and periodically sampled for bacteria and bioluminescence. /end Termination of the Experiment At the conclusion of the experiment, all plant material remaining at the test site will be incorporated into the soil and the test site watered thoroughly for 1 week. Plant parts and soil removed from the test site for laboratory analysis will be autoclaved prior to disposal. Plant samples removed during the course of the experiment and not autoclaved will be returned to the test site and buried in the disposal area to allow for natural decomposition. Survival of the recombinant bacterium in soil will be minimized by irrigating the field after termination of the experiment. Irrigation of the field will also encourage decay of plant debris. There is no evidence showing prolonged survival of Xanthomonads in irrigation water. /end