PLANTS - NON-PATHOGENIC MICROBES MODULE EXAMPLES test organisms The Bradyrhizobium japonicum strains proposed for testing, four recombinant and two wild-type strains, are listed below: Description Strain Number ----------- ------------- I-110 (wild-type) BJB1000 (host) I-110 ê BJB1001 I-110 BJB1003 BJB2000 (wild type) BJB2000 (host) BJB2000 ê BJB2001 BJB2000 BJB2003 /end source of donor DNA Strains BJB1001 and BJB2001 do not differ from their wild-type parents, except that they contain a single copy of the ê antibiotic resistance marker in the chromosome. The ê element contains a gene which confers resistance to streptomycin and spectinomycin. This gene, aad A, was originally isolated from the R-factor plasmid R100.1 of incompatibility group FII, which was obtained from a clinical isolate of Shigella flexneri. The aad A gene encodes a single polypeptide, which confers resistance to these antibiotics by enzymatically adenylating the antibiotic molecules, thereby abolishing their toxicity. Prentki and Krisch bracketed the gene with transcription termination signals from bacteriophage T4, embodied in polylinkers of known sequence. /end vector(s) The integration vector used is derived from pMR19 of Legocki et al. which was in turn derived from pJN13 of Yun et al. Plasmid pJN13 contains a 13.5 kb KpnI fragment of the B. japonicum I-110 chromosome, cloned into the PstI site of pBR325, as well as the RP4 oriT locus, cloned into the EcoRI site. These insertions disrupt the ampicillin resistance gene and chloramphenicol resistance gene, respectively. Legocki et al. modified pJN13 by moving the oriT sequence from the EcoRI site to the BamHI site, restoring the chloramphenicol resistance gene at the EcoRI site. Since the BamHI site is within the tetracycline resistance gene, this makes pMR19 tetracycline-sensitive and chloramphenicol- resistant. pMNR19 has been modified by the insertion of the Tn5 nptII (kanamycin resistance) gene, excised from Tn5 as a HindIII-salI fragment, into the salI site. The resultant vector, pMNR19K, is Tcs, Kmr and Cmr. /end exact location(s) The proposed field test site is located on the Ben Hur farm, a 2100-acre farm located in East Baton Rouge Parish (County), just beyond the city limits of Baton Rouge, Louisiana. The Parish is a region where "maturity group VI" soybeans are being grown. The immediate area around the Ben Hur farm is rural, consisting mainly of cattle and dairy operations. /end summary statement (Institution name) proposes to conduct field tests of wild type and genetically engineered Bradyrhizobium japonicum strains to assess the influence of recombinant DNA and enhanced symbiotic effectiveness on the ability of B. japonicum (serogroup 110) to colonize soil. The survival of wild-type and recombinant cells in the rhizosphere will be compared at various times after their introduction as seed inocula. Treatments will include inoculation with preparations containing only wild-type and recombinant strains. These experiments will assess the influence of the soybean host on soil colonization by B. japonicum by comparing survival of inoculant strains in plots planted to soybeans to that in plots maintained as bare fallow. Soybeans will be planted in all plots during the second year and grain yeild, N assimilated occupancy into above ground biomass, nodule numbers and size, and nodule occupancy by the inoculant strains of soybeans grown after soybeans will be compared to those of soybeans grown after fallow. These experiments will be conducted at least for 5 years because similar experiments showed that applicable colonization was not evident until 4-7 years after application of inocula. /end purpose The purpose of our ecological studies of the proposed field site is to observe the persistance of Bradyrhizobium japonicum in Louisiana soils. We will assess the influence on the soil colonization ability of B. japonicum of (1) recombinant DNA and enhanced symbiotic effectiveness, and (2) the soybean host. Field observation in Louisiana suggests that the presence of the soybean plant may stimulate the development of soil populations capable of establishing effective symbiosis with the plant. The data obtained from this long term study (minimum 5 years) will contribute to our understanding of rhizobial colonization and will aid in assessing the persistence and dispersal of recombinant strains in the soil. /end ADD description of the methods Provide a description of the methods which were used to construct the recombinant organism. Appropriate diagrams, construction description should be appended to this application. The following items are representative of material that should be considered here: - construction details of any marker elements - construction of plasmids - helper plasmids used - methods of crossing the plasmids into the host organism (e.g. triparental crosses) - how integrants are isolated - what screening process is used - what does the screening identify - how site specific integration is confirmed e.g. southern blots. /end amount and nature Gene transfer should not be of concern for the recombinant strain because the introduced genes are not plasmid-borne and do not contain transposition functions. Their incorporation into the bacterial chromosome should render them genetically stable. To test the stability of the inserted genes, the four recombinant strains were grown in liquid cultures without antibiotics and used to inoculate soybean plants. About 96 nodules formed by each strain were surface-sterlized and crushed onto media with and without antibiotics. In all cases, bacteria from the nodules displayed the expected resistances. In a second series of experiments, bacteria from crushed nodules formed by strain BJB1001 were streaked for single colonies on non-selective media, and about 50 colonies were then patched onto antibiotic media. All colonies displayed the appropriate phenotype. The result of both the experiments demonstrate the stability of the inserted genes even in the absence of selected pressure. These results are consistent with the studies of rifampicin resistant R. meliloti. Bromfield et al. found no evidence of genetic interchange, including plasmid transfer, in nodules formed by paired competitive strains. Hybridization analyses were made of DNA isolated from 26 randomly-selected BJB1001 and BJB2001 transconjugants that showed the presence of the appropriate marker gene resistances and the absences of the vector backbone resistance. Since no vector sequences were found in the transconjugants, there is an excellent correlation between the absence of vector DNA as judged by hybridization, and the absence of the vector as judged by the vector antibiotic sensitive phenotype. /end other genetic sequences The integration vector used has the following features: (a) 13.5 kb fragment of a conversion from the B. japonicum 1-110, (b) a unique HindIII site for insertion of desired genes flanked by contiguous regions of B. japonicum DNA, (c) selectable antibiotic resistances, (d) an oriT sequence, to allow the vector to be mobilized into B. japonicum in a triparental cross, and (e) an origin of replication (oriV) which is functional only in E. coli and not B.japonicum, so that the vector is unable to replicate in B. japonicum. When mobilized into B. japonicum by conjugation, the DNA inserted in the HindIII site is integrated into the chromosome through a double recombination event between the flanking regions of homology on the plasmid and their native locus on the B. japonicum chromosome. The integration vector used was derived from pMNR19 of Legocki et al. which was in turn derived from pJN13 of Yun et al. /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 the following: 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. Provide a brief description. Shipping and containment example Rhizobia will be transported in sealed containers following all applicable regulations and guidelines, including those of the USDA APHIS under the Plant Pest Act. /end design of the experiment Two types of trials are proposed to be undertaken at the Ben Hur Farm. The first, a "strain comparison" test, is designed to assess the effect of the strain on the soybean yield. The second a "strain competition" test, is a smaller scale test designed to assess the competitive ability of the recombinant strains BJB1001 and BJB1003 vs. their wild-type parent. The two soybean cultivars which will be used ("Centennial" and "Davis") are public varieties grown extensively for soybean production in the southeastern U.S. 1. STRAIN COMPARISON TEST The following sixteen treatments, including four genetically-engineered B. japonicum strains, will be used in this test: 1. BJB1001 with Braxfon 2. BJB1000 with Centennial 3. BJB2000 with avis ......................... ......................... ............. and so on. A randomized complete design totalling 18 treatments per replicate will be used. Each treatment will be replicated six times. The total area treated with recombinant rhizobia is 0.32 acres and the trial is laid out over a total area of approx. 2.4 acres. Two plots per replicate (treatments 17 and 18) will receive granular nitrogen fertilizer in place of rhizobia. This will enable comparisons to be made that will indicate whether the availability of nitrogen affected plant growth. Each treatment will be planted in 4 row * 40'' plots using 36" row spacing. After emergence plots will be trimmed to a length of 32'' to eliminate irregularities in the stand caused by starting and stopping the planting equipment. Plants which appear outside the designated plot area will be removed. Seeds will be sown at a rate of about 8-10 per foot. The plots will be separated from each other by a 6''wide buffer strip, down the center of which will be sown a single row of uninoculated soybean. The entire experiment will also be surronded by a 9'' wide perimeter strip of 4 rows of uninoculated soybeans. The uninoculated soybean buffer strip will help prevent cross-contamination between plots. In treatments 1-12 inclusive, the inoculum will be coated on to the seed prior to sowing using vermiculite as the carrier. No agent will be used to adhere the carrier to the seed. The cultures will be added to a predetermined quantity of sterilized (microwave at full power for 10 minutes), washed, ground vermiculite in the lab. The inoculum will be shaken with the seed in a sealed container to give approx. 10 million cells per seed. The seed used for treatments 13, 15, 17 and 18 will be coated with inoculum-free vermiculite. 2. STRAIN COMPETITION TEST ( ........similar to above). /end 22. (consequences) When addressing the potential environmental effects of your field test, the following list of topics may be germane: 1. Effect on human health: effects on public health worker safety and exposure. 2. Environmental fate: persistence in the environment competition dispersal horizontal gene transfer. 3. Environmental effects: target plants non-target plants soil microflora other non-target organisms (e.g. animals). Provide information on any special environmental conditions that may prevail at the test site. effects on human health Effects on public health. No hazards attributable to rhizobia have been documented in the literature. Rhizobia have been used for most of this century in commercial agriculture without any effect on public health. It appears that rhizobia are not pathogenic to humans, and the effect on the public health of proposed field test should be negligible. Worker safety and exposure. In current commercial use, rhizobia are grown in large-scale fermentors, and are then combined with a peat-based carrier for application to seeds just before planting. Therefore, seed handlers come into contact with large numbers of bradyrhizobium. No negative effects on workers associated with such large-scale handling of rhizobia have been reported. /end transmitted to other species The test strains are expected to show similar environmental persistence to their wild-type parents. XYZ's greenhouse experiments, in which non-sterile soil from the proposed test site was inoculated with the four PMN strains or their wild-type parents, demonstrated decreased persistence of all strains over time. The log of the population density decreased linearly with time over the 8-week period. No differences were observed between the test strains and their wild-type parents. These results of persistence studies in Baton Rouge soil agree with the studies performed by XYZ with soil collected in Iowa and Wisconsin which were not free of indigenous B. japonicum. In those studies, strains BJB1001 and BJB2001 exhibited persistence profiles similar to those observed in the Baton Rouge soil. Some of these data were presented in the May ##, 19## document. These data show that the test strains behave like the wild-type strains, and corroborate the results of many studies which have addressed Rhizobium or Bradyrhizobium persistence in the soil. In addition to persistence in soil under greenhouse conditions, other studies have shown that commercial strains also have limited survivability in the soil under field conditions. Indeed, persistence at levels above the initial inoculum size has not been reported for any Bradyrhizobium species in unamended non-sterile soil. Persistence of rhizobia in the soil depends on environmental factors, including temperature, soil moisture, clay content, soil acidity, the persistence of other soil microorganisms, and nutrient availability. Reports of higher levels of persistence in sterile soils compared to that in non-sterile soils implies that competition and possibly predation by other soil microflora are important factors limiting survival in natural environments. Nutrient availability is affected by the presence of plants and especially host plants; rhizobia increase cyclically from 100 - 1000 cells/g soil in the absence of soybeans to 1000000 cells/g in the disintegrating taproot rhizoshperes of mature soybeans after the harvest. Persistence of rhizobia in non-soil environments is even less than that in soil. Studies have been made of the survival of R. melitoli and R. leguminosarum in lake water and sewage. While R. melitoli was able to grow and persist in sterile lake water or sterile sewage, cell numbers gradually declined under non-sterile conditions, generally reaching a steady state level of persistence at 2-3 orders of magnitude below that of the initial inoculum. Bradyrhizobium japonicum is also unable to increase population numbers in sterile water, but remains viable and able to nodulate soybean roots for longer than one year. Under saturated soil conditions of rice fields where few aerobic microbes survive, B. japonicum population levels are maintained and are viable. Thus, we expect the test strains to persist in the soil to the same extent as the wild-type strains. /end EX: Dispersal. Vertical migration of modified organisms through the soil is unlikely. Information in the literature indicates a substantial reduction in microbial colonization of soil much below 10 inches, mainly because of temperature effects, lack of nutrients, and depletion of oxygen. Madsen and Alexander have shown limited vertical motility for B. japonicum in the soil. Unless aided by water percolation or by other carriers such as burrowing worms, viable B. japonicum cells were not transported below 2.7 cm in moist soil after seven days. Data from the P87-568/559/570 field test showed no evidence of vertical (or horizontal) movement through the soil. Therefore, it is reasonable to predict that few, if any, inoculant organisms will be found much below the plant rhizosphere. Other data reviewed by Madsen and Alexander indicate that rhizobia may be susceptible to dispersal by wind or water. Such dispersal could only occur as a result of unusual climatic conditions at the test site. Storms with high winds occasionally occur in the region of the test site, but usually wind speeds are low and very heavy wind-driven rain storms seldom occur. Dispersal via water should not be a problem. The field is surrounded by a 6-inch dike that prevents runoff. Although the test site is in the flood plain region of the Mississippi River, it is adequately protected by leeves from major flooding. Data submitted for the P87-568/569/570 field test showed limited aerial dissemination of R. melitoli from the site. This occurred only at planting when the rhizobia are added to the soil, and in very low numbers that should not pose any risk. In the proposed B. japonicum test, the rhizobia will not be spray-applied, therefor significantly limiting the potential for aerial dispersal at the start of the test. In addition, data submitted for the P87-568/569/570 field test showed no evidence of horizontal dissemination of R. meliloti through the soil. Therefore, we conclude that no significant dispersal of the PMN strains from the test site is expected. /end monitoring plan A microbiological monitoring program will be performed to assess the behavior of the Bradyrhizobium strains introduced in the soil. Of the B. japonicum strains proposed for use in the field test, in situ populations of a control strain (BJB1000), and two genetically modified strains (BJB1001 and BJB1003) will be enumerated through the field season. The monitoring will be limited to the strains comparison trial, except for aerial dispersal and nodule occupancy which will also be assessed for the strains competition trial. Populations in the soil will be enumerated via selective isolation on antibiotic-amended agar plates of Rhizobium defined Medium (RDM) parent strain. PHENOMENA TO BE ADDRESSED 1. Persistence of the inoculant population in the plant rhizosphere and rhizoplane; 2. Vertical Dispersal of the inoculant population, down through the A and B soil horizons and possibly into the C horizon (fragipan); 3. Horizontal Dispersal via either motility along the soil organic horizon, or transport by animal or windborne vectors; 4. Nodulation of the soybean plant roots. /end PROCEDURE FOR SAMPLE COLLECTION Samples will be taken from the strains comparison only. They will be collected at a number of locations within the test site, both within plots and at various distances away ???? from the edge of the plots (described under separate ???? headings below). Soil samples will be placed in sterile Whirl-Pak bags for transport back to the laboratory in [city]; plant samples will be placed in Whirl-Pak or Zip-Loc bags, depending on the size of the sample. The tool used to collect the sample will be disinfected with 70% ethanol after each sampling. /end EMERGENCY RESPONSE 24. (border areas) Will the test area, including #mborder areas#m, be monitored for volunteers after the experiment is complete? EX: EMERGENCY PROCEDURES: Workers will be instructed to report any irregularities in the field tests as soon as possible. Only authorized persons are to report potential problems to the relevant government agencies. Emergency termination procedures would only be undertaken after consultation with the government agencies. If emergency termination is deemed necessary, the tests can be terminated by application of the fumigant methyl bromide to the portion of the trial affected. The field will be covered with a plastic sheet or tarpulin during fumigation to prevent disemination of the gas. Licensed applicators are commonly used for this procedure. A 2:1 mixture of methyl bromide and chloropicrin, more effective than methyl bromide alone, is recommended by the manufacturer. The recommended application rate is 500 pounds per acre, which would be effective to fumigate the soil to a depth of 20-24 inches. Termination would involve killing growing soybeans with an appropriate herbicide followed by deep tillage and application of "67-33" under plastic tarpaulins. /end MAINTENANCE sprayed with disinfectant This would be an extraordinary precaution to prevent test organisms from escaping the area on tools or equipment. Farm machinery (such as the plot planter and harvester) coming into contact with cells will be thoroughly washed in the rinse area after each use with 50% ethanol and rinsed with water. /end Termination of the Experiment Procedures for routine termination of the field test: Both the strain comparison field test and the strain competition test are proposed to run for the season, about 20-22 weeks. XYZ reserves the right to terminate the experiment before that time. Each trial will be terminated by multiple tilling of the plot following harvest of the soybeans. About four weeks prior to harvesting of the trial, a post-harvest monitoring plan will be devised based on the results obtained to that point in the trial. The monitoring plan will be devised in consultation with the EPA. /end