
             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                        BJB1001
     I-110                         BJB1003
     BJB2000 (wild type)           BJB2000 (host)
     BJB2000                      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  antibiotic resistance marker in the chromosome.  The 
     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
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