TERRESTRIAL
MICROBIAL ECOLOGY/BIOTECHNOLOGY
PROGRAM
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TERRESTRIAL
MICROBIAL ECOLOGY/BIOTECHNOLOGY PROGRAM
BRIEFING BOOK
MARCH 1986
Corvallis Environmental Research Laboratory
Toxics/Pesticides Branch
Corvallis, Oregon 97333
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Table of Contents
Page
Microbial Ecology at CERL 1
Laboratory Physical Components 2
Recent Accomplishments of the Program 3
Research Activities
- Inhouse 4
Extramural 10
Personnel
- EPA 20
- Visiting 36
Appendix 40
1. Interim Protocol for Testing the Effects of Microbial Pathogens
on Predatory Mites (Acarina: Phytoseiidal).
2. Draft - Interim Protocol for Testing the Effects of Microbial
Pathogens on the Common Green Lacewing (Chrysoperla carnea
(Stephens) (Neuroptera: Chrysopidae)
3. Draft-Interim Protocol for Testing the Effects of Microbial
Pathogens on Predatory Hemipterans (Hemiptera: Lygacidae).
4. Progress Report - Methods for Assessing Fate of Genetically
Engineered Microorganisms in Soil (8/1/85 - 1/31/86).
Investigators: J. M. Tiedje and B. H. Chelm, Michigan State
University.
5. Progress Report - Transformation Studies: Fate and Effects of
Genetically-Engineered Microbes in Terrestrial Environments.
Investigator: G. Stozky, New York University, NYC
6. Draft - Interim Protocol for Oral Exposure of Avian Species to
Microbial Pest Control Agents
7. Draft-Interim Protocol for Intraveneous Exposure of Avian
Species to Microbial Pest Control Agents.
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Table of Contents
Page
Microbial Ecology at CERL
Laboratory Physical Components
Recent Accomplishments of the Program
Research Activities
- Inhouse
- Extramural
Personnel
- EPA
- Visiting
Appendix
1. Interim Protocol for Testing the Effects of Microbial Pathogens
on Predatory Mites (Acarina: Phytoseiidal).
2. Draft - Interim Protocol for Testing the Effects of Microbial
Pathogens on the Common Green Lacewing (Chrysoperla carnea
(Stephens) (Neuroptera: Chrysopidae)
3. Draft-Interim Protocol for Testing the Effects of Microbial
Pathogens on Predatory Hemipterans (Hemiptera: Lygacidae).
4. Progress Report - Methods for Assessing Fate of Genetically
Engineered Microorganisms in Soil (8/1/85 - 1/31/86).
Investigators: J. M. Tiedje and B. H. Chelm, Michigan State
University.
5. Progress Report - Transformation Studies: Fate and Effects of
Genetically-Engineered Microbes in Terrestrial Environments.
Investigator: G. Stozky, New York University, NYC
6. Draft - Interim Protocol for Oral Exposure of Avian Species to
Microbial Pest Control Agents
7. Draft-Interim Protocol for Intraveneous Exposure of Avian
Species to Microbial Pest Control Agents.
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8. Report - Assessment of Methods for the Detection, Identification,
and Enumeration of Genetically-Engineered Bacteria in Soil (2/86).
9. Draft Report - Conjugal DNA Transfer Among Bacteria: Techniques,
Issues, and Funding Relevant to the Release of Genetically Engineered
Bacteria.
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MICROBIAL ECOLOGY AT CERL
Overall Mission
The Microbial Ecology/Biotechnology Team is responsible for developing
test methods based on research and databases concerning the potential
risks of microbial pest control agents and genetically-engineered
microorganisms on terrestrial ecosystems and effects of hazardous
pollutants on microbial processes, activities, and interactions with
other living components.
The activities include:
A. The development of methodologies to assess environmental risks from
genetically-engineered microorganisms (OTS,OPP).
B. The development of scientific protocols for testing the effect of
biological control agents to non-target organisms (OPP).
C. The development of environmental risk models to estimate and predict
the impact of xenobiotic chemicals to microbial populations involved
in nutrient transformations important to plants (OTS, OPP).
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MICROBIAL ECOLOGY/BIOTECHNOLOGY
Laboratory Physical Components
1. Main Laboratory
a. Three P2 biotechnology laboratories
b. Soils microbiology/microbial ecology laboratory.
c. Enzymology/analytical biochemistry laboratory
d. Virus laboratory for biological control agent research.
e. Insectory
2. Microcosm Containment facility
3. Avian Test facility
4. Greenhouse/plant toxicology laboratory
5. Soils preparation laboratory
6. Tissue culture containment facility
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Recent Accomplishments of the Program
I. Biotechnology
Aided in the preparation of the report:
1. Aided in the preparation of the report: Research Needs in Biotech-
nology the and Environment, 1985, Office of Public Sector Programs,
American Association for the Advancement of Science, Washington, DC
2. Gave presentations on EPA's coordinated biotechnology plan for
Congress, OPTS and ORD.
3. Evaluated four methods of measuring DNA transfer between candidate
GEMs for use in the terrestrial environment.
4. Demonstrated applicability of "simple" and "complex" microcosms
for simulating environmental conditions for detecting and
measuring DNA transfer events.
5. Invited to Fourth International Conference on Safety Evaluation
and Regulation; Safety Evaluation in Biotechnology.
6. Establishment of FY 85 extramural program to support the first
projects dealing directly with established agency risk assessment
needs in terrestrial biotechnology (Michigan State; New York
University).
7. Sponsored summer workshop to develop program plans for FY 86
extramural biotechnology research program in both terrestrial
and aquatic areas.
II. Biological Control Agents
1. Developed interim protocols for biological control agents
a. Predatory mites—reviews completed
b. Predatory Chrysopidae (lacewing) — in review
c. Predatory Lygaeidae—in review
d. Predatory Hymenopterans—in preparation
e. Predatory Coleoptera—in preparation
2. Developed quantitative MPCA inoculation/artifical diets to
culture nontarget beneficial arthropods.
3. Completed construction of working instrumentation for Electronic
Bioassay System to evaluate effects of MPCA's on insects.
4. Completed initial steps for a computer searchable data base
on effect of biological agents on arthropods.
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5. Published manuscript entitled "Effects of MPCAs on Non-Target
Beneficial Arthropods" by Flexner, Lighthart and Croft; in press:
Agriculture, Ecosystems, and Environment
6. Completed interim protocols on oral and intraveneous exposure
of avian species to MPCAs. Protocol on inhalation is under
development.
7. Established tissue culture facility for propagating baculoviruses.
III. Microbial Ecology
1. Developed new research laboratory for studying the effect of chemicals
on microbial initiated processes and biodegradation.
2. Recruiting technical help for establishing research in studying
microbial mediated transformations in soil.
4'
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INHOUSE RESEARCH
IN MICROBIAL ECOLOGY/BIOTECHNOLOGY
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PROJECT TITLE:
Fate and genetic stability of genetically-engineered
microorganisms in the terrestrial environment.
PRINCIPAL INVESTIGATORS: Ramon Seidler, Mike Walter and Arlene Porteous
INSTITUTION: Corvallis Environmental Research Laboratory
OBJECTIVES: To develop test methods for studying the genetic stability of
GEMs, particularly those impacting terrestrial environments.
In the development of these test methods, a combination of
laboratory as well as simple and complex microcosms approaches
are envisioned.
Further objectives include determining applicability of
laboratory techniques in quantitatively detecting trans-
conjugants which may form in microcosms, and ascertaining
whether indigenous recombinants which received engineered DNA
can in turn become donors of this engineered DNA to
indigenous species. Further subobjectives include determining
whether a generalized technique can be developed to detect
transfer of DNA from GEM species into certain components of
the natural flora. The stability of engineered DNA within a
GEM is influenced by a complex array of factors, both
biological as well as physical/chemical. A longer term
objective is to address the effects of GEM DNA stability on
control of substrate utilization specificity and gene
expression within transconjugants.
OUTPUTS: Special Report: Conjugal DNA transfer among bacteria:
issues, and findings relevant to the release of GEMs.
techniques,
Special Report: A data base for detecting, identifying, and
enumerating GEMs released into terrestrial environments.
Journal Article: An evaluation of four techniques for the
detection and enumeration of transconjugants in laboratory
media.
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PROJECT TITLE:
Survival and fate of genetically-engineered microorganisms
in the insect-plant ecosystem.
PRINCIPAL INVESTIGATORS: John Armstrong, Guy Knudsen
INSTITUTION: Corvallis Environmental Research Laboratory
OBJECTIVES: The primary objective of this research is to study the survival,
fate, and genetic stability of engineered microbes (GEMs) in
insects feeding on plants that have been sprayed with GEMs.
The objective includes the development of test methods to
evaluate factors important in the survival and fate of
specific groups of GEM species appropriate to those that
industry is developing for release into the plant/terrestrial
ecosystem. Understanding the role of the insect gut and how
that ecosystem affects the survival, genetic stability and
fate of GEMs are significant subobjectives. The role of the
plant system and the physical/chemical environs (daylight
hrs, humidity, temperature) are fundamental parameters which
can impact GEM survival and thus genetic stability.
OUTPUTS: Journal Article: Fate and survival of recombinant bacteria and
recombinant DNA in insects.
Journal Article: Regrowth of recombinant bacteria and
persistence of DNA in insect frass.
Test methods for studying the fate and genetic stability of
GEMs in the insect/plant ecosystem.
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PROJECT TITLE: Microbial Ecology/Risk Assessment
PRINCIPAL INVESTIGATOR: Charles Hendricks
OBJECTIVE: .Prepare necessary planning documentation to develop a
terrestrial microbial ecology program on risk assessment.
This is to include appropriate material on the in situ
treatment of hazardous wastes.
INSTITUTION: Corvallis Environmental Research Laboratory
ACCOMPLISHMENTS: Toward the goal of establishing a new laboratory
in terrestrial microbial ecology, the following has
been accomplished.
1. Completed nine month sabbatical leave at the
Department of Plant & Soil Biology, University
of California, Berkeley
2. Completed transfer to CERL
3. Draft manuscript - Growth
Measurements of Terrestrial Microbial
Species by a Continuous-Flow Technique
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PROJECT TITLE: Effects of Microbial Pesticides on Non-Target, Beneficial
Arthropods
PRINCIPAL INVESTIGATORS: Bruce Lighthart and Brian Croft
INSTITUTION: CERL, OSU
OBJECTIVES: To develop tests to evaluate the effects of microbial pest
control agents (MPCA) on beneficial arthropods taken from
five insect and mite orders.
To use the above information in conjunction with a literature
data base and expert information in a decision support system
to help decide the risk(s) inherent in the use of potential
MPCAs.
OUTPUTS:
1. Optimized protocols for each of five tested insects.
2. Journal Articles: Effects of stress conditions on the infectious
processes of entomopathogenic viruses, bacteria, fungi and protozoa.
3. Software: A user friendly microcomputer program to assist personnel
in evaluating the risk(s) involved with the use of a potential MPCA.
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PROJECT TITLE: Development and Validation of Protocols for Safety
of Microbial Pest Control Agents to Avian Species
PRINCIPAL INVESTIGATORS: M. D. Knittel and A. Fairbrother
INSTITUTION: Corvallis Environmental Research Laboratory
OBJECTIVES: The project has two objectives: (1) Development and
validation of interim protocols for the intravenous, oral
and respiratory inoculation of test avian species with
MPCA; and (2) the development of In Vitro test protocols
to determine safety of MPCA to avian species.
OUTPUTS: 1. Interim protocol for the oral evaluation of safety of
MPCAs to avian species.
2. Iterim protocol for the intravenous evaluation of safety
of MPCAs to avian species.
3. Interim protocol for the respiratory evaluation of safety
of MPCAs to avian species. Draft ready for review.
4. Journal article on In Vitro testing of MPCAs.
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EXTRAMURAL RESEARCH
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TITLE: Fate and effects of genetically-engineered microbes in terrestrial
environments
PRINCIPAL INVESTIGATORS: Guenther Stotzky
INSTITUTION: New York University
STATE DATE:
OBJECTIVES:
June 1985
RESOURCES: $228,000
The objectives are to develop test methods and protocols for
evaluating the fate, survival, and genetic stability of GEMs
released to terrestrial ecosystems. The overall objectives
are being achieved through analyses of growth and survival
of many species of GEMs released into many soil types
collected from around the world. Broth sterile and natural
soils are used. Genetic stability involving the transfer
of naked DNA molecules released from GEMs as well as the
role of bacterial viruses in the genetic transfer processes
will also be evaluated.
OUTPUTS:
Journal article on the survival and genetic transfer by GEMs in natural
terrestrial environments.
Journal article on the fate in soil of a recombinant plasmid carrying a
Drosophila gene.
Journal article on the transformation and transduction processes in soils
which influence the genetic stability of GEMs.
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TITLE: Methods for assessing fate of genetically engineered microorganisms
in soil
PRINCIPAL INVESTIGATORS: James Tiedje and Barry Chelm
INSTITUTION: Michigan State University
AWARD DATE: July 85
RESOURCES: $135,000
OBJECTIVE:
The primary objective is to establish and verify test methods
which would describe the fate and survival of GEMs in soil
ecosystems. The subobjectives involve the development of a
method for the quantitative recovery of GEMs from soil
followed by an enmasse extraction of DNA and probing
specific sequences; develop a soil microcosm that minmics
the natural environment in order to soil parameters which
influence survival of GEMs.
OUTPUTS:
Journal article on a method for the recovery of GEMs from soil for DNA
probe analyses of genetically-engineered DNA sequences.
Journal article on the use of a soil column technique for evaluating
chemical factors influencing the fate and survival of genetically-
engineered microbes in soil.
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TITLE: Evaluation of terrestrial microcosms for assessing the fate and
effects of genetically engineered microorganisms on ecological
processes
PRINCIPAL INVESTIGATOR: Peter Van Voris
INSTITUTION: Battelle Memorial Institute Northwest
AWARD DATE: New Estimated June 1986 RESOURCES: $196,000
OBJECTIVE: To evaluate and modify the existing EPA/OTS terrestrial
microcosm test system and test protocol such that they can
be used to determine the environmental fate and ecological
hazards of genetically engineered organisms. The intact
soil-core microcosm design closely represents natural
terrestrial ecosystems and when coupled with appropriate
test protocols can be used to define and limit risks
associated with the deliberate release of GEM's.
OUTPUTS:
Validated protocol of a soil core to test the fate and effects of GEMs
on ecological processes.
Journal article on the use of soil cores for studying GEM effects on
plants.
Journal article on test methods for studying the effects of GEMs on
soil/plant nutrient cycles.
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TITLE: Effect of Foreign Genes on Virulence of Pathogenic Fungi
PRINCIPAL INVESTIGATOR: 0. C. Yoder
INSTITUTION: Cornell University
AWARD DATE: New Estimate June 1986 RESOURCES: $226,931
OBJECTIVES:
Determine the effect of foreign pathogenicity genes on the virulence,
host-specificity, and/or survival of saprophytic and pathogenic
filamentous fungi.
Develop a test method to determine exchange of recominant DNA among
fungi.
Determine the effect of heterologous "neutral" genes on the virulence
of a pathogen carrying this DNA in its genome.
OUTPUTS:
Journal article on the influence of foreign pathogenic genes on the
virulence, host-specificity, and survival of saprophytic fungal species.
Journal article on the effects of foreign "neutral" genes on the
virulence and physiology of pathogenic fungal species.
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TITLE: Effects of released recombinant streptomyces on the microbiolgy
and ecological processes of a soil ecosystem
PRINCIPAL INVESTIGATOR: Donald Crawford
INSTITUTION: University of Idaho
AWARD DATE: New Estimated June 1986 RESOURCES: $174,130
OBJECTIVE:
To develop protocols for assessing the effects of gram positive bacteria
on ecological processes in the soil.
Determine the effects of genetically altered Streptomyces on native gram
positive organisms in the soil.
Determine whether the Strepytomyces recombinants effect major ecological
processes with an emphasis on elucidating their long-term effects on the
cycling of organic carbon.
OUTPUTS:
Journal article on the persistence and fate of recombinant lignin bio-
degrading Streptomyces released to terrestrial ecosystems.
Test method for evaluating effects of recombinant lignin biodegraders
on the cycling of carbon in terrestrial environments.
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TITLE: Effects «f genetically-engineered microbes on ecological
processes in soil.
PRINCIPAL INVESTIGATOR: Guenther Stotzky
INSTITUTION: New York University
AWARD DATE:
OBJECTIVES:
New Estimated June 1986
RESOURCES: $270,775
Develop test methods based upon enzymatic, nutritional,
physiological, functional, and taxonomic criteria for
evaluating the potential effects of GEMs on ecological
processes. Determine which, if any, enzymatic assays
are sensitive, economical, and convenient for detecting
effects from GEMs; develop a series of physiological
and nutritional tests for indexing microbial species
diversity effects of GEMs; evaluate various broad,
functional kinetic activities such as nitrification,
respiration, dinitrogen fixation, etc., which may be
influenced following the release of GEMs into an
ecosystem.
OUTPUTS:
Journal article on the influence of GEMs released into terrestrial
ecosystems on enzymatic processes in soil.
Journal article on the microbial species diversity index to detect
potential influences of GEMs on terrestrial microflora.
Journal article on series of test to measure the effects of GEMs on
key functional processes occurring in soil.
Special report on an index of appropriate measurements for evaluating
the effects of GEMs on biochemical and taxonomic features of the
terrestrial ecosystem.
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TITLE: Impact of energy controlled survival on the stability of plasmid
DNA in genetically-engineered microorganisms.
PRINCIPAL INVESTIGATOR: Richard Morita
INSTITUTION: Oregon State University
AWARD DATE: New Estimated June 1986
OBJECTIVES:
RESOURCES: $199,917
Determine if energy-controlled survival has an impact on the
maintenance of foreign (engineered) DNA, especially plasmid
DNA, in prokaryotic cells. Determine whether the loss of
plasmid DNA is a generalized phenomenon common to many
candidate GEM species and whether this loss of plasmid DNA
is related to plasmid in group, size, and metabolic functions
encoded; determine how long a GEM must be starved before loss
of plasmid; determine the relative relationship between the
net available nutritional energy in a given terrestrial
enviroment and the loss of the plasmidS; what relative energy
level is necessary for the maintenance of plasmid DNA
molelcules; is there any relationship between cell deatth
and plasmid loss; ascertain whether there are correlations
between nutrition and physiological conditions and loss of
plasmid DNA.
OUTPUTS:
Journal article on the relationship between the available energy in the
terrestrial environment and the persistence of GEMs.
Journal article on the maintenance of recorabinant DNA in GEMs as a
function of plasmid size, function, and available energy in the
ecosystem.
Journal article on the morphological changes during long-term starvation
survival and the loss of plasmid DNA.
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TITLE: Evaluation of novel methods for detection of recombinant DNA
in the soil environment
PRINCIPAL INVESTIGATOR: Betty Olson
INSTITUTION: University of California, Irvine
AWARD DATE: New Estimate June 1986 RESOURCES: $198,775
OJECTIVE: Develop a generalized test method for optimal detection
enumeration of GEMs in terrestrial ecosystems. The objective
will be acieved through an investigation of the efficacy or
riboprobes, nonradioactive probe analysis, direct detection
of R-DNA in soil, and a validation of these methods in a
terrestrial microcosm.
OUTPUTS:
Journal article comparing conventional DNA probes with riboprobes in the
detection of GEMs in terrestrial environments.
Journal article on a novel technique for the direct detection of R-DNA
in soil samples.
Journal article on validation of new methods for detecting GEMs in a
terrestrial ecosystem.
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TITLE: Ice nucleation and bioluminescence as sensitive tools for
detection
PRINCIPAL INVESTIGATOR: Trevor Suslow
INSTITUTION: Advanced Genetic Sciences
AWARD DATE: New Estimate June 1986 RESOURCES: $165,393
OBJECTIVE: To determine the feasibility of using the bioluminescent
(lux) gene and ice nucleation active gene (INA) as markers
to detect genetically engineered microorganisms in
terrestrial environments. The system, if successful, will
allow us to rapidly, conveniently and sensitively assess
the fate of GEMs in the environment.
OUTPUTS:
Method to incorporate the Lux and INA genes into GEMs for the purpose
of detection within a controlled environment.
Journal article on the use of lux genes in the detection of GEMs
released to plant roots.
Journal article on the fate of Pseudomonas in a terrestrial microcosm
as measured by lux genes and INA gene expression.
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TITLE: Determination of the fate and effects of released recombinantly
generated microorganisms and their parental strains compared
with measurements made in contained environments
PRINCIPAL INVESTIGATOR: Steven Lindow
INSTITUTION: University of California, Berkeley
AWARD DATE: New Estimated June 1986 RESOURCES: $196,235
OBJECTIVE:
To determine the fate of introduced microorganisms on treated plants,
soil and other habitats following application of such organisms at
concentrations used in microcosm studies and used in typical release
scenarios in the field.
To determine the effects of variable inoculum concentrations of introduced
microorganisms on population size and changes in associate micro and
macroflora on plants and in the soil.
To measure spacial flux of microorganisms from sites of introduction and
determine if such measurements predict resultant spacial distributions
of organisms on adjacent plants or in habitats.
Compare field behavior of wild-type and in vitro constructed strains
under variable biological and physical environments in the field.
OUTPUTS:
Test method for estimating fate of GEMs released to plant surfaces.
Journal article on observations on the fate and spatial distributions
of GEMs released to plants and soil environments.
Journal article on a comparison of survival, population densities and
fate of GEMs released into microcosms and into the field.
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EPA SCIENTISTS
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Ramon J. Seidler
Research Mlcrobiologist
Team Leader Microbial Ecology/Terrestrial
Biotechnology Program
EDUCATION
EXPERIENCE
PROFESSIONAL
AFFILIATIONS
Ph.D. — University of California, Davis
B.S. — California State University, Northridge
1968
1964
Research Microbiologist
Corvallis Environmental Research Laboratory
Toxics/Pesticides Branch
1984-Present
1970-1984
Full, Associate, Assistant Professor
Department of Microbiology
Oregon State University
Corvallis, Oregon
Microbial ecology and genetics of pathogens, indicator
organisms.
Sabbatical Leave 1978-1979
University of Maryland
with R. R. Colwell
Molecular systematics, ecology marine bacteria
Postdoctoral Fellow
National Institute of Health
Univeristy of Texas
M. 0. Anderson Hospital and Tumor Institute
Houston, Texas
New methods for molelcular systematics
American Society for Microbiology
American Academy for Microbiology
Society of Sigma Xi
Phi Kappa Phi
Gamma Sigma Delta
1968-1970
21
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EDUCATION
EXPERIENCE
PROFESSIONAL
AFFILIATIONS
Anne Fair-brother
Research Biologist
Ph.D. — University of Wisconsin, Veterinary Science 1985
M.S. — University of Wisconsin, Veterinary Sciences 1982
D.V.M.— University of California, Davis 1980
1976
B.S. — University of California, Davis
Wildlife Biology
1986-Present
Research Biologist
Corvallis Environmental Research Laboratory
Toxics/Pesticides Branch
Define endpoints resulting from exposures of nontarget
avian species to microbial pest control agents;
develop assays to detect immunological responses of
wildlife to toxic chemicals.
Graduate Research Assistant 1980-1985
University of Wisconsin
Interactions of pathogens, toxicants, and temperature
and their effects on growth, reproduction, metabolism
and immune responses to deer mice.
Reserach Assistant 1976-1980
University of California, Davis
Ectoparasite reinfestations and annual cycles on
deer mice.
Laboratory Technician 1972-1976
University of California, Davis
School of Veterinary Medicine
Bovine transplacental transmission of IBR & BVD
viruses; antibiotic transmission in milk; genetics
of hoof overgrowth in Holstein-Friesian dairy cattle.
American Society of Mammalogy
American Veterinary Medical Association
Association of Wildlife Veterinarians
Wildlife Disease Association
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Charles W. Hendricks
Research Mlcrobiologist
EDUCATION
EXPERIENCE
Ph.D. — Colorado State University, Microbiology
M.S. — Colorado State University, Microbiology
B.S. — Colorado State University, Bacteriology
1966
1964
1962
Research Microbiologist 1985-Present
Corvallis Environmental Research Laboratory
Toxics/Pesticides Branch
Effect of xenobiotic chemicals on soil microorganisms.
Visiting Scientist 1985
Department of Plant and Soil Biology
University of California, Berkeley
Research on mlcrobial growth in soil
Environmental Scientist 1980-1985
Office of Environmental Processes and Effects Research
Washington, D.C.
Research planning for toxics and pesticides.
Microbiologist 1974-1980
Office of Drinking Water/EPA
Washington, D.C.
Sanitary microbiology regulations development.
Assistant Professor 1967-1974
Department of Microbiology
University of Georgia, Athens
Microbial ecology of sanitary microorganisms.
Microbiologist 1966-1967
NAS-NRC Postdoctoral Fellow
Ft. Detrick, Maryland
Regulatory mechanisms of toxin synthesis by
Staphylococcus
PROFESSIONAL
AFFILIATIONS American Society for Microbiology
American Academy for Microbiology
Sigma Xi
23
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Bruce Lighthart
Research Microbiology
EDUCATION
EXPERIENCE
PROFESSIONAL
AFFILIATIONS
Ph.D. — University of Washington, Seattle
M.S. — San Diego State University
B.S. — San Diego State University
1967
1961
1959
1978-Present
Research Microbiologist
Corvallis Environmental Research Laboratory
Toxics/Pesticides Branch
Research concerned with pollutant effects on soil
microbiota; effects of microbiological pesticides on
non-target, beneficial insects; airborne genetically
engineered microorganisms.
Director and Assistant Professor 1969-1973
Institute for Freshwater Studies
Department of Biology
Western Washington State University, Bellingham
Research on the limnology of Lake Whatcom,
and survival of airborne bacteria in urban air
pollutants; and teaching oceanography, microbial
biology, limnology and biochemistry.
Assistant Professor of Applied Microbiology
University of Washington, Seattle
Research and teaching of microbiology.
1967-1969
American Association for the Advancement of Science
(member-at-large, Pacific Station)
Sigma Xi
American Society for Microbilogy
Society for General Microbiology
American Society for Limnology and Oceanography
Plankton Society of Japan
Water Pollution Control Association
Air Pollution Control Association
Society for Invertebrate Pathology
24
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John L. Armstrong
Molecular Ecologist
EDUCATION Ph.D. — University of Washington, Microbiology 1968
B.A. — University of California, Microbiology 1964
EXPERIENCE Molecular Ecologist 1985-Present
Corvallis Environmental Research Laboratory
Toxics/Pesticides Branch
Research scientist in Biotechnology Group
Research Associate 1983-1985
Oregon State University
With Dr. G. S. Beaudreau; studied molecular biology
of bacterial toxins.
Instructor in Microbiology
Oregon State University
Team-taught one term of introductory microbiology
1983
Research Associate 1980-1983
Oregon State University
With Dr. R. J. Seidler; studied antibiotic resistant
bacteria in water.
Development Disabilities Services Coordinator 1978-1979
Medford, Oregon
Coordinated services for mentally disabled people
Instructor and Director 1971-1978
Clifton, Arizona
School and workshop for mentally disabled people.
Assistant Professor of Biology
Grinnell College, Iowa
Lecturer in Biology
Reed College, Oregon
1969-1971
1968-1969
25
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Paul T. Rygiewicz
Reearch EcologistS
EDUCATION
EXPERIENCE
PROFESSIONAL
AFFILIATIONS
Ph.D. — University of Washington
Tree/Plant Physiology
M.S. — University of California, Berkeley
Forestry/Forest Products
B.S. — University of Illinois
Forestry
Research Ecologist
Corvallis Environmental Research Laboratory
Toxics/Pesticides Branch
Plant and soil microbial ecophysiology
of xenobiotic chemicals
Assist. Research Soil Microbiologis
Dept. of Plant and Soil Biology
University of California, Berkeley
Soil microbial ecophysiology in forest
and oak/grassland and ecosystems
Research Associate
Centre National de Recherches Forestieres
Nancy, France
Biochemistry of nitrogen assimilation
in mycorrhizal fungi
Research Associate
College of Forest Resources
University of Washington
Mineral nutrient uptake of coniferous
species
Research Assistant
Dept. of Forestry
University of California, Berkeley
Ecophysiology of xylem formation
1983
1976
1974
1985-Present
1984-1985
1983-1984
1979-1983
1978-1979
1974-1976
Research Wood Technologist
ITT Rayonier, Inc.
Wood quality related to pulp and paper quality
American Society of Plant Physiology
Ecological Society of America
Phytochemical Society of America
American Association for the Advancement of Science
Sigma Xi, Xi Sigma Pi, Gamma Sigma Delta
26
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Martin D. Knittel
Microbiologist
EDUCATION Ph.D. — Oregon State University, Microbiology
M.S. — Oregon State University
Microbiology
B.S. — Willamette University
EXPERIENCE Microbiologist
Corvallis Environmental Research Laboratory
Toxics/Pesticides Branch
1965
1962
1955
1980-Present
1974-1980
Microbiologist
Corvallis Environmental Research Laboratory
Western Fish Toxicology
Research on stress induced disease in salmonid fishes
Research Microbiologist
Corvallis Environmental Research Laboratory
Waste Treatment Branch
Research on coliforms in pulp mill effluents
1971-1974
Senior Engineer 1969-1971
Jet Propulsion Laboratory
Pasadena, California
Research on survival of microorganisms in space
Microbiologist 1966-1969
Corvallis Environmental Research Laboratory
Eutrophication Branch
Research on development of an algal assay test for
eutrophic lakes.
Senior Bacteriologist 1965-1966
Norwich Pharmacal Co.
Norwich, New York
Research on mode of action of nitrofurian compounds
on bacteria.
Research Assistant 1963-1965
Department of Microbiology
Oregon State University
Research on generic validness among members of
genus streptococcus
Aquatic Biologist
Oregon Fish Commission
Clackamas, Oregon
Research on diseases of fish
1962-1963
27
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Aquatic Biologist 1962-1963
Oregon Fish Commission
Clackamas, Oregon
Research on diseases of fish
Teaching Assistant 1959-1961
Department of Microbiology
Oregon State University
Teaching of laboratory sections of General Microbiology
Medical Laboratory Technician 1958-1959
Doctor's Clinic
Salem, Oregon
Routine midical lab procedures
Medical Reserach Lab Technician 1956-1957
U.S. Army Medical Research Lab
Technician support to G.I. physiology research lab
Bacteriologist 1955-1956
Oregon Department of Agriculture
Division of FOod and Dairy
Salem, Oregon
Bacterial counts on foods for compliance with state
regulations.
PROFESSIONAL
AFFILIATIONS Sigma Xi
Phi Sigma
New York Academy of Science
American Association for Science
American Society for Microbiology
28
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L. Arlene Porteous
Biological Laboratory Technician
EDUCATION
B.S. — Seattle Pacific University, Microbiology
1967
EXPERIENCE
PROFESSIONAL
AFFILIATIONS
Biological Laboratory Technician 1985-Present
Corvallis Environmental Research Laboratory
Toxics/Pesticides Branch
Research technician testing and evaluating the stability
of DNA transfer in environmental related samples and
providing analytical support for biotechnology projects
in evaluating genetically engineered microorganisms and
their effect on the environment.
Research Assistant 1984-1985
Oregon State University
Animal Science Department
Research microbiologist testing and evaluating the
microbial quality and stability of liquefied fish
protein, a high protein supplement used in animal feed.
Microbiolgist 1974-1980
U.S. Food and Drug Administration
Los Angeles District Office
Analytical microbiologist testing and evaluating the
microbial quality of food, drugs and cosmetics.
Sampling and testing interstate and import samples for
the presence of microbiological pathogens and bacterial
toxins.
Microbiologist 1968-1974
U.S. Food and Drug Administration
National Center for Antibiotic Analysis
Analytical microbiologist testing and determining
antibiotic potency levels in certifiable human and
veterinarian drug products. Evaluating and testing
methods for determining antibiotic residues in animal
feeds, tissues, serum and food for human consumption.
American Society for Microbiology
29
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David W. Schmedding
Analytical Chemist
EDUCATION B.A. — California State University, Chemistry 1969
EXPERIENCE Analytical Chemist 1983-Present
Northrop Services, Inc.
Presently involved in analytical methods development
and feed preparation and treatment level verification
for toxicants used in LC5Q> 1.059, induced tolerance,
and reproductive tests both at CERL and WFTS.
Research Chemist 1970-1983
Department of Agricultural Chemistry
Oregon State University
With a variety of pesticide and industrial chemical
research projects. Areas of research include nuclear
magnetic resonance studies on the binding of toxicants
to biomembranes, physical property determinations for
chemicals of environmental concern, determination of
adsorption isotherms for various PCBs and pesticides
on sand, soil, and clay surfaces, and correlations of
physical properties with bioaccumulation. Developed
a method to measure the evaporative loss rate of
aerial agriculture sprays as modified by various
adjuvants. Work with toxicants led to an involvement
in a U.S.-USSR joint research project on substituted
aniline compounds and their mammalian toxicity as
related to physical constants. A cooperative research
project with the University of Miami School of
Medicine led to the solution of the delayed neuro-
toxicological symptomology and resultant deaths of
victims of accidentalD ingestions of high partition
coefficient organophosphate insecticides. The
interest in organophosphates led to the development
of a Knudsen effusion apparatus for determining vapor
pressures of "non-volatile" compounds of interest for
use in environmental models on bioaccumulation.
Research Chemist 1969-1970
Friden Research
Where work involved computerized formulation and
preselection of fluorescent inks, intrinsic viscosity,
UV quenching, and volatility determinations with
subsequent plant production and screening.
30
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Jerome J. Wagner
Associate Scientist/Chemist
EDUCATION B.A. — Linfield College, McMinnville, Oregon 1967
EXPERIENCE Associate Scientist/Chemist 1977-Present
Toxics/Pesticides Branch
(IAG) Corvallis Environmental Research Laboratory
Ames Laboratory (DOE)
Iowa State University
Ames, Iowa
Responsible for the chemical analyses of major, trace
and ultra-trace levels of metals in environmental
samples, using Inductively Coupled Plasma Atomic
Emission Spectroscopy at CERL.
Research Assistant 1968-1977
Oregon State University
School of Oceanography
Radioecology Department
Responsible for radiochemical analyses using gamma-ray
spectroscopy and trace metal analyses using Atomic
Absorption Spectrophotometry of ecological samples.
Other responsibilities included operation and
maintenance of computers and supervising a small
chemical analytical laboratory.
Sanitarian II 1967-1968
Yamhill County Health Department
McMinnville, Oregon
Public health official with duties such as inspecting
various public facilities to assure compliance with
State of Oregon public health related statues.
PROFESSIONAL
AFFILIATIONS Society for Applied Spectroscopy
31
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Suean Ott
Research Associate
EDUCATION Corvallis High School 1972
LBCC - Certified Assistant Laboratory Animal Tech. 1973
LBCC - Certified Laboratory Animal Tech. 1977
EXPERIENCE Associate Scientist with NSI June 1982-Present
(IAG) Corvallis Environmental Research Lab
Toxics/Pesticides Branch
Ames Laboratory (DOE)
Iowa State University
Ames, Iowa
Responsible for chemical analyses of major, trace and
ultra-trace metals in the environment using Induc-
tively Coupled Plasma Atomic Emission Spectroscopy.
Assistant Laboratory Tech at OSU June 1972-March 1982
32
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Katharine A. Harbour
Biological Aid
EDUCATION B.S. — Oregon State University, Microbiology 1981-1986
— Blue Mountain Community College, Medical 1978-1980
EXPERIENCE Biological Aid 1985-Present
Corvallis Environmental Research Laboratory
Toxics/Pesticides Branch
Media preparation, quality assurance tests, bacterial
plasmid extractions, bacterial mating experiments and
growth curves.
Columbia Medical Laboratories 1984-1985
Drawing venous blood samples, Hematology and Urinalysis
testing, glassware cleaning, receptionist work and
miscellaneous paperwork.
El Centre Community Hospital
Phlebotomist/Lab Assistant
Calexico Hospital
Phlebotomist/Lab Assistant
OSU Microbiology Department
Microbiology Lab Assistant
Hermiston Veterinary Clinic
Veterinary Assistant
1982-1983
1981
1978-1979
33
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Valerie J. Prince
Laboratory Assistant
EDUCATION Oregon State University BS expected June 1986
Senior in Microbiology
EXPERIENCE Laboratory Assistant January 1986-Present
Corvallis Environmental Research Laboratory
Toxics/Pesticides Branch
Prepares and provides media, buffers, and other
microbiological materials necessary for experiments
performed by the Biotechnolgy Team
PROFESSIONAL
AFFILIATES American Society of Medical Technologists
34
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Karen J. Armstrong
Biological Lab Aid
EDUCATION B.S. — Oregon State University, Microbiology March 1986
EXPERIENCE Biological Lab Aid May 1985-Present
Corvallis Environmental Research Laboratory
Toxics/Pesticides Branch
(1) Responsible for the preparation of media, buffers,
and solutions necessary for environment research;
(2) Assist in the evaluation of four common methods
to evaluate conjugal DNA transfer utilizing plamids
from several incompatability groups with laboratory
strains and environmental isolates as recipients.
35
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VISITING SCIENTISTS
-------
Michael V. Walter
Post Doctoral Research Associate
EDUCATION Ph.D. — University of North Dakota
M.A. — St. Cloud State University
B.A. — Northland College
EXPERIENCE Post Doctoral Research Associate
Corvallis Environental Research Lab
Toxics/Pesticides Branch
Biotechnology Team
Teaching Assistant
University of North Dakota
Teaching Assistant
St. Cloud State Univ.
Associate Director of Admissions
Northland College
PROFESSIONAL
AFFILIATIONS North Dakota Academy of Science
American Society of Microbiology
1985
1982
1978
1985-Present
1982-1985
1980-1982
1978-1980
37
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Alan R. Barker
Research Associate
EDUCATION
EXPERIENCE
Ph.D. — University of Utah, Biochemistry
B.S. — University of Utah, Biology
B.S. — University of Utah, Chemistry
Research Associate
Oregon State University and
Corvallis Environmental Research Laboratory
Research scientist investigating the enzymology,
regulation and genetic stability of 2,4-D
degradation.
1982
1976
1976
1985-Present
Research Associate 1983-1985
Oregon State University
Department of Nitrogen Fixation
Research scientist involved in characterization of
hydgogenase in R. japonicum and E.coli.
Research Associate 1982-1983
Washington State University
Department of Agronomy and Soils
Research scientist responsible for purification and
characterization of NADPH-nitrate reductase in barley.
Graduate Research Assistant 1977-1982
University of Utah
Department of Biology
Graduate research in the enzymology and physical
properties of nitrogenase in A. vinelandii.
38
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Guy R. Knudsen
EDUCATION
EXPERIENCE
Ph.D. — Cornell University, Plant Pathology
M.S. — Cornell University, Plant Pathology
B.S. — University of New Hampshire, Forestry
1984
1981
1978
1985-Present
Associate Reserach Scientist (Post-Doc)
New York University and
Corvallis Environmental Research Laboratory
Toxics/Pesticides Branch
Biotechnology Group: Fate and Genetic Stability of
Recombinant Bacteria
Research Associate 1983-1985
USDA-ARS Tobacco Research Lab
Oxford, NC
Biocontrol and computer simulation modeling of foliar
diseases of peanuts and tobacco.
Research Associate
Department of Plant Pathology
North Carolina State University
PROFESSIONAL
AFFILIATIONS American Phytopathological Society
1983-1985
39
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APPENDIX
-------
28 January 1986
Interim Protocol for Testing the Effects of Microbial Pathogens
on Predatory Mites (Acarina: Phytoseiidae)*
INTRODUCTION
Registration of Microbial Pest Control Agents (MPCAs) under Section Three
of the Fungicide Insecticide Rodenticide Act requires that the susceptibility
of nontarget species be tested. Among nontarget species important in diverse
agricultural crop systems are predatory mites in the family Phytoseiidae.
These mites are important predators of spider mites (Acarina: Tetranychidae).
While the family Phytoseiidae is estimated to include ca. 1000 species, there
are 3 species that are most common and representative of biological control
agents used in agricultural cropping systems in the U.S.: Amblyseius fallacis
(Garman), Metaseiulus occidental is (Nesbitt), and Phytoseiulus persimilis
Athias-Henriot.
This document will outline proposed methods of evaluating the effects of
MPCA's on predators. These methods will have to be adapted to specific
microbial agents, depending on the mode of action of the MPCA. In addition,
caution should be exercised in interpreting the results of such tests in
predicting the effects of the MPCAs under field conditions. Precise correla-
tion in dosage relationships between laboratory and field effects are rare.
Laboratory data are usually obtained under optimal conditions so that complete
* Disclaimer: This interim protocol was developed on the basis of current
knowledge of testing the toxicity of chemical pesticides to the Phytoseiidae.
Modifications may be required as experience in testing microbial pest control
agents is acquired.
1
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coverage is attained, prey are provided as needed, and other environmental
impacts such as UV irradiation and rain are excluded. Thus, laboratory data
may overestimate or underestimate pathogenicity. Impacts under field condi-
tions are easiest to predict in the extremes, i.e., if the MPCA is highly
pathogenic or nearly nonpathogenic in the laboratory at the proposed field
rates.
MPCAs are likely to be directed against both insect and spider mite pests.
MPCAs could, therefore, have both direct and indirect effects on predatory
mites. Direct effects occur by contact, ingestion, or through systemic effects
from feeding on infected prey (spider mites). Indirect mortality can occur in
the MPCA is more pathogenic to the spider mite prey than to the predators since
these three speices are obligatory predators. Lack of prey results in preda-
tory death or dispersal from the site. Thus, classical serial dilutions of the
pathogen to produce LCso determinations with the predatory mite(s) may not be
particularly useful if the pathogenic effects is due to indirect mortality.
Mites in the family Phytoseiidae are primarily predaceous in habit,
although some species will feed and reproduce on pollen. Sometimes predators
probe leaf tissue for moisture but none is known to reproduce or develop on
plant tissue. Ingestion of prey involves preoral digestion and sucking the
liquid foods into the predator's digestive tract. Phytoseiids lack eyes, and,
as chelicerate arthropods, have chelicerae and palps for mouthparts. Their
fundamental body organization includes the gnathosoma (the mouth-bearing
portion of the body) and the idiosoma. Phytoseiids have 4 pair of legs as
nymphs and adults, but only 3 pair as larvae. The life cycle is: egg, larva,
protonymph, deutonymph, and adult. At 25°C it takes about one week to complete
the life cycle. Phytoseiids are dimorphic, with males about 2/3 the size of
females. The sex ratio is commonly about 2.5 females to 1 male, but may vary.
2
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Mating is required before egg deposition occurs. Pregnant (gravid) adult
females are commonly used for testing; when well fed, females deposit approxi-
mately 2 eggs per day. Eggs are oval and hatch within 3-4 days at 25°C.
Larvae (with 3 pair of legs) molt to protonymphs within about 24 hours.
The three recommended phytoseiid species feed on a variety of spider mite
species, but all do well on the two-spotted spider mite, Tetranychus urticae
Koch. All stages (eggs, larvae, nymphs and adults) of T. urticae are consumed
by the active stages of these phytoseiids. Good color photographs of M_.
occidental is females and eggs, as well as of spider mites, are presented in:
"Grape Pest Management," Publ. 4105, Division of Agricultural Sciences,
University of California, Berkeley, California 94720, 312 pp., 1981.
All three phytoseiid species have strains which have acquired resistance
to various insecticides and fungicides. No acquired resistance to MPCAs is
known in the family, although a rickettsial-like disease and two viruses have
been found in M_. occidental is. The fungal pathogen Hirsutella thompsom'i is
pathogenic to spider mites, and to the citrus rust mite (a mite of the family
Enophyidae). It is reported to be pathogenic to predatory mites in the field,
although no laboratory assays have been conducted to confirm this. So, it is
possible that certain MPCAs will be directly pathogenic to M_. occidental is and
to other phytoseiids. v
TEST PROCEDURES
A. Summary of Tests:
The tests are designed to determine if direct or indirect mortality
results from exposure to MPCAs. Test 1 assays direct pathogenic effects on
adult female predators and impact on their fecundity. Test 2 assays survival
of immature predatory mites, another test of direct mortality. Test 3 assays
3
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the effects on spider mite prey, and this test is most important if the MPCA
appears nonpathogenic to the predators it assays for indirect pathogenicity in
Test 1 and 2. The test species recommended is either Metaseiulus occidental is
or Amblyseius fallacis. These two species are preferred because they are key
predators in diverse agricultural corps, and because they are commercially
available. £. persimilis is commercially available but very difficult to
contain in test arenas (unless caged) because of its high rate of activity. If
one species is to be tested, M_. occidental is is recommended because it is
readily available, has a wide crop range, and is easy to rear and test. There
is no reason at this time to believe that different species of phytoseiids
differ in their suscpetibility to MPCAs, but comparative tests have not been
done. [M_. occidental is (and their prey, T. urticae) can be obtained from
commercial sources such as Integrated Orchard Management, 3524 West Fairview
Avenue, Visalia, California 93277 (209)625-5199; and Biotactics, Inc., 7765
Lakeside Drive, Riverside, California 92509 (714)685-7681.]
Test 1: Gravid females of M_. occidentalis are placed on untreated bean
leaf discs provided with the two-spotted spider mite (Tetranychus urticae) as
prey. The MPCA is applied and mortality of the predator is assessed after 48
hrs. If no mortality occurs, monitoring should continue up to 7 days post
treatment. The number of eggs produced by M_. occidentalis is counted after 48
and 120 hrs. Eggs should be smashed or removed during counts. Initial doses
used should utilize the formulated MPCA at the proposed field rate, 10X and
1/10X the proposed field rate, as well as a water control. If mortality to
adult females occurs at these doses, a serial dilution of the MPCA is made and
a full dose response can be established (see Methods for details).
Test 2; The second test involves placing gravid M_. occidentalis females
on bean leaf discs provided with all stages of the two-spotted spider mite as
4
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prey, removing the predator females after 24 hr, treating the leaf discs (and
predator eggs) with the MPCA and observing whether the predator eggs deposited
hatch and develop successfully to the adult stage. (Eggs hatch at 25°C within
48-72 hrs after deposition. Adults should be present within 7 days.)
Test 3; Indirect toxicity is assessed by recording the effect of the MPCA
at the field rate, 1/10X, and 10X the field rate on all stages of the two-
spotted spider mite prey. Since phytoseiids require prey to survive and
reproduce, high toxicity to the prey can result in starvation of the predator
or lead to dispersal out of the area in search of food.
B. Test Species:
Either M_. occidental is or £. fallacis are suitable for testing. Both have
been used for testing toxicity of chemicals and therefore an extensive knowl-
edge of laboratory rearing is available.
MPCAs obtained from the manufacturer should be tested in the formulation
expected to be used under commercial conditions. Doses should be made up fresh
the day of the tests using distilled water.
Ideally, an inhouse colony of M_. occidental is or A. fallacis will be
established for use in MPCA testing. To maintain predator colonies, prey must
also be maintained, preferably the two-spotted spider mite, J. urticae. Stock
colonies of T. urticae should be maintained on bean plants grown in vermiculite
or a mixture of soil and vermiculite to which a complete fertilizer is added.
J. urticae should be reared in a different area from where the predators are
reared to avoid contamination, and personnel should not re-enter the area where
T. urticae are reared after handling predators.
M_. occidental is colonies can be cultured on paraffin-coated black paper
discs placed on water-soaked cotton. Small tufts of cotton provide a site for
5
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egg deposition by females. J. urticae can be added as needed by brushing
spider mites from the foliage of the bean plants, using either a mite brushing
machine (available from Leedom Engineering, Route 1, Box 325, Twain Harte,
California 95383) or by brushing individual leaves with a soft camel's hair
paint brush 1-3 inches wide.
M_. occidental is females used for a test should be the same age, randomly
selected and placed on the test bean (Phaseolus vulgaris L.) leaf discs.
Numbers tested for each test dose should consist of a minimum of 40, but 100 is
necessary if there is substantial variability. Usually, 5 or 10 females are
placed on a single bean leaf disc.
C. Test Conditions:
Bean leaf discs should be cut from healthy, green foliage using a cork
borer, sized 12 to 15. The leaf disc should be placed, bottom side up, on a
petri dish or plastic sandwich box filled with absorbent cotton soaked with
distilled water. Leaf discs should be held at 25°C at 50-75% R.H. under a long
daylength (16-18 hrs light). JT. urticae should be provided for predators in .
tests 1 and 2 by using leaves already infested to make leaf discs. Test 3
involves treating bean leaf discs infested with all stages of T. urticae; no
predators are added.
D. Number of Predators in Treatment Groups:
The number of test predators per treatment rate should be no less than 40
and preferably 100, with the same number in each control group. Usually, 5 or
10 females are placed on each leaf disc for tests 1 and 2. After females have
been removed from discs in test 2, the number of predator eggs present should
be counted and the location of each egg marked by placing a dot of India ink
6
-------
near it to identify the location so that eggs that fail to hatch can be iden-
tified. A total of 40-100 eggs per test dose should be evaluated.
E. Preparation and Quantification of Test Materials:
The MPCA should be prepared using the manufacturer's directions. Ini-
tially, rates tested should include the field rate, 1/10 the field rate, and 10
times the field rate. If mortality is observed at these rates in test 1 or 2
then a dose response should be conducted using at least 4 treatment rates and a
water control. If no mortality is observed in tests 1, 2, or 3 at the initial
rates, the value of a dose response is dubious and need not be performed.
F. Application Methods;
Phytoseiid mites and spider mites have been tested using a variety of
application methods including slide dip, leaf spray, and leaf dip. In some
tests predatory mites are placed on the dried residues; in other cases they are
sprayed using a Potter spray tower (available from Burkhard Manufacturing Co.,
Ltd., Woodcock Hill, Rickmansworth, Hertfordshire, WD3 1P5, England) or other
spray device. In general, application of the spray to the predators after
being placed on bean leaf discs with their prey provides the most useful
information as 1t approximates the field condition. If a Potter spray tower is
not available to apply the MPCA, an inexpensive alternative Involves the use of
a propellent spray apparatus such as Crown* Spray-tool power pak (Crown Indus-
trial Products Co., Hebron, Illinois 60034), which contains a chlorofluoro-
carbon under pressure (62 Ibs at 70°F). Separate plastic tubes and bottles
should be kept for each MPCA used to reduce the likelihood of contamination of
the holding container with previously-used MPCA. The leaf discs should be
sprayed to drip. Place the discs in a vertical position, hold the spray power
-------
pak about 12 inches away from the leaf discs and spray for approximately 5
seconds to obtain a complete coverage. When performing any of the microbial
procedures used in this protocol, it is redommended that the Class II contain-
ment procedures be followed as specified in "Biosafety in Microbiological and
Biomedical Laboratories" (Eds. J. H. Birchardson and W. Emmett Berkley, 1984,
U.S. Department of Health and Human Services, Center for Disease Control,
Atlanta, Georgia).
G. Post Treatment Observations:
Test 1; Adult predator females should be evaluated after 48 hrs for
mortality; numbers of eggs deposited by the females should be determined by
counts under a dissecting microscope after 48 and 120 hrs. Eggs should be
removed so the next count records new eggs deposited. The number of females
that are absent or have run off the disc should be recorded, but these data
typically are included as "dead" when determining dose response data. Death is
assumed if the female is unable to move when gently touched with a fine brush.
In some cases, mortality can't be determined within 48 hrs and observa-
tions should be continued every 24 hrs up to 7 days subsequent to the test.
After 7 days, the quality of the bean leaf discs is poor, and reliable data are
not obtained.
Test 2: Tests involving predator eggs (deposited by gravid females within
a 24-hr period) should be monitored 72 hrs after treatment to determine the
number of eggs that have hatched and the number of larvae that are dead, alive,
absent, or off the disc. Such counts should be repeated until it is clear from
the control discs that no more eggs will hatch. Then, the discs should be held
until the larvae have reached adulthood on the control discs. Determine the
number of adult males and females present on each disc. If the MPCA has
8
-------
affected devlopmental rate, this may be measured by comparing the number of
adults on control discs with the number present on treated discs. Predators
should become adults within 7 days at 25°C.
General: Leaf discs containing both adult and immature predators should
be checked daily to be sure sufficient ]_. urticae prey are available. Preda-
tors will die or run off the discs if prey becomes scarce. Additional prey can
be added to the discs as needed by brushing on small quantities.
Test 3: Check the discs with ]_. urticae every 48 hrs for one week;
determine whether all stages are alive and whether new eggs have been depos-
ited. Continue observations for 7 days; the quality of the leaf tissue will
deteriorate after that so that spider mites may disperse or die due to poor
leaf quality.
H. Reporting:
Test data should be recorded as described above. Also included should be
notes on behavioral changes observed, such as a tendency for treated predators
to be excessively active. Data submitted should include the following:
1. Name of the test, sponsor, test laboratory, study director, principal
investigator and dates of testing.
2. A detailed description of the test MPCA, including the formulation and
concentration. Include the type of dilutions carried out.
3. Detailed information about species of predators used in the test, their
scientific name, source (supplier or inhouse colony), and history of any
indigenous disease, if known. Age of test subjects, test conditions
(temperature, lighting level and duration, and relative humidity) should
be included.
-------
4. Describe the test substrates, dimensions, number of test predators per
leaf disc, and number of replicates per dilution of MPCA.
5. The percentage of test subjects dead at each dose after each observation
period should be reported.
6. If a dose response test is conducted, the statistical test used should be
reported (logit or probit). Minimal data reported would include 1059 and
LCgo with their confidence intervals, the slope (and confidence interval)
and intercept.
7. Any deviation from this test protocol should be reported, as well as
anything unusual about the test such as temperature fluctuations, disease
problems in the control predators, etc.
10
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USEFUL REFERENCES
Phytoseiid Biology and Ecology
Hoy, M. A., Ed. 1982. Recent Advances in Knowledge of the Phytoseiidae, Div.
Agric. Sciences, University of California, Berkeley, Publ. 3287, 92 pp.
McMurtry, J. A., C. B. Huffaker, and M. van de Vrie. 1970. Ecology of
tetranychid mites and their natural enemies: A review. I. Tetryanychid
enemies: their biological characters and the impact of spray practices.
Hilgardia 40(11):331-390.
Krantz, G. W. 1978. A Manual of Acarology, second edition, Oregon State
University Book Stores, Corvallis.
Microorganisms Associated with the Phytoseiidae
Hess, R. T., and M. A. Hoy. 1982. Microorganisms associated with the spider
mite predator Metaseiulus (=Typhlodromus) occidental is; Electron micro-
scope observations. J. Invert. Pathol. 40:98-106.
Sutakova, G., and F. Ruttgen. 1978. Rickettsiella phytoseiuli and virus like
particles in Phytoseiulus persimilis (Gamasoidea: Phytoseiidae) mites.
Acta Virol. 22:333-336.
Microorganisms Associated with Mites in General
Surges, H. D., and N. W. Hussey, Eds. 1971. Microbial Control of Insects and
Mites, Academic Press, London.
11
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Krieg, A. 1968. Effectiveness of Bacillus thuringiensis exotoxin on
Tetranychus telarious (Acarina: Tetranychidae). J. Invert. Pathol.
12:478-480.
Reed, D. K. 1981. Control of mites by non-occluded viruses, pp. 472-431.
In: Surges, H. D. Microbial Control of Pest and Plant Diseases 1970-
1980, Academic Press, NY.
Steinhaus, E. A. 1949. Principles of Insect Pathology. McGraw Hill, New
York, 757 pp.
12
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7 February 1986
Interim Protocol for Testing the Effects of Microbial Pathogens
on the Common Green Lacewing, Chrysoperla carnea (Stephens)
(Neuroptera: Chrysopidae)1
Introduction
Registration of Microbial Pest Control Agents (MPCAs) under Subsection M
of the Fungicide, Insecticide, and Rodenticide Act as amended (PL 92-516,
94-140, 95-396; Sept. 30, 1978) requires that the susceptibility of nontarget
species be tested. Among nontarget insect species beneficial in field and
orchard crop systems are predatory Neuroptera. Because of its worldwide
distribution, high densities in orchards and field crops, wide prey range, and
availability from commercial sources, the common green lacewing, Chrysoperla
carnea (Stephens) is an important species for laboratory study.
This document outlines proposed methods of testing the susceptibility of
this predator to MPCAs. Because the lacewing 1s a nontarget organism, it is
not likely to be killed by the MPCA, and so a classic serial dilution of the
pathogen is not likely to produce a classic LDso, LDgn,, and straight slope of
mortality. Instead, the researcher simply may have to show that a range of
concentrations approximating field rates does not significantly alter the -
survival, reproduction, and predatory capabilities of the lacewing.
1 Disclaimer: This interim protocol was developed on the basis of current
knowledge of testing the toxicity of chemical pesticides to the Phytoseiidae.
Modifications may be required as experience in testing microblal pest control
agents is acquired.
-------
These methods will have to be adapted to specific mlcrobial agents,
depending upon the MPCA mode of action. Interpretations of such test results
in predicting the effects of the MPCAs under field conditions require caution.
Precise correlations in dosage relationships between laboratory and field
pathogenicity are rare. Laboratory data are usually obtained under optimal
conditions: complete coverage is attained, prey are provided as needed, and
other environmental impacts such as UV irradiation and rain are excluded.
Thus, laboratory data usually overestimate pathogenicity. Impacts under field
conditions are easiest to predict in the extremes, i.e., if the MPCA is highly
pathogenic or mildly pathogenic in the laboratory at the proposed field rates.
MPCAs are not likely to be directed against the common green lacewing
since its prey are found in completely different taxonomic groups. The lace-
wing is in the order Neuroptera, while its prey includes mites (Acarina),
aphids, whitefly, and psyllids (Homoptera), and eggs and small caterpillar
larvae (Lepidoptera).
Since lacewing eggs and pupae are not readily penetrated by chemicals,
they are rarely affected by chemical pesticides (reviewed by Bigler, 1984;
Grafton-Cardwell and Hoy, 1985). Thus, eggs and pupae are not likely to be
significant targets of MPCAs. The adult £. carnea are nectar feeders, non-
predacious, and disperse widely. Since they do not stay in one place, contact
infection is probably minimal; the greatest infective potential would occur
with MPCA ingestion during feeding on plant nectar. The primary stage affected
both by contact and oral infection would be the predacious £. carnea larvae,
which would be resting on and searching treated plant tissue and feeding on
infected prey. Ingestion of prey involves piercing the integument of the prey
and sucking liquid foods. Larvae may also occasionally feed on nectar and
water. Additional pathogenic effects for the larval stage are those which are
2
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sublethal or more long-term and result in decreased predation rates as larvae,
slower development, Incomplete pupation, unsuccessful emergence as adults, and
lowered fecundity and fertility.
Various life stages of £. carnea have been tested with several MPCAs to
detect the adverse effects of immediate contact exposure as well as those of
long-term exposure. Suter (1978) and Franz et^ al_. (1980) treated a glass plate
with a suspension of Bacillus thuringiensis and examined feeding rates of the
larvae and fertility of adults resulting from larvae reared on this plate. IJ.
thuringiensis had little or no effect on either of these parameters. Wilkinson
iet^ al_. (1975) treated filter paper with a suspension of JJ. thuringiensis or NPV
of Heliothis and placed larvae and adults in petri dish cages with the treated
paper. No significant mortality resulted from this contact exposure. Since
the target host may have to feed to become infected with viral and bacterial
MPCAs, it is likely that the predator must also ingest the MPCA to become
infected. Therefore, simple treatment of the substrate and measurements of
contact mortality such as those done with chemicals (e.g., chlorinated hydro-
carbons, organophosphorus, and carbamate pesticides) may not be the most
realistic tests for these groups of MPCAs.
Other researchers have attempted to design tests which include consumption
of the MPCA by the predator larvae. Hassan and Groner (1977) produced a three-
level test in which lacewing larvae were (1) directly sprayed with a suspension
of NPV of Mamestra brassicae, (2) placed on dried residues of the NPV, and (3)
fed for 3 days on artificial diet containing the NPV. The larvae were then
placed in untreated containers with untreated host and the fecundity of the
adults resulting from the treated larvae, food consumption rate of the larvae,
and % egg hatch of the resulting adults were measured. The NPV of M. brassicae
had no significant effect on these parameters. Salama et _al_. (1982) had a
3
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two level test in which lacewing larvae were (1) fed for 7 days on Aphis
durante sprayed with a suspension of 13. thuringiensis. and (2) fed for 7 days
on Spodoptera littoral is (Lepidoptera) fed for 24 hours an artificial diet
containing B_. thuringiensis. After 7 days, the larvae were fed untreated host;
it was found that their larval duration was significantly longer and consump-
tion rate significantly lower. Umarov et jil_. (1975) found no mortality when
larvae were exposed to plants or prey sprayed with jj. thuringiensis. Wilton
and Klowden (1985) fed solubilized crystals of ti. thuringiensis to newly adult
£. carnea and observed no mortality.
From these experiments using varied methods for detecting immediate and
long-term toxic effects of MPCAs, five tests were selected for this protocol.
Test Procedures
A. Summary of Tests:
Test 1 — Adults
Since £. carnea adults actively move from plant to plant, they contact
treated surfaces for short periods. Thus, their dermal exposure is less than
that of larvae. In addition, adults preen themselves and feed on plant nectar,
making oral inoculation the most likely route and the most conservative test
for this stage of C. carnea. The suggested method for testing adults is
described by Wilton and Klowden (1985). Newly emerged £. carnea adults are
held by the wings with forceps and offered 1 microliter of solubilized MPCA or
heat-inactivated MPCA. Adults drink readily upon emergence. After the drop is
ingested, the test insects are placed either individually or as treatment
groups in cages with food and a water source and checked for mortality after 48
and 72 hours. If no mortality occurs after 72 hours, observations should be
made daily for up to 7 days. Equal numbers of males and females should be
-------
tested. Initial doses used should utilize the formulated MPCA at the proposed
field rate, lOx, and l/10x the proposed field rate, as well as a heat-inacti-
vated control. If mortality to adults occurs at these doses, a serial dilution
of the MPCA is made and a full dose response can be established.
Test 2 — Adults
Fungal pathogens may penetrate the adult lacewing integument and so a
contact toxicity test was designed for this group.
A 1-ounce cup (or other suitable disposable container) is sprayed or
dusted with the MPCA or inactivated MPCA. Adults are provided food and water
and mortality is assessed after 48 and 72 h. If no mortality occurs after 72
hours, observations should be made daily for 7 days. Equal numbers of males
and females should be tested. Initial doses used should utilize the formulated
MPCA at the proposed field rate, lOx, and l/10x the proposed field rate, as
well as an inactivated control. If mortality to adults occurs at these doses,
a serial dilution of the MPCA is made and a full dose response can be estab-
lished.
Test 1 -- Larvae
The first test consists of dipping or spraying? the test cage with a
suspension of the MPCA or heat-inactivated MPCA and placing first Instar £.
carnea larvae on the dried residues with untreated prey. It is important to
treat every surface that the larvae will contact, since larvae will avoid
resting on repellent chemicals. After 7 days, the larvae are moved to
untreated dishes and fed untreated prey until pupation. Percentage larval
2 Precautions should be taken to avoid breathing the aerosol, I.e., carry out
spraying in a Class II containment hood.
-------
mortality, mean larval duration, percentage successful emergence of adults, and
number and percentage hatch of eggs deposited by 20 mated females for a 2-week
period (which were exposed to the MPCA as larvae) are measured. Initial doses
should use the formulated MPCA at the proposed field rate, lOx, and l/10x the
proposed field rate, as well as a heat-inactivated control. If mortality to
larvae occurs at these doses, a serial dilution of the MPCA is made and a full
dose response can be established. x
Test 2 — Larvae
This method is described by Salama et^ al_. (1982). Two-day-old C_. carnea
larvae are fed for 7 days on eggs, larvae, or pupae of the target host which
have been sprayed2 with a suspension of an MPCA or heat-inactivated MPCA. If
the target host is not available, the next best hose choice is one in the same
insect order which is also susceptible to the MPCA (commonly used prey are
neonate larvae of Lepidoptera, lepidopterous eggs, or aphids). After 7 days,
the larvae are fed untreated prey until pupation. Percentage larval mortality,
mean larval duration, percentage successful emergence of adults, and number and
percentage hatch of eggs deposited by 20 mated females for a 2-week period
(which were exposed to the MPCA as larvae) are measured. Initial doses should
use the formulated MPCA at the proposed field rate, lOx and l/10x the proposed
field rate, as well as a heat-inactivated control. If mortality to larvae
occurs at these doses, a serial dilution of the MPCA is made and a full dose
response can be established.
Test 3 — Larvae
The most rigorous test is to assay the effect of £. carnea larvae feeding
on target hosts which have ingested the MPCA organism. However, target hosts
6
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may be difficult to obtain, inoculate, rear, and feed to the lacewing larvae.
Caution should be used if artificial diets are used instead of live prey,
because the diets are difficult to produce and will reduce the vitality and
survival of lacewing larvae (Tulisalo in Canard et al., 1984). This confuses
insect mortality because of the inadequate larval nutrition.
The method chosen for this protocol is described by Salama et^ ^U (1982).
The £. carnea larvae are fed for 7 days on the target host, which has been fed
for 24 h on an artificial diet containing the MPCA at a specific concentration.
The control £. carnea receive prey which have been fed artificial diet contain-
ing the heat-inactivated MPCA. After 7 days, the lacewing larvae are fed until
pupation on untreated diet. Percentage larval mortality, mean larval duration,
percentage successful emergence of adults, and number and percentage hatch of
eggs deposited by 20 mated females for a 2-week period (which were exposed to
the MPCA as larvae) are measured. This test requires that an artificial diet
be available for the target host. Artificial diets are not available for some
insect groups such as aphids; in this case, the lacewing larvae could be fed
the MPCA directly in lacewing artificial diet without involvement of the target
host. Initial doses should utilize the formulated MPCA at the proposed field
rate, lOx and l/10x the proposed field rate, as well as a heat-inactivated
control. If mortality to larvae occurs at these doses, a serial dilution of
the MPCA is made and a full dose response can be established.
B. Test Species:
The common lacewing, Chyroperla carnea (previously known as Chrysopa
carnea) is available as eggs from many commercial biological control suppliers
(e.g., 1. Bioinsect Control, 1710 S. Broadway, Plainview, Texas 79072;
2. Peaceful Valley Farms, 11173 Peaceful Valley Road, Nevada City, California
7
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95959; 3. Biogenesis, Inc., Lake Road, P.O. Box 36, Mathis, Texas 78368).3
Ideally, an inhouse colony of lacewings should be maintained so that the diet
and rearing conditions can be kept uniform. However, £. carnea have voracious
appetites, are cannibalistic, and so must be fed every 48 hours. Thus, great
time and expense may be required to maintain a colony of lacewings and suffi-
cient live prey to feed them. If eggs are obtained from commercial sources,
the larvae must be fed as soon as they hatch and should be used for testing ca.
2 days after hatching. It is important to test lacewing larvae as first
instars because the longer they are reared as a group, the higher cannibalism
rate. Even if cannibalistic attacks are not fatal, the vigor of the test
animals is reduced. Also, when treated as first instar larvae, subsequent
larval, pupal, and adult stages can be observed for sublethal effects.
An excellent summary of the techniques for rearing lacewings is found in
the Biology of Chrysopidae (Canard et al. 1984). Eggs and larvae of the potato
tuberworm, Phthorimaea operculella, and grain moth eggs such as Anagasta
kuhnielja (Zeller) or Sitotroga cerealella (Olivier) are commonly used as food
for £. carnea larvae. The adults rear extremely well on a diet of water and a
food source consisting of 1 gram yeast hydrolysate (Yeast Products, Inc.,
Clifton, New Jersey) + 6 grams Formula 57 (Qualcepts Company, Edina, Minnesota)
+ 10 grams honey + enough water to make a paste.
C. Test Conditions:
During testing, both larvae and adults should be kept in a growth chamber
(16-hour photoperiod, 50-80% RH, 22-26°C). Temperature and humidity should be
adjusted within these ranges for optimal effectiveness of the MPCA. Adults
3 Mention of trade names or commercial products does not constitute endorsement
or recommendation for use.
8
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should be provided food (1 g yeast hydrolysate + 6 g Formula 57 + 10 g honey +
water) and water-soaked cotton. Adults can be grouped in a petri dish half or
other cage setup such as 8 to 16 ounce plastic cartons. The adult cage should
have a mesh opening for airflow.
Larvae must be provided prey (grain moth eggs, aphids, and/or neonate
Lepidoptera larvae) every 48 to 72 hours. A water source is not needed.
Larvae must be placed individually in cages such as 1-ounce plastic cups with
tight-fitting lids (e.g., 1-oz. Serco Cups, manufactured by S. E. Rykoff and
Co., California) to prevent escape and cannibalism. Larvae should be 2 to 4
days old at the start of the test.
Regular disinfection of cages must be done; equipment, instruments, and
supplies must be sterilized and aseptic techniques used when indicated.
Stock colonies must be separated physically (separate laboratory or build-
ing) from test area, where contact with MPCAs could occur.
As a general rule, if larval or adult control mortality is greater than
20% or successful pupation is less than 75%, then the colony is diseased or
inappropriately fed, invalidating test results.
D. Number of Individuals in Treatment Groups:
There should be at least 40 adult £. carnea per treatment rate, with the
same number in the control group. Usually 5 individuals are placed in each
test cage and 2 to 3 groups of 15 to 20 adults are tested on different dates.
For the larval testing, a minimum of 100 larvae should be tested per treatment
rate since larvae are more plentiful than adults and since the tests measure
long-term effects. Usually, 20 to 25 larvae are tested per treatment and the
tests are replicated 4 times.
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E. Preparation and Quantification of Test Materials:
The MPCA should be prepared using the manufacturer's directions. Ini-
tially, rates tested should include the proposed field rate, 1/10 the fjeld
rate, and 10 times the field rate. If significant mortality is observed at
these rates, than a dose response should be conducted using at least 4 treat-
ment rates and a water control. If no mortality is observed at the initial
dosages, the value of a dose response is dubious and a dose response test need
not be performed.
When performing any of the microbial procedures used in this protocol, it
is recommended that the Class II containment procedures be followed as speci-
fied in Biosafety in Microbiological and Biomedical Laboratories (Birchardson
and Barkley, 1984).
F. Application Methods:
Test 1 — Adults
The formulated MPCA is drawn up into a microliter pipet and the newly-
emerged adult lacewing is held gently by the wings and allowed to feed on a
1-microliter droplet.
Test 2 — Adults
The adult cage, which may be a petri dish or disposable plastic cup with
lid, should be sprayed, dipped, or dusted with the MPCA so that the entire
inside surface is treated. The cage is air-dried for 1 hour. One to 5 adult
lacewings and food and water are then placed inside the cage.
10
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Test 1 — Larvae
The larval cage, which may consist of a 1-ounce plastic cup with lid,
should be dipped in or sprayed^ to runoff with a suspension of the MPCA so that
all of the inside surface of the cup and lid is treated. The cage is drained
and then air-dried for 1 hour. One lacewing larva and untreated prey are then
placed inside the cage.
Test 2 -- Larvae
The target host or other suitable prey for C_. carnea are sprayed using a
Potter spray tower (Burkard Manufacturing Company, Ltd., Woodcock Hill,
Rickmansworth, Hertfordshire WD3, 1P5, England) or other spray device. If a
Potter spray tower is not available, an inexpensive alternative involves the
use of a propellent spray apparatus such as Crown SprayTool Power Pak (Crown
Industrial Products Company, Hebron, Illinois 60034) which contains a chloro-
fluorocarbon under pressure (62 pounds at 70°F). Separate plastic tubes and
bottles should be kept for each MPCA concentration to prevent contamination due
to residues. The target host should be sprayed for 1 second from a distance of
8 inches. All sprays should be conducted in a laminar flow hood and the test
personnel should wear gloves, laboratory coats, and masks. Air exhausted from
the spray areas must not be recirculated. Appropriate hoods with HEPA filters
must be used. A minimum class II hood is needed. Waste MPCAs should be
disposed of properly. The treated target host is then fed to 2-day-old lace-
wing larvae.
11
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Test 3 -- Larvae
Specific doses of the formulated MPCA are incorporated into target host
artificial diet or lacewing artificial diet. The target host or lacewing diet
is then fed to 2- to 4-day-old lacewing larvae.
G. Post Treatment Observations
Tests 1 and 2 — Adults
Adult lacewing females and males should be evaluated after 48 and 72
hours. Death is assumed if an individual is unable to move when gently touched
with a brush. If there is no mortality after 72 hours, observations should be
continued for 7 days.
Tests 1, 2, and 3 — Larvae
Lacewing larvae should be evaluated for mortality after 48 and 72 hours.
Death is assumed if the larva is unable to walk when gently touched with a
brush. Records should be kept of percentage larval mortality, mean time in
days until pupation, % successful emergence as adults, and number and percent-
age hatch of eggs deposited by 20 pairs of mated 10-day-old females. £. carnea
must mate to produce fertile offspring and do not reach peak oviposition until
approximately 1 week after emergence. Therefore, sexed pairs should be caged
upon emergence and records of number and % hatch of eggs should be initiated 10
days after emergence. Number and percentage egg hatch should be measured for 2
week s.
H. Reporting:
Test data should be recorded as described and include:
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1. Name of the test, sponsor, test laboratory, study director, principal
investigator, and dates of testing.
2. A detailed description of the test MPCA, including the formulation concen-
tration, and lot number or batch number. Include the type of dilutions
carried out.
3. Detailed information about the source of lacewings used, and history of
indigenous disease, if known. Age of test subjects, test conditions
(temperature, lighting level and duration, and relative humidity) should
be included, as well as the exact method of dosing.
4. Describe the test substrates, dimensions, number of test predators per
cage, and number of replicates per dilution of MPCA.
5. The percentage of test subjects dead at each dose after each observation
period should be reported.
6. For long-term tests of larvae, the mean duration in days of the larval
period, the percentage successful emergence of adults, and the number of
eggs and the % egg hatch of 20 pairs of adults should be reported.
7. If a dose response is conducted, the statistical test used should be
reported (logit or probit). Minimal data reported would include the LCso
and confidence interval, the slope, and intercept.
8. Any deviation from this test protocol should be reported, as well as
anything unusual about the test such as temperature fluctuations, disease
problems in the predators, etc.
13
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REFERENCES
Bigler, F. 1984. Biological control by chrysopids: integration with pesti-
cides, pp. 233-246. Jj^ Biology of Chrysopidae, M. Canard, Y. Semeria,
and T. R. New (eds.), Dr. W. Junk Publishers. The Hague, The Netherlands.
Birchardson, J. H., and W. Emmett Barkely (eds.). 1984. Biosafety in Micro-
biological and Biomedical Laboratories. U.S. Department of Health and
Human Services, Center for Disease Control, Atlanta, Georgia.
Canard, M., Y. Semeria, and T. R. New (eds.). 1984. Biology of Chrysopidae.
Dr. W. Junk Publishers, The Hague, The Netherlands. Chapter 8. Biological
and integrated control by chrysopids. 8.1 Mass rearing techniques, by U.
Tulisalo, pp. 213-220.
Franz, J. M., H. Bogenschutz, S. A. Hassan, P. Huang, E. Nation, H. Suter, and
6. Viggiani. 1980. Results of a joint pesticide test programme by the
working group: pesticides and beneficial arthropods. Entomophaga
25:231-236.
Grafton-Cardwell, E. E., and M. A. Hoy. 1985. Intraspecific variability in
response to pesticides in the common green lacewing, Chrysoperla carnea
(Stephens) (Neuroptera: Chrysopidae). Hilgardia 53:1-32.
Hassan, S. A., and A. Groner. 1977. Die Wirkung von Kernpolydern (Baculovirus
Spec.) aus Mamestra brassicae auf Trichogramma cacoeciaeps (Hymenoptera:
Trichogrammitadae) und Chrysopa carnea (Neuroptera: Chrysopidae).
Entomophaga 22:281-288.
14
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Salama, H. S., F. N. Zaki, and A. F. Sharaby. 1982. Effect of Bacillus
thuringiensis Berl. on parasites and predators of the cotton leafworm
Spodoptera UttoraTIs (Boisd.). Z. Angew. Entomol. 94:498-503.
Suter, H. 1978. Prufung der Einwirkung von Pflanzenschutzmltteln auf die
Nutzarthropoden, Chrysopa carnea Steph. (Neuroptera: Chrysopidae) --
Methodic und Ergebnisse. Schweizerische Landwirtschaftliche Forschung
17:37-44.
Umarov, S. A., Nilova, G. N., and I. D. Davlyatov. 1975. The effect of
entobakterin and dendrobacillin on beneficial arthropods (in Russian).
Zashch. Rast. 3:25-36. (English abstract translation in Rev. Appl. Ent.
A64:6920, 1976).
Wilkinson, J. D., K. D. Biever, and C. M. Ignoffo. 1975. Contact toxicity of
some chemical and biological pesticides to several insect parasitoids and
predators. Entomophaga 20:113-120.
Wilton, B. E., and M. J. Klowden. 1985. Solubilized crystal of Bacillus
thuringiensis subsp. israelensis: effect on adult house flies, stable
flies (Diptera: Muscidae), and green lacewings (Neuroptera: Chrysopidae).
J. Am. Mosq. Control Assoc. 1:97-98.
15
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Interim Protocol for Testing the Effects of Microbial Pathogens
on Predatory Henri pterans (Henri ptera: Lygaeidae)
INTRODUCTION
Registrations of Microbial Pest Control Agents (MPCAs) under
subsection M of the Fungicide Insecticide Rodenticide Act requires that
the susceptibility of nontarget species be tested. Among nontarget
species important in various agroecosystems are predatory true bugs in
the Geocorinae subfamily of the family Lygaeidae. These true bugs are
Important predators of mites and numerous insect pest species, especially
pests in their egg or early larval stages. As such geocorines,
especially members of the genus Geocoris are important agents of
biological control of pest insects. In the southern and southwestern
agroecosystems of the U.S., £. punctipes 1s one of the most economically
Important members of this genus.
This document will outline proposed methods of testing these
geocorine predators with MPCAs. These methods will have to be adapted to
specific microbial agents, depending upon the mode of action of the
MPCA. Caution should be exercised in Interpreting the results of such
tests In predicting the effects of the MPCA's under field conditions.
Precise correlation in dosage relationships between laboratory and field
tox1c1t1es are rare. Effects of enviornmental factors in the field are
difficult to duplicate under laboratory conditions. Laboratory data may
overestimate or underestimate toxicity.
MPCAs may have direct and indirect effects on predatory Insects.
They may be directly toxic to predators, and/or may adversely affect
predators by reducing the numbers of their food supply. They may even
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have a temporarily beneficial effect upon the predators by weakening or
killing prey, making these prey more susceptible to predator attack or
scavenging. In any of these cases, classical serial dilutions of the
pathogen to produce 1650 determinations with the predator may not be
useful if the effect is due to indirect mortality.
The geocorines are primarily predaceous lygaeids with some plant
feeding as a means of getting water. Damage to plant tissue is
negligible: the value of geocorines as predators of crop pests is
great. As heteropterans, Geocoris punctipes and other ja. spp. probe
foods with their piercing/sucking mouthparts. Mobile prey are paralyzed
by injection of a venom, and body fluids and liquified body parts are
ingested through the beak of these insects.
As heteropterans Gi. punctipes is a hemimetabolous insect with 3
stages in its life cycle: egg, nymphal and adult stages. The nymphs
undergo 5 molts in becoming adults. The life cycle at 27°C for £.
punctipes fed budworm or boll worm eggs is about 22 days from egg to
adult, 7 days of egg development and in excess of 120 days as adults.
Females that have been adults for 2 - 4 days can lay 1 - 5 eggs/day for
more than 100 days. Healthy adult males weigh from 3 to 5 mg; females
weigh from 4 to 7 mg. Both adults and nymphs are strongly predaceous on
numerous species of insect and mite prey. There is no existing
literature on the effects of MPCA's on J3. punctipes or any geocorine
lygaeid for that matter.
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TEST PROCEDURES
Summary of tests.
Rickettsias, protozons, viruses, and bacteria most often enter
insects orally (via os); therefore the tests recommended for these MPCAs
is through food contamination. Fungi often infect insects through the
cuticle, so topical application is recommended for these MPCAs. Specific
techniques from Poinar and Thomas, 1978 are recommended for inoculation
and diagnosis of each agent.
The tests are designed to test direct mortality, the indirect
mortality tests being those applied to various geocorine prey of numerous
taxa. The species of predator described here, Geocoris punctipes, is
among the most widely distributed, abundant, polyphagous and economically
important geocorines (predaceous lygaeids). While they are not available
from commercial suppliers, they are easily obtained from agricultural
fields and readily cultured by methods described by Champ!ain and Sholdt
(1967) on natural diet or by Cohen (1985) on artificial diet.
Test 1 assays the toxicity of ingested MPCAs to adult females. Test
2 assays the toxicity of topically or externally applied MPCAs. Tests 3
and 4 pertain to ingested and externally applied MPCA's in nymphal Gu
punctipes.
TEST 1; Adults of both sexes are placed in containers with artificial
prey that have been inoculated with the MPCA. Containers are also
equipped with cotton wadding as oviposition sites and free water in a
sponge-filled Petri dish (Cohen, 1985). After 72 hours, cotton wadding
is removed for inspection and counting of eggs. Every 3 days, diet
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capsules should be replaced; and eggs should be counted. This procedure
should be repeated 3 times for a total of 9 test days. Eggs should be
removed and discarded with each count. Initial doses used should be at
the proposed field rate for that MPCA, 10X and 1/1 OX the proposed field
rate as well as a control with no MPCA. A statistical test (ANOVA) must
be used to compare numbers of eggs from treated vs control individuals.
Should any adverse response be noted at any level of MPCA concentration,
a serial dilution of MPCA is made and a full dose response can be
established.
TEST 2; The floor of a 1 gallon ice-cream carton used as a rearing cage
should be sprayed with MPCA at field concentration, 10X and 1/1 OX field
concentration and an unsprayed control should be concurrently tested.
Diet and water should be provided, and 6 adult 05. punctipes, 3 of each
sex, should be placed in each of the containers. Mortality should be
measured at the end of a 48 hour exposure period. Surviving £. punctipes
should be re-located in unsprayed containers (free of MPCA) that are
marked to indicated MPCA exposure history (field, 10X, 1/10X and control)
and allowed to oviposition for 5 days (120 h). Cotton wadding in 3X3 cm
sheets should be placed in cages as an oviposition substrate. At the end
of the oviposition period eggs on sheets and in cages should be counted
and incubated for 10 days at 27°C and 70-80% RH. At the end of this
incubation period eggs should be inspected so that hatch (fertility)
counts can be made. Statistical tests (ANOVA) should be used to compare
means of % survival, fecundity, and fertility.
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TEST 3: Nymphal Development with MPCA in Diet
Newly hatched 1st instar nymphs of £. punctipes are provided water
and units of artificial diet for 15 days. Diet units should, be changed
every third day. By the end of this period all surviving nymphs should
have reached the 3rd instar nymphal period. Ten of these 3rd instar
nymphs should be placed in a 1 gallon rearing contained with diet units
contained IX, 10X, 1/10X field concentrations of the MPCA. Water should
be provided as well as 3 2X10 cm strips of tissue paper to provide hiding
places in order to reduce cannibalism. Diet units should be changed
every other day. At the end of 3 weeks, counts of surviving individuals
should be made and % adult eclosion determined.
TEST 4: Nymphal Development with MPCA on Container Surface
Test cage is dipped or sprayed* with MPCA or heat-inactivated MPCA
solution. Surfaces of cages should be IX, 10X, or 1/1 OX field
concentrations or control (heat-inactivated MPCA) treatment. The
conditions should otherwise duplicate those of Test 3 with 3rd Instar
nymphs used to start the test and 3 weeks of exposure. Counts of
surviving individuals and % adult eclosion should be made for statistical
comparison (by ANOVA). Where positive tests are Indicated, serial
dilutions of MPCA and full dose response should be established.
*Precautions should be taken to avoid breathing the aerosol, i.e., carry
out spraying in laminar flow hood.
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Test Species
The predator, Geocorls punctlpes can be kept In the laboratory via
an inhouse colony maintained on artificial diet made of beef liver,
hamburger and sucrose solution packaged in stretched paraff 1m diet units
as described by Cohen, 1985. Cultures of £. punctlpes or other species
of Geocoris can be started from field-collected samples. The use of an
artificial diet obviates rearing of prey as food sources both of nymphal
and adult stages, greatly simplifying tests of MPCAs.
MPCAs obtained from the manufacturer should be tested in the
formulation expected to be used under commercial conditions.
Test Conditions
During testing, as in rearing, both adults and nymphs of §.
punctlpes (or other geocorines) should be kept 1n a growth chamber at
27°C^ 1°C, 40 - 50% RH with a 16 hour photophase. Because of a constant
potential of cannibalism In J5. punctlpes. crowding should be avoided
(I.e., no more than 5 individuals/500 ml of rearing space). Also strips
of tissue paper wadded and placed on the cage bottom are helpful in
averting cannibalism. For purposes of standardization, strips of the
same dimensions and the same number should be provided in all tests.
Containers such as 1 pint Ice cream cartons with tops replaced by organdy
screen are suitable for groups of 5 or 6 £. punctlpes. Petrl dishes (7-9
mm diameter) fitted with a sponge are adequate watering devices.
Stretched parafilm feeding units (Cohen, 1985) are essential for allowing
feeding.
-------
Number of Predators In Treatment Groups
The number of test predators per treatment should be no less than 40
and preferably 100, with the same number In each control group.
Locations of eggs should be marked with India ink to simplify counting.
This is especially important with eggs deposited into cotton wadding. A
total of 40-100 eggs per test dose should be evaluated.
Preparation and Quantification of Test Materials
The MPCA should be prepared using the manufacturer's directions.
Initially, rates tested should include the proposed field rate, 1/10 the
field rate, and 10X the field rate. If significant mortality or
reduction in fecundity or fertility is observed at these rates, then a
dose response should be conducted using at least 4 treatment rates and a
water control.
Application Methods
MPCAs can be added to the diet during diet preparation when the
sucrose/H20 solution is added to the homogenized meat mixture.
Calculations of the appropriate dilution are based on the fact that the
sucrose solution constitutes 6% of the final diet on a volume/weight
basis.
Application of MPCAs for tests involving dried residues is achieved
by spraying appropriate doses on the cage surface using a Potter spray
tower (available from Burkard Mfg. Co. Ltd., Woodcock Hill, Rickmanworth,
Hertfordshire, WD3 IPS, England) or a substitute such as a propellent
spray apparatus such as CrownR Spray-Tool Power Pak (Crown Industrial
-------
8
Products Co., Hebron, 111. 60034), which contains a chlorofluorocarbon
under pressure (62 Ibs. at 70°F). Separate plastic tubes and bottles
should be kept for each MPCA used to reduce the likelihood of
contamination of the holding container with previously-used MPCA. All
sprays should be conducted in a laminar flow hood and the test personnel
should wear gloves, laboratory coats, and masks. Care should be taken to
avoid contamination of personnel and the environment. Waste MPCAs should
be properly disposed of.
Post Treatment Observations
Test 1 — Adults
Survival of adults should be monitored 48 hours after onset of
exposure to diets or substrates treated with MPCAs. Egg production
(fecundity) should be monitored 1 week after exposure and egg hatch
(fertility) should be monitored until 10 days after the last eggs were
laid.
Survival and adult eclosion of nymphal (i. punctipes should be
monitored for each treatment both in nymphs given MPCA treated diet and
in surface-exposed individuals. Mean % survival and mean % adul.t
eclosion should be monitored after 3 weeks of exposure to dietary or
surface-contacted MPCA's.
-------
Reporting:
Test data should be recorded as described above. Data submitted
should include the following:
1. Name of the test, sponsor, test laboratory, study director, principal
investigator, and dates of testing.
2. A detailed description of the test MPCA, including the formulation of
concentration. Include the type of dilutions carried out.
3. Detailed information about the source of G. punctipes used, and
history of indigenous disease, if known. Age of test subjects, test
conditions (temperature, lighting level and duration, and relative
humidity) should be included.
4. Describe the test substrates, dimensions, number of test predators
per cage, and number of replicates per dilution of MPCA.
5. The percentage of test subject dead at each dose after each
observation period should be reported.
6. For long-term tests of nymphs, the mean duration in days of the
nymphal period, the percentage successful emergence of adults, and % egg
hatch of 20 pairs of adults should be reported.
7. If a dose response is conducted, the statistical test used should be
reported (logit or probit). Minimal data reported would include the 1650
and confidence interval, the slope, and intercept.
8. Any deviation from this test protocol should be reported, as well as
anything unusual about the test such as temperature fluctuation, disease
problems in the predators, etc.
-------
10
References
Champlain, R. A., and L. L. Sholdt. 1967. Life history of Geocoris
punctipes (Henriptera: Lygaeidae) in the laboratory"Ann.
tntomol. Soc. Am. 60:881-3.
Cohen, Allen C. 1985. Simple Method for Rearing the Insect Predator
Geocoris punctipes (Heteroptera: Lygaeidae) on a Meat Diet.
d. Econ. Entomol. 78: 1173-75.
Cooper, D. J. 1981. The Role of Predatory Henri ptera in Disseminating a
Nuclear Polyhedrosis Virus of Heliothis Punctiger.
J. Aust. ent. Soc. 20: 145-50.
Dunbar, D. M., and 0. G. Bacon. 1972a. Influence of temperature on
development and reproduction of Geocoris atricolor, G. pal
and JG. punctipes from California"! Environ. Entomol.~"1:596
Dunbar, D. M., and 0. G. Bacon. 1972b. Feeding, development and
reproduction of Geocoris punctipes (Heteroptera: Lygaeidae
eight diets. Ann. Entomol. Soc. Am. 65:892-5.
Poinar, G. 0., Jr. and G. M. Thomas. 1978. Diagnostic Manual for the
Identification of Insect Pathogens. Plenum Press, New York
218 pp.
Steinhaus, E. A. 1949. Principles of Insect Pathology. McGraw Hill,
New York, 757 pp.
Wilkinson,J. D., K. D. Biever, and C. M. Ignoffo. 1975. Contact
Toxicity of Some Chemical and Biological Pesticides to Several
Insect Parasitoids and Predators. Entomophaga 20(1):113-120.
Wilkinson, J. D; K. D. Biever, and C. M. Ignoffo. 1979. Synthetic
Pyrethroid and Organophosphate Insecticides Against the
Parasitoid Apanteles marginiventris and the Predators Geocoris
^_^ :onvergen!
J. Econ. Entomol. 72(4):473-75.
punctipes. Hippodamfa convergens, and Podisus maculiventris.
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Progress Report 'to EPA
8/1/85 to 1/31/86 (first 6 months)
Methods for Assessing Fate of Genetically Engineered Microorganisms in Soil
Grant No. CR812476-01
J.M. Tiedje and B.K. Chelm, Co-Principal Investigators
Michigan State University
Extraction, purification and probing soil DNA
We have developed a protocol for the extraction of total bacterial DNA
from soil. Previously used protocols for the extraction of bacterial DNA
from soil involve the use of trichloroacetic acid treatment, followed by
ethanol/ether extraction of soil bacterial fractions filtered through acid-
washed diatomaceous earth (1), or purification of DNA from detergent, and
high-salt by hydroxyapatite column chromatography (2). However, because we
anticipate the necessity of isolating bacterial DNA of sufficient purity
for use in hybridization studies from large numbers of soil samples, it was
desirable to develop a procedure that would allow the simultaneous
processing of multiple soil samples and yield high molecular weight DNA, in
a concentrated and purified form, in a short period of time.
The DNA isolation protocol which we have developed involves an initial
separation of soil bacteria from fungal biomass and soil debris. This
separation involves multiple rounds of homogenization of the soil into a
buffered salt solution followed by fractionated centrifugation. This
fractionated centrifugation technique was developed by Goksoyr and co-
workers (3) and involves first a low-speed centrifugation step. This low
speed centrifugation pellets the fungal biomass and the soil debris in the
homogenate while leaving the soil bacteria in the supernatant. A second
high-speed centrifugation step pellets the soil bacteria contained in the
supernatant from the low-speed centrifugation. The pellet from the low-
speed centrifugation is extracted a second and third time to enhance the
yield of soil bacteria. Using this protocol, more than 50% of the bacteria
contained in the soil sample are isolated after three rounds of
homogenization/centrifugation. Additional rounds of
homogenization/centrifugation serve to isolate additional soil bacteria but
in increasingly lower proportions (3,4). It appears that-the distribution
of the different bacterial types remains constant through eight rounds of
homogenization/centrifugation such that the bacterial fraction, after the
first round of purification, is presumed to be as representative of the
entire bacterial population present in the soil as the combined bacterial
fraction after eight rounds of purification (4). Since three rounds of
purification yields about 50% of the bacterial population and is
representative of the entire bacterial population, and further rounds of
purification yield diminishing amounts of bacteria, we used three rounds of
purification by fractionated centrifugation in our protocol.
The purified bacterial fraction is then lysed using a protocol which
is derived from, and includes the salient features of, several individual
lysis protocols designed for use with various type of bacteria. We have
combined the important features of several lysis protocols in an effort to
insure the maximal disruption of the various types of bacteria present in
the natural soil population. The purified bacterial fraction is first
washed in 2% sodium hexametaphosphate pH 8.5 and then washed twice in
Crorabach's buffer (Tris-HCl 33 mM, EDTA 1 mM, pH 8..5) as described by V.L.
Torsvik (2). The cells are then incubated for 10 min in Crombach's buffer
brought to 1 M with NaCl. This incubation in high concentrations of NaCl
is necessary for the efficient lysis of slow-growing species of RhLzobia.
-------
(5). The cells are then collected by centirifugation and resuspended in TS
(Tris-HCl 50 mM, NaCl 50 mH, pH 8.5) to which Sarkosyl is added to a final
concentration of 0.1%. Prewashing in 0.1% Sarkosyl allows for more
efficient lysis of lysozyme refractory strains of bacteria, presumably by
making the cell wall more susceptible to attack by lysozyme by some, as
yet, unknown mechanism (6). The cells are again collected by
centrifugation and resuspended in a sucrose solution (sucrose, 0.75 K;
Tris-HCl, 50 mM, pH 8.5; EDTA, 10 mM) and incubated on ice for 20 min.
Lysozyme is then added to a final concentration of 5 mg/ml, followed by
incubation at 37°C for 1 h. Pronase is then added to a concentration of
0.625 mg/ml followed by incubation at 37°C for 1 h. Following the 37°C
incubation, the mixture is heated to 60°C at which time Sarkosyl is added
to a final concentration of 1% followed by incubation for 10 min at 60°C.
The lysate mixture can now be kept on ice overnight.
The lysate mixture is then centrifuged at 10,000 rpm at 4°C for 30
min. The supernatant from this centrifugation is used to prepare cesium
chloride-ethidium bromide gradients for equilibrium centrifugation.
Following equilibrium centrifugation the total bacterial DNA can be
visualized as a discrete band in the gradient using long-wave ultraviolet
light. The DNA band is fractionated from the gradient and extracted in
isopropanol, precipitated, by ethanol and extracted with phenol according
to standard techniques. DNA purified as just described is at least 47
kilobases long and is not subject to degradation under standard conditions
for nuclease digestion. The addition of exogenous restriction
endonucleases under the same conditions shows that the DNA can readily be
digested to completion.
We are currently performing hybridization experiments on DNA isolated
from soil in order to determine the lowest level at which a particular DNA
sequence can be present and still be detectable. These experiments involve
combining genomic DNA isolated from a Bradyrhizobium japonicum strain which
contains the gene for kanamycin resistance in its chromosome with the DNA
proportions such that there is essentially an end-point dilution of the B.
japonicum DNA into the soil DNA. The DNAs are digested with the
restriction endonuclease Hind III then subjected to agarose gel
electrophoresis followed by Southern transfer to nitrocellulose filters.
The filters are then probed using pkC7 DNA which has been alpha labeled
with p by nick-translation. pKC7 contains the gene for kanamycin
resistance. In this way, we will be able to determine the lowest limit at
which the DNA sequence must be present in order to be detected by
hybridization.
DNA hybridization as a method for quantitation of a dechlorinating isolate
in mixed communities.
The dechlorinating bacterium, DCB-1, is able to remove halogen atoms
from the aromatic ring of some toxic compounds. Because of this
dechlorination property, it is the type of organism that will be
"engineered" for proposed release to treat hazardous wastes. DCB-1 is a
member of an anaerobic consortium able to completely degrade
chlorobenzoate. The other members of the consortium include a benzoate
degrading organism and methanogens. Quantitation of various members of the
consortium is complicated by the dependence of the benzoate degrader on the
methanogen, and the strict anaerobic requirements of all the members.
Therefore, the purpose of these experiments is to adapt.existing DNA-
hybridization techniques to quantitation of DCB-1 in these unique cultures.
Since DCB-1 is a very unusual bacterium with no known relatives, its DNA
may be somewhat more unique than for many natural isolates. Since the
-------
consortium is a simple, defined interacting microbial community, it can
serve as a model for development of these techniques for more complex
systems. In addition, we are interested in looking for the presence of
DCB-1, or a similar organism, in other anaerobic samples enriched for
degradation of halogenated toxic compounds.
DNA was first isolated from anaerobic enrichments and pure cultures.
The isolated DNA was purified and spotted onto a nitrocellulose filter.
Genomic DNA from DCB-1 was nick-translated to serve as a radioactive probe
for similar DNA on the filter. The DNA was hybridized to the filter and
the amount of hybridization was quantified by autoradiography. Intensity
of the autoradiographic signal corresponds with the degree of hybridization
and indicates the amount of like DNA in the samples.
A variety of cultures were probed with the dechlorinator DNA. These
included the consortium of which DCB-1 is known to be a member, a
chlorophenol degrading enrichment, a phenol enrichment, and lambda DNA.
The phenol enrichment and lambda DNA each served as negative controls. The
phenol enrichment is not enriched for degradation of halogenated aromatic
compounds, and does not express dechlorination activity.
We are .in the process of probing DNA isolated from a sediment enriched
for the bromobenzoate degradation. In addition, we will receive cultures
which express dehalogenase-like activity from other sources (e.g. Germany
and Battelle Laboratories, Columbus, OH).
Table 1 shows the results of our first two experiments.
Table 1. Percent hybridization of DCB-1 DNA to various cultures.
Source of DNA ] % hybridization to DCB-1 DNA
DCB-1* 100, 100
Consortium* 50, 50
Chlorophenol Enrichment
Phenol Enrichment 3
Lambda* 0, 0
**
Bromobenzoate Enrichment N.D.
Methanogen and Benzoate Degrader N.D.
(from consortium)
Methanogen only N.D.
(from consortium) __
Results from two independent experiments.
^
Not enough DNA was extracted from this culture to obtain a reliable
estimate.
•Jf-Jf
N.D. Not determined. We are in the process of analyzing these cultures.
The chlorobenzoate-degrading consortium which includes DCB-1 as a
member has 50% DNA hybridization to the dechlorinator (Table 1). It will
be interesting to see the results of hybridization to the DNA of the
benzoate-degrading organism, and the methanogen. This should tell us the
amount of hybridization to other members of the consortium due to similar
DNA sequences in these organisms. It should then be possible to more
accurately quantitate DCB-1 in the consortium.
DNA hybridization is a useful method for quantitatiorr of the- presence
and concentration of DCB-1 in various anaerobic cultures. This method
should also prove to be a useful technique for quantitation of other
organisms in other complex environments, such as soil.
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Development of a soil core microcosm
We have been testing, modifying and retesting soil core designs that
allow the detection of survival, transport and plasmid survival.and
transfer of introduced soil bacteria. In our proposal we had fL argued that
conventional selective markers were the only established reliable and
sensitive method for monitoring organism fate. Therefore, one aspect of
our research program was to further develop the use of this methodology as
well as to use it for comparison with the gene probe methodology. Our
approach uses the following principles:
1. The test method stresses reliability which in its broadest sense
means good recoverability of viable GEMs or of the novel gene(s) and good
reproducibility of environmental conditions. It also stresses cost-
effectiveness so that many different GEMs or simulated conditions can be
evaluated at one time.
2. Optimum conditions for survival and genetic transfer between
microorganisms are maintained to ensure a worst case scenario. Also,
natural soil bacteria have been used as test organisms as these have the
best chance for survival.
3. Genetic donor and recipient bacteria are added to the soil. Both
have chromosomal antibiotic resistance markers for easy selection and
enumeration. Use of two organisms in one column allows obtaining .
information on fate and survival of two organisms rather than one as well
as provides an optimum recipient to increase the chance of obtaining
genetic exchange. The donors contain high-frequency of transfer
conjugative plasmids which offer the best route for genetic transfer
between bacteria.
The outline of the experiment is given in Fig. 1 where C0101 is the
genetic donor of plasmid pDGlOl and CB101 is the potential recipient.
After addition to soil the bacteria are enumerated by selective plating
using resistance markers contained by each and the combined resistance
markers of the transconjugant progeny.
Our microcosm involves use of a packed soil column (Fig. 2) to which
the donor and recipient bacteria are added. The core has two injection
ports for the bacteria. The donor is injected in the top, the recipient in
the bottom. Thus, the donor must be physically moved through the core by
water percolation to reach the recipient for DNA transfer to take place.
This method overcomes the problem of mixing large numbers of donors and
recipients together and thereby enhancing conjugation by artificially
creating a close physical presence. Bacteria were collected from the core
either by percolating water through and collecting the effluent and by
sacrificing the cores and extracting the bacteria by blending soil samples
in a diluent which dislodges the bacteria from soil particles. This method
also allows us to discern the effect of percolation on bacterial movement
from the point of inoculation. Some of these cores were planted to wheat
and others to corn to determine the effect plant roots had on transport,
survival and genetic exchange.
We have approached the question of the fate of the GEM by using a
tiered system of analysis. The sequence outlined below offers a cost-
effective approach. The initial screening for GEM survival is inexpensive
and may preclude more difficult and expensive procedures used for genome
detection and identification of GEM physical location.
-------
Non-viable
I. Establish viability of GEM £> Rapid death
Viable ,
Non-detectable
II. Establish fate of genome of interest -^. Genome loss
1 precludes need to
Genome detected assess physical
location of GEM
III. Establish physical location of GEM.
We have gone through this approach using a natural soil bacterium,
CorynebacCerium flaccumfaciens, to assess its usefulness:
I. Viability of GEM-Extraction of bacteria by blending soil samples
and enumeration by selectively plating demonstrated an ideal population
number for the bacterium of approximately 10 -10 /g soil (Fig. 3). This
number remained constant for a period of over 3 months. We successfully
extracted 10-100% of added bacteria. The method of recovery by percolation
yielded approximately 1% of the introduced number but has allowed us to
follow bacterial translocation through soil. We have demonstrated that
plant roots enhance movement by 2X the number of bacteria obtained in the
effluent without plant roots (Fig. 4).
II. The genes of focus confer resistance to heavy metals and are
located on a high frequency of transfer plasmid in the donor bacterium.
The genome remained stable within the donor population even without
selective pressure. The genome also was transferred to recipient bacteria
at low frequencies.
Our immediate goal for the future is to establish the detection limits
for bacteria and for genomes in soil. To presume loss of a gene or of the
GEM, it must be non-detectable under sufficiently sensitive conditions. By
our extraction method we hope to reach the point of detecting 1 bacterium/g
soil out of a natural population of 10 /g soil. We also plan to include
soil derived Pseudomonas and Rhizobium strains in our soil core system in
the near future.
REFERENCES
1. Lid Torsvik, V. and Goksoyr, J. 1977. Soil Biol. Biochem. 10:7-12.
2. Lid Torsvik, V. personal communication.
3. Faegri, A., Lid Torsvik, V. and Goksoyr, J. 1976. Soil Biol.
Biochem. 9:105-112.
4. Bakken, L.R. 1985. Appl. Environ. Microbiol. 49:1482-1487.
5. Denarie, J., Boistard, P., Casse-Delbart, F. Atherly, A.G., Berry,
J.O. and Russel, P. 1981. pp. 225-246. In K.L. Giels and A.G.
Atherly (eds.) International Review of Cytology. Supplement 13,
Biology of the Rhizobiaceae. Academic Press.
6. Schwinghamer, E.A. 1980. FEMS Microbiol. Lett. 7:157-162.
-------
Figure 1 .
OUTLINE OF EXPERIMENT
C0101
Smr Tcr
pDG10l(ArsarArsir)
CB101
Rfr Nar
SOIL COLUMN
„
BACTERIA EXTRACTED
SELECTIVE PLATING
CB101
TRANSCONJUGANT
1. PLASMID ISOLATION ;-:
2. COLONY HYBRIDIZATION
-------
Figure 2
INJECTION PORT
(DONOR)
INJECTION PORT
(RECIPIENT)
WIRE MESH
-------
Bacterial Survival in Soil Cores
1E+09 -•
1E+08 •-
* cells/g soil 1E+07--
1E+06--
1E+05-.
1E+04
0x5
1 1.5 2
Time (weeks)
2.5
-------
Figure 4
BACTERIAL RECOVERY FROM SOIL CORES
108
x CO (wheat)
o—-CO (no wheat)
o CB (no wheat)
x CB (wheat)
DAYS
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ASA -- CSSA -- S-SSA Title-Summary No. S3-31
Iplete two (2) copies.
he interpretive summary you prepare is designed for people who are not familiar with your
ork, yet are interested in what you are doing. Please make sure that your summary is
Ilearly written and easy to understand by nonscientific people. Be sure to indicate the
roper address to use in requesting a news release about your research. Keep the interpretiv
ummary simple! This is not an abstract.
Title: A Soil Column Method for Assessing the Effect of Plant Roots on Movement and Plasmid
Transter or Conjugative Bacteria
^hors: P.F. DWYER*, J.R. KNUTSON, C.W. RICE, and J.M. TIEDJE
itjHmary:
• With the advent of molecular engineering the production of bacteria with novel
genotypes has become more than academic. Soil is the environment most conducive to
viability for many types of bacteria. We have, therefore, attempted to devise a test
method to concurrently assess both the fate of a bacterium added to soil and the
frequency of transfer of the novel genotype contained by that bacterium. As our test
organisms we have used bacteria harboring plasmids with genetoypes that are not
commonly found among soil bacteria. We used bacteria with plasmid-coded traits
because plasmids theoretically represent the greatest possibility for gene transfer.
The test method assesses population changes, ie. whether the bacterium will die-
off or become established in soil. It is important to have a high sensitivity of
detection for viable cells. Thus, we used antibiotic resistant bacteria for the
purpose of selective plating of viable cells on appropriate media. To assess gene
transfer we have designed our method such that both a plasmid-donor and plasmid-
recipient strain are present in the soil. But to ensure that the method of addition
does not prejudice gene transfer the two strains are physically separated and the
plasmid-donor must be transported by water-saturated flow through the soil to effect
transconjugation (i.e. plasmid transfer). Our experiments have demonstrated three
things (1) our bacterial strains rapidly die off after addition to soil (2)
transconjugation occurs at low detectable frequencies before the die-off, (3)
bacterial movement through soil is facilitated by the presence of plant roots.
ilinq address for your employer's office Mailing address for corresponding author:
oublic relations:
rce Charles Downs, Frf-it-n-r TTT P,.Mir p»Tat-tnnc Name Mr. Daryl F. Dwyer _
dress ns T.-mrnn Hall _ Address Dept. of Microbiology and Public
Michigan Sl-afe TTm'vPTgi fy _ - - Hpalt-T-i^ M-ioV<
-------
ABSTRACT FORM —AGRONOMY ABSTRACTS
American Society of Agronomy — Crop Science Society of America — Soil Science Society of America
Type one perfect copy following closely the directions below.
Division and Title-Summary No. 53—31
A Soil Column Method for Assessing the Effect of Plant Roots
on Movement and Plasmid Transfer of Conjugative
Bacteria. D.F. DWYER*. J.R. KNUTSON, C.W. RICE, and
J.M. TIEDJE, Michigan State Univ.
A test method was devised for assessing the fate of plasmid-
containing bacteria added to soil as to (a) survival, (b)
movement with water percolation, and (c) frequency of
plasmid transfer. The plasmid-donor was CorynebacCerium
flaccumfaciens subsp. oortiL (C0101) which harbors plasmid
(pDGlOl) that encodes resistance to arsenite and arsenate.
The plasmid-recipient was, C. flaccumfacLens subsp. oorti
(CB101). Both had chromosomal markers for antibiotic
resistances allowing for selective plating of donor (Tet,
Str, arsenite, arsenate), recipient (Nal, Rif, Amp) or
transconjugants (Nal, Rif, Amp x arsenite, arsenate). The
donor was added to the top layer of a packed-soil column,
the recipient to the bottom; some columns contained wheat.
The bacteria were leached through the column. The added
bacterial strains were detected in the effluent for at least
3 weeks. The minimum number detectable represented 200
CFU/gra soil. Root channeling increased the number of j
bacteria in the effluent. Transconjugants occurred at low j
frequencies in columns with or without wheat. |
Mailing Address of Corresponding Author:
Title and name
Department
University or other organization
Street or P.O. Box if needed
City, State, ZIP CODE
Mr. Daryl F. Dwyer
Dept. of Microbiology and Public Health
Michigan State University
East Lansing, Michigan 48824
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(over)
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PROGRESS REPORT G. Stotzky
CR812484
Transformation Studies
Preliminary transformation studies are being conducted with Bacillus subtilis strain
IS-75 as the recipient (obtained from Dr. Issar Smith, Public Health Research Institute of
the City of New York; PHRI) and B. subtilis strain BD307 (obtained from Dr. David
Dubnau, PHRI) as the donor of the transforming DNA. Strain IS-75 is auxotrpphic for
histidine (hisAl), leucine (leuAS), and methionine (metBS), and strain BD307 is
auxotrophic for pyrimidine (pyr26) and leucine (Ieu2).
DNA from BD307 was purified, using standard techniques (e.g., Dubnau et al.,
1971), and its efficiency of transformation was measured by addine various amounts, in a
volume of 8 ;ul, to a constant quantity (100 jil containing ca. 7 x 10° cells) of competent
IS-75 cells (the time of competency was determined earlier in growth studies), and the
transform ants were enumerated, after incubation for 30 min at 33 C with shaking, on
Spizizen agar augmented with histidine and leucine (i.e., transformation of the gene
coding for methionine was measured). Although the transformation of other
combinations of the three auxotrophic markers of IS-75 could have been studied, the
efficiency of transformation of these genes on DNA from BD307 would probably be
equal, and therefore, for these preliminary studies, only the transformation of the gene
coding for methionine was measured. The transformation frequency (TF; number of
transformed cells X 100/number of total cells) showed a direct linear relationship to the
concentration of DNA added (Fig. 1). In subsequent experiments, lO^ig DNA (in 8;ul)
was used. However, as this concentration of DNA was diluted in subsequent experiments
(e.g., by the addition of clay suspensions, DNase solutions), the effect of dilution on the
TF was determined. Even a ten-fold dilution yielded respectable and easily measurable
TF values (Fig. 2). N
To determine the effect of the clay mineral, montmorillonite (M), on the TF,
various amounts of the clay (Bentonite, Fisher Scientific Co., which contains only M and
a small amount of mica) were reacted with 10,ug DNA for 4 h on a rotating wheel at
33 C, and the M-DNA mixture was then incubated with 100 ^il of competent cells for 30
min at 33 C with shaking. There was a linear decrease in TF as the concentration of the
clay was increased (Fig. 3). It has not been established whether this decrease was the
result of the adsorption and binding of the DNA on M, of a competition between M and
DNA for DNA-binding sites on the recipient cells, or of other factors. However, when M,
DNA, and cells were incubated directly for 30 min, the TF was similar to that obtained
after reaction of M and DNA for 4 h before incubation with the cells, suggesting that
surface interactions between M and DNA were less important in reducing the TF than
were possible steric effects of the clay at the cell surface. The reasons for the decrease
in TF caused by M will be determined in future studies.
DNA and M (at a ratio of lO^ug DNA/mg M) were reacted for 4 h, as above, and a
100 ;ul aliquot containing 10;og DNA and 1 mg M was incubated with 100 .ul of cells for 30
min at 33 C with shaking, and the number of transformants was enumerated. The
remaining M-DNA mixture was then centrifuged (40,000 g for 10 min, refrigerated), a
100 ,ul aliquot of the supernatant was incubated, as above, with 100 ;il of cells, and the
TF was determined. On the basis of the TF, the amount of DNA actually present was
estimated from a standard curve (Fig. 1) of the TF in the absence of clay (S in Fig. 4).
-------
The clay pellet was then resuspended in deonized water to yield 1 mg M/100^1, and 100
}A of the suspension was incubated, as above, with 100 ^ul of cells, and the TF was
determined. The amount of DNA associated with M was deduced by subtracting the
amount of DNA apparently present in supernatant 1 (S.) from the amount originally
added (i.e., 10 ,ug), and this value was plotted against the TF obtained (P^ in Fig. 4). The
pellet was then centrifuged, the TFs of the supernatant ($2) and the subsequently
resuspended pellet (?2) were determined, as above, and the procedure was repeated once
more (83, P3). These results showed again that the presence of M reduces the TF, that
this reduction is linear and independent of the concentration of DNA, and that the DNA
associated with M neither loses its transforming ability nor is tightly bound. The latter
was further shown by the high amount of DNA that was recovered from M by the three
sequential washings of the clay-DNA mixture (Fig. 5).
To determine whether the presence of M affects the degradation of DNA by DNase,
10,ug DNA and 1 mg M (in 80,ul) were reacted for 4 h, as above, various concentrations
of DNase (bovine pancreas DNase 1; Sigma) (in 20^11) were then reacted with the M-DNA
mixture for 30 min at 37 C without shaking, and the 100 ;ul mixture was then incubated
with 100>il of cells for 30 min at 33 C with shaking. The addition of DNase, even at
DNase:DNA ratios of 1:1, 10:1, and 100:1, had no significant effect on the TF in the
absence of M. Only at a ratio of 1000:1 was there a decrease in the TF to undeteetable
levels. Similar results were obtained in the presence of M, except that the TF values
were several logs lower, reflecting the previously observed reduction in TF in the
presence of M, and the data were more variable. These preliminary studies suggested
that M does not affect the degradation of DNA by DNase. However, the conditions used
in these studies were not conducive to the enzymatic activity of DNase (e.g., no buffer
and no Mg were added), which probably explains the low activity of DNase at DNase:DNA
ratios below 1000:1.
All of the above studies, with additional permutations, are being repeated.
Furthermore, other clay minerals (e.g., kaolinite, attapulgite), with different cations on
the exchange complex, will be evaluated in these and other transformation systems.
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G. Stotzky
CR812484
Transduction Studies
The initial transduction studies are being conducted with a temperature-sensitive
strain of the generalized transducing bacteriophage, Pl::Cm cts. This strain is
essentially temperate at 37 C and lytic at 42 C, carries a gene conferring resistance to
chloramphenicol (Cm), has a broad host range, and in the prophage stage, remains
autonomous in the cytoplasm, thereby mimicking plasmids and bridging to our earlier
studies with true plasmids.
Lysates of phage PI have been prepared by two methods: 1) heat induction of a
lysogenic strain of Escherichia coli DU1040(P1) and 2) infection of E. coli W3110 with
phage PI in liquid culture followed by heat induction of lysis. Both methods produced
lysates containing liters as high as 10 PFU/ml.
Lactose-positive strains of E. coli have been verified for the absence of phage PI
and, if Ivsogenic for phage PI, have been cured. For example, E_. coli K802 was cured by
growth at 30 C until log phase followed by culture at 42 C. The lysate was then plated
on MacConkey Agar (MAC), incubated at 37 C, and resultant colonies were replica plated
to MAC containing 30 ug/ml Cm. Colonies that appeared to be cured (i. e., growth on
MAC but not on MAC + Cm) were subcultured on MAC and verified as E. coli by the
Enterotube II system (Roche Diagnostics Inc.). These cultures will be verified as being
cured of PI by their resistance to lysis at 42 C. Numerous strains of E_. coli that are
nonlysogenic for phage PI are now available.
The multiplicity of infection (MOD for the transduction of resistance to Cm by
phage PI to E_. cob' W3110 m vitro was then determined, to provide guidelines for the
ratios of the~phage and the~bacterium to be used in soil studies. The transduction
frequencies for different MOIs were:
MQI Transduction frequency (%)
9.5 6.6 x 10"1
6.6 5.1 x 10";
2.7 4.8 x 10~;
0.27 <: 1.0 x lO'1*
On the bases of these data, studies on transduction in soil have been initiated, using
E_. coli W3110 and Pseudomonas fluorescens PAO1 and phage PI at an MOI of 3, in both
sterile and nonsterile soil. These studies have been designed to determine the 1) survival
of the recipient bacterial species, 2) survival of the phage, 3) transduction of introduced
bacterial species, and 4) transduction of indigenous bacteria. As these studies have been
initiated concurrent with the preparation of this biannual progress report, watch the
annual report for the results of these and subsequent studies.
-------
Publications supported, in part, by Cooperative Agreement No. CR812484
Stotzky, G. and Babich, H. 1986, Survival of, and genetic transfer by, genetically
engineered bacteria in natural environments. In Advances in Applied
Microbiology, A. I. Laskin, ed. Academic Press, N.Y. pp. 93-138 (in press).
Stotzky, G. 1986. Influence of soil mineral colloids on metabolic processes, growth,
adhesion, and ecology of microbes and viruses. In Interaction of Soil Minerals
with Natural Organics and Microbes, P. M. Huang et al., eds. Soil Science Society
of America, Madison, WI. pp. 305-428 (in press).
Devanas, M. A., Rafaeli-Eshkol, D., and Stotzky, G. 1986. Survival of plasmid-containing
strains of Escherichia coli in soil: effect of plasmid size and nutrients on
survival of hosts and maintenance of plasmids. Applied and Environmental
Microbiology (in press).
Devanas, M. A. and Stotzky, G. 1986. Fate in soil of a recombinant plasmid carrying a
Drosophila gene. Current Microbiology (in press).
Devanas, M. A., Harsell, C., Wu, C., and Stotzky, G. 1986. Plasmid transfer in
Escherichia coli in sterile and nonsterile soil.. Abstract, Annual Meeting of the
American Society for Microbiology (in press).
Stotzky, G. and Devanas, M. A. 1986. Fate and potential ecologic effects of genetically
engineered microbes in soil. Abstract, International Congress of Soil Science
(submitted).
-------
1986 ASM ANNUAL MEETING
Washington, D.C. 23-28 March 1986
Official Abstract Form
(Read all instructions before typing)
! .( ; Plasmid Transfer in Escherichia coli in Sterile '
''. •>"ll's " ; and Nonsterile Soil. MONICA A. DEVANASl*. j
'CYNTHIA HARSELL1, CYNTHIA WU1, and G. STOTZKY2, Rutgers !
Univ., New Brunsvick, NJ1, and NYU, NY, NY2 |
i Strains of E. coli [DUIOUO(pRR226), DU10^0(pDU202),
• and PRCU8?, a lac+ C600] were introduced separately into
sterile and nonsterile soil, with and without nutrients (5%
Luria Broth). The numbers of colony forming units of in-
troduced strains, transconjugants, and indigenous soil mi-
. crobes were determined on nonselective and selective me-
dia. The survival of the donor and recipient strains in
. sterile soil was reduced under low moisture content (1.6%;
.the -33 kPa potential was 25%} and low nutrient levels, (in-
' oculated in saline), which apparently limited the poten-
tial for conjugation as none was observed. Plasmid trans-
fer at a frequency of 10 transconjugants/recipient cell
was observed in sterile soils at 25% moisture when nutri-
ents were added. In nonsterile soil at 25% moisture and
without nutrients, competition with indigenous soil mi-
crobes reduced the survival of the donors and recipients,
. and no plasmid transfer was observed. When nutrients were
: added with the inoculum, there was a rapid growth of the
introduced strains, during the first 2k h, but no plasmid
transfer was observed, and the numbers of both donors and
recipients subsequently declined rapidly.
Instructions
Indicate below the subject category number from the list on p. iv, check your poster or slide session preference, complete the
check list on the reverse side of this sheet, and sign your name in the space provided.
Indicate category number from page iv
Category number
Poster/Slide Session Preference , ,
Because of the flexibility in programming afforded by poster sessions, the Program Committee will attempt to schedule all
abstracts which (i) are considered by elected divisional officers to be of acceptable quality and (ii) conform to rules established by
the Program Committee. The decision of whether an abstract is scheduled in a slide or a poster session will be made by the elect-
ed Program Committee, which will be guided (but not bound) by the preference of the authors. Approximately 75% of the
abstracts will be scheduled in poster sessions. By submitting an abstract, the author agrees that the paper will be presented as
scheduled. ~ . .
Please check one: S Poster session preferred D Slide session preferred Q No preference
Please provide telephone number of signing author (201 ) 932-8906 '
Area code " •
vii '
-------
INTERIM PROTOCOL FOR ORAL EXPOSURE OF AVIAN
SPECIES TO MICROBIAL PEST CONTROL AGENTS
M. D. Knittel
Toxics and Pesticides Branch
Corvallis Environmental Research Laboratory
Corvallis, Oregon
-------
INTERIM PROTOCOL FOR ORAL EXPOSURE OF AVIAN
SPECIES TO MICROBIAL PEST CONTROL AGENTS
INTRODUCTION
Registration of Microbial Pest Control Agents (MPCAs) under Subsection M
of the Fungicide Insectide Rodenticide Act requires that the susceptibility of
nontarget species be tested. Among the nontarget hosts chosen for testing are
the avian species. The suggested test birds are either the Bobwhite quail or
the Mallard duck. These avian species have been chosen largely because of the
history of use in chemical toxicity testing and are adapted to laboratory
rearing.
This document will outline proposed test methods for oral exposure of
birds to an MPCA and includes a summary of the observations of symptoms which
must be made during the test.
Wild bird populations will be orally exposed to MPCAs by consuming
infested pests. The classic serial dilution of the pathogen to produce an LD5Q
determination most likely will not be realized because the MPCA is being tested
in a nontarget host. For this reason the proposal is to expose the test brids
to a single high dose, based on the field application rate of the MPCA. The
route chosen is oral inoculation. In this manner the MPCA is introduced
directly into the alimentary canal. A large dose should provide every oppor-
tunity for the MPCA pathogen to establish an infection, if it is capable.
-------
Definitions of Terms Used in This Interim Protocol
Dorsal: Located near or on back of an animal or one of its parts.
Histopathology: A branch of pathology that deals with tissue changes associ-
ated with disease.
Indigeneous disease: Disease carried by the host and specific for the host.
May be subclinical or in a carrier state.
LDso: The dose of pathogen which is fatal to 50% of the test animals.
Maximum hazardous dose: Doses of the MPCA should be multiples of maximum
amount of active ingredient expected to be available to nontarget species
in the environment. This amount can be based on the per acre application
rate or amount of MPCA contained in the target host at the time of death.
Necropsy: Autopsy, or examination of internal organs of the body to determine
cause of death.
Nephelometry: Measurement of numbers of particles by amount of light scatter-
ing.
Primary defense barriers: Outer dermal or mucous membranes of animal that act
as a mechanical barrier to infection.
Secondary defense barriers: Internal body mechanism against infection such as
phagocytic cells and nonspecific immune responses.
Target host: The host from which the MPCA was isolated and the one intended
for control.
Trituration: Grinding or crush as in a mortor with a pestle or in a tissue
homogenerator.
Ventral: On or belonging to lower or anterior surface of an animal, side
opposite the back.
Virulence: Disease-producing power of a microorganism.
-------
Test Procedures
A. Summary of Test: The test is designed to determine if the MPCA is infec-
tive and/or pathogenic to avian species. Because the MPCA is pathogenic
for hosts phylogenetically far removed from the test species, exposure is
by a large dose administered by the direct oral route. The test species
is either Bobwhite quail or the Mallard duck. These two species were
chosen because colonies have been established for chemical
toxicity research and are adapted to laboratory rearing.
A single large dose is administered by oral gavage into the crop or
preventriculus and the birds observed for death or illness due to infec-
tion. If death or illness is observed, then a serial dilution of the MPCA
is made and several groups of birds are orally inoculated to establish an
LD5o. If death or illness is not observed, birds are sacrificed at the
end of observation period and a necropsy performed and tissues taken for
histopathology and isolation of the test MPCA.
B. Test Species: Either Bobwhite quail or Mallard duck are suitable for the
test. Both have been used for testing toxicity of chemicals and, there-
fore, an extensive knowledge of laboratory rearing is available.
An inhouse colony of known pedigeree and indigeneous disease history
will be established. If test birds are purchased from a supplier, a
disease free certification will be required. Birds for a single test will
be of the same age, randomly selected, and placed in cages. Numbers of
birds for each test should consist of a minimum of 10 for each dilution of
the MPCA plus a control group.
1. Identification: Each bird will be identified with a leg band number.
-------
2. Husbandry: Current acceptable practices of good husbandry will be
followed at all times (Case and Robel, 1974).
3. Temperature: Room temperature should be maintained at 25°C +_ 2°C.
4. Relative Humidity: Relative humidity of 30 to 80 percent will be
maintained during study. Adequate ventilation will be by use of
exhaust fans.
5. Feed: All birds will be provided a ration suitable for rearing or
maintaining study species. The feed will be free of any antibiotic
medication prior to and during study. Ration should be provided ad
libitum. Feed consumption during the study will be recorded.
6. Water: Water will be available ad libitum during rearing and study.
Water will be changed as often as required to provide potable water.
During study of MPCA infection the amount of water used will be
recorded.
7. Photoperiod: Light and dark period should be maintained at 12 h
light 12 h dark.
8. Age: Birds for test will be at least 16 weeks old at the start of
the experiment.
C. Cages: Wire cages 30 x 24 x 10" are used to house the experimental quail.
The cages should have wire floors to allow droppings to pass through to a
catch pan beneath. Cages for Mallard ducks should be 27.5 x 39.25 x 9.5
inches for ducklings 14 days old and older. Adult duck require 5.4 square
feet per bird which would be a cage about 7 times the size used for the
young ducklings (192 x 276 x 66 inches).
-------
D. Number of Birds In Treatment Groups: The number of test birds per treat-
ment group should be no less than 10 with the same number in each control
group. However, if the number of birds is increased the results become
more statistically significant. The proposed acute exposure, maximum
hazardous dose, require that a group of 10 birds inoculated orally with a
number of the MPCA pathogen equivalent to some multiple of the per acre
application rate or amount contained in target host at death. A control
group would receive an equivalent inoculum which has been inactivated with
heat (autoclaving, 15 Ibs/in2 248°F for 15 minutes).
E. Preparation and quantification of inoculum
Bacteria: Bacterial MPCAs are grown in a liquid medium that best supports
growth, i.e., the medium for growth of Bacillus thuringiensis (Faust and
Traverse, 1981). Temperature and aeration during incubation should be
optimum for the bacterium under study to provide sufficient cell numbers
at the end of the incubation time. The incubation time should also be
optimum for the particular species of bacterium being studied.
The bacterial cells should be removed from the culture medium by
centrifugation at 10,000 RPM, 10 minutes at 4°C, and washed by resuspen-
sion and sedimentation with phosphate buffered saline at least twice. The
inculum size should be large and correspond to a multiple of the per acre
application rate or that found in the target host at the time of death.
Bacterial cell numbers should be adjusted to the above level either by
standard bacterial plate count or by nephelometry.
Fungi: Culture Growth and Quantisation: Growth of fungi culture for
inoculation can follow that outline above for bacteria using appropriate
5
-------
medium, temperature, and aeration to obtain sufficient growth (Lewis e*
al., 1958). If the spore is the infective stage, standardization will be
by microscopic enumeration in a hemocytometer or by plate count of
inoculum on appropriate medium. If the mycelium is the infective stage,
then the inoculum should be quantitated by the dry weight of the harvested
and washed mycelium.
Virus: The inoculum for virus infectivity studies should be grown in cell
culture if such a system is available. If not, the inoculum is prepared
from host tissue by trituration and purified from cell debris by centrifu-
gation. Bacterial contamination from the host may present problems upon
inoculation into the test birds and should be suppressed by the use of
antibiotics such as streptomycin and penicillin.
If the virus is occluded, separate inoccula of occluded and non-
occlued virus should be made and used in separate infectivity studies.
Quantification of the virus inoculum can be based either on tissue
culture infective doses (TCIDso), plaque assay, or lethal dose in the
target host (Groner et^ a_L, 1984).
Protozoan: The inoculum should be prepared from pure culture if possible.
In this way, extraneous contaminating microorganisms can be eliminated or
minimized. If it must be prepared from infected host, it should be
prepared in such a manner as to eliminate contaminating microorganisms.
This may be accomplished by repeated centrifugation followed by resuspen-
sion and incubation of the inoculum with antibiotics to suppress or
eliminate contaminating microorganisms.
-------
Quantification of inoculum can be by direct microscopic count in a
hemocytometer, or by 1059 determination in a susceptible host (Kudo,
1966).
F. Oral Inoculation: The dose of MPCA will be administered by oral gavage
with a syringe fitted with a stainless steel canula. Dose will be placed
into crop or preventriculus.
G. Post Inoculation Observation:
1. Time: The inoculated birds should be observed daily for signs of
illness and any mortalities recorded. The length of observation
period should be no less than 30 days.
2. Food and Water: Food and water should be provided ad lib. A daily
measurement of the amount of food and water consumed during the
observation period should be made. Signs of illness can be reflected
in loss of appetite and increase in water consumption. Recovery from
the illness will be seen in a recovery of appetite and drop in water
consumption.
3. Signs: The inoculated birds should be observed daily for signs of
illness. Among general symptoms of disease are the following:
labored breathing, ruffled feathers, drooping wings, listlessness,
head lowered, eyes closed, increased water consumption, mucous
discharge from mouth, diarrhea, loss of weight, coughing, gasping,
respiratory difficulty, and weakness. The control group should also
be observed for similar signs and the two groups compared.
-------
H. Necropsy: At the end of the observation period, all remaining birds
should be sacrificed and a necropsy performed. The internal organs should
be examined for any overt lesions. Samples of tissue should be removed
for attempt to isolate the MPCA and for histopathological preparation and
examination.
I. Bioassay of Inoculum: It is recommended that the experimental inoculum be
assayed in the target host at the same time as it is inoculated into the
birds. This will assure that the inoculum has not lost any virulence due
to preparation.
J. Reporting: The sponsor should submit to the USEPA all data developed by
the test that are suggestive or predictive of the infectivity of the MPCA
for avian species. Included are any behavioral changes during the
observation period, gross lesion of any internal organs, results of
attempts to reisolate the MPCA from sampled organs, and results of the
histopathologic examination of tissues. The reporting should follow the
requirements set out in Part 792 of the "Good Laboratory Practice
Standards" and should also include the following:
1. The name of the test, sponsor, testing laboratory, study director,
principle investigator and dates of testing.
2. A detailed description of the test MPCA, such as type (virus,
bacteria, etc.), source of inoculum (i.e., pure culture, infected
host, etc.), preparation of inoculum, and standardization of the
dose. Description should also include the type of resuspension fluid
and its composition.
8
-------
3. Detailed information about species of birds used in the test, their
scientific name, source of test birds (supplier or in-house colony),
and history of any indigenous disease. The sponsor should also
include age of test subjects, ambient conditions, i.e., temperature,
room lighting level, and photoperiod.
4. Description of the test cages, dimensions, number of test birds per
cage, number of replicates per dilution of MPCA.
5. Number of MPCAs dosed into each bird and method of verification,
microscopic count, plate count, or tissue culture infective doses.
6. The percentage of test birds affected by treatment at each observa-
tion period must be recorded.
7. Results of bioassay of inoculum in the target host, time of first
mortalities, time of last mortalities, and LD50 determination with
statistical treatment must be required.
8. Any deviation from this test guidelines and anything unusual about
the test, e.g., temperature fluctuations, disease problems in flock,
etc. must be reported.
-------
References
Surges, H. D., G. Croizer, and J. Huber. 1980. A review of safety tests on
baculovi ruses. Entomophaga £5_, 329-340.
Case, R. M., and R. J. Robel. 1974. Bioenergetics of the bobwhite. J. Wild.
Manage. 38, 638-652.
Faust, R. M., and R. S. Travers. 1981. Occurrence of resistance to neomycin
and kanamycin in Bacillus popilliae and certain senotypes of Bacillus
thuringiensis: Mutation potential in sensitive strains. J. Invert. Path.
34, 113-116.
Groner, A., R. R. Granados, and J. P. Burand. 1984. Interaction of Autographa
californica nuclear polyhedrosis virus with two nonpermissive cell lines.
Invervirol. 2U 203-209.
Kudo, R. R. 1966. Protozoology. Chapter 43. pp. 1057-1088. Charles C.
Thomas Publisher. Springfield, Illinois. 5th ed.
Lewis, G. M., M. E. Hopper, J. W. Wilson, and 0. A. Plunkett. 1958. Introduc-
tion to medical mycology. Year Book Publishers, Inc. Chicago, Illinois.
pp. 382-418. 4th ed.
10
-------
INTERIM PROTOCOL FOR INTRAVENEOUS EXPOSURE OF AVIAN
SPECIES TO MICROBIAL PEST CONTROL AGENTS
M. D. Knittel
Toxics and Pesticides Branch
Corvallis Environmental Research Laboratory
Corvallis, Oregon
-------
INTERIM PROTOCOL FOR INTRAVENEOUS EXPOSURE OF AVIAN
SPECIES TO MICROBIAL PEST CONTROL AGENTS
INTRODUCTION
Registration of Microbial Pest Control Agents (MPCAs) under Subsection M
of the Fungicide Insectide Rodenticide Act requires that the susceptibility of
nontarget species be tested. The avian species are among the nontarget hosts
chosen for testing. The suggested test species is either the Bobwhite quail or
the Mallard duck. These avian species have been chosen largely because of
their history of use in chemical toxicity testing and their adaptability to
laboratory rearing.
This document will outline proposed methods of exposing the test birds to
the MPCA, route of inoculation, and observation of symptoms during the test.
Because the MPCAs are microbial pathogens, but being tested in nontarget
species, effects may be limited to symptoms other than overt mortality. The
classic serial dilution of the pathogen to produce an LD50 determination will
probably not be possible. For this reason, the experiments will expose the
test birds to a high dosage of the MPCA by the most direct route: intravenous
inoculation. This introduces the MPCA directly into the blood stream, bypass-
ing the primary barriers of body defenses and allows it access to the internal
organs. A large dose would overwhelm the secondary body defense mechanisms,
providing an opportunity to establish an infection, if it is pathogenic to the
avian species.
-------
Definitions of Terms Used in This Interim Protocol
Dorsal: Located near or on back of an animal or one of its parts.
Histopathology: A branch of pathology that deals with tissue changes associ-
ated with disease.
Indigeneous disease: Disease carried by the host and specific for the host.
May be subclinical or in a carrier state.
Intravenous: Located within or going into the veins.
LD50: The dose of pathogen which is fatal to 50% of the test animals.
Maximum hazardous dose: Doses of the MPCA should be multiples of maximum
amount of the per acre field active ingredient expected to be available to
the nontarget species in the environment. This amount can be based on the
per acre application rate or amount of MPCA contained in a target host at
time of death.
Necropsy: Autopsy, or examination of internal organs of the body to determine
cause of death.
Nephelometry: Measurement of numbers of particles by amount of light scatter-
ing.
Primary defense barriers: Outer dermal or mucous membranes of animal that act
as a mechanical barrier to infection.
Secondary defense barriers: Internal body mechanism active against infection
such as phagocytic cells and nonspecific immune responses.
Target host: The host from which the MPCA was isolated and the one intended
for control.
Trituration: Grinding or crushing as in a mortor with a pestle or in a tissue
homogenator.
Ventral: On or belonging to lower or anterior surface of an animal, side
opposite the back.
-------
Virulence: Disease-producing power of a microorganism.
Test Procedures
A. Summary of Test: The test is designed to determine if the MPCA is infec-
tive and/or pathogenic to avian species. Because the MPCA is pathogenic
for hosts phylogenetically far removed from the test species, exposure
will be by a large (maximum) dose administered by a direct route such as
intravenous (I.V.). The test species is either Bobwhite quail or the
Mallard duck. These two species were chosen because colonies have been
established for chemical toxicity research and they are adapted to labora-
tory rearing.
A single large dose is injected I.V. into the wing vein and the birds
observed for death or illness due to infection. If death or illness is
observed, then a serial dilution of the MPCA is made and several groups of
birds are injected I.V. to establish an LD5Q. If death or illness is not
observed, birds are sacrificed at the end of the observation period, a
necropsy performed, and tissues taken for histopathology and isolation of
the test MPCA.
B. Test Species: Either Bobwhite quail or Mallard duck are suitable for the
test. Since both have been used for testing toxicity of chemicals, an
extensive knowledge of laboratory rearing exists.
An inhouse colony of known pedigree and indigenous disease history
will be established. If test birds are purchased from a supplier, a
disease free certification will be required. Birds for a single test will
be of the same age, randomly selected, and placed in cages. Numbers of
-------
birds for each test should consist of a minimum of 10 for each dilution of
the MPCA plus a control group.
1. Identification; Each bird will be identified with leg band number.
2. Husbandry: Current acceptable practices of good husbandry will be
followed at all times (Case and Robel, 1974).
3. Temperature: Room temperature should be maintained at 25°C, +_ 2°C.
4. Relative Humidity; Relative humidity of 30 to 80 percent will be
maintained during study. Adequate ventilation will be assured by use
of exhaust fans.
5. Feed: All birds will be provided a ration suitable for rearing or
maintaining study species. The feed will be free of any antibiotic
medication prior to and during the study. Rations should be provided
ad libitum. Feed consumption during the study will be recorded.
6. Water: Water will be available ad libitum during rearing and study.
Water will be changed as often as required to provide potable water.
During study of MPCA infection the amount of water used will be
recorded.
7. Photoperiod: Light and dark period will be maintained at 12 h light
12 h dark.
8. Age: Birds for test will be at least 16 weeks old at the start of
the experiment.
C. Cages: Wire cages 30 x 24 x 10" are used to house the experimental quail.
The cages should have wire floors to allow droppings to pass through to a
catch pan beneath. Cages for Mallard ducks should be 27.5 x 39.25 x 9.5
inches for ducklings 14 days old. Adult ducks require 5.4 square feet per
-------
bird which would be a cage about 7 times the size used for the young
ducklings (192 x 276 x 66 inches).
D. Number of Birds in Treatment Groups: The number of test birds per treat-
ment group should be no less than 10, with the same number in each control
group. However, a larger number of birds will increase the statistical
significance of the results. The proposed acute exposure, maximum
hazardous dose, requires that a group of 10 birds be injected intraven-
ously with a number of the MPCA pathogen equivalent to 10 to 100 times the
per acre rate applied to a 70 kg man. A control group would receive an
equivalent inoculum which has been inactivated with heat (autoclaving, 15
Ibs/in2 248°F for 15 minutes).
E. Preparation and Quantification of Inoculum
Bacteria: Bacterial MPCAs are grown in a liquid medium that best supports
growth, i.e., the medium for growth of Bacillus thuringiensis (Faust and
Traverse, 1981). Temperature and aeration during incubation should be
optimum for the bacterium under study to provide sufficient cell numbers
at the end of the incubation time. The incubation time should also be
optimum for the particular species of bacterium being studied.
The bacterial cells should be removed from the culture medium by
centrifugation at 10,000 RPM, 10 minutes at 4°C, and washed by resuspen-
sion and sedimentation with phosphate buffered saline at least twice. The
inoculum size should be large and correspond to a multiple of the per acre
application dose or amount contained in host at death. Bacterial cell
numbers should be adjusted to the above level either by standard bacterial
plate count or by nephelometry.
-------
Fungi: Culture Growth and Quantification: Growth of fungi for inocula-
tion can follow that outlined above for bacteria using appropriate medium,
temperature, and aeration to obtain sufficient growth (Lewis et al.,
1958). If the spore is the infective stage, standardization will be by
microscopic enumeration in a hemocytometer or by plate count of inoculum
on appropriate medium. If the mycelium is the infective stage, then the
inoculum should be quantitated by the dry weight of the harvested and
washed mycelium.
Virus: The inoculum for virus infectivity studies should be grown in cell
culture if such a system is available. If not, the inoculum is prepared
from host tissue by trituration and purified from cell debris by centrifu-
gation. Bacterial contamination from the host may present problems upon
inoculation into the test birds and should be suppressed by the use of
antibiotics such as streptomycin and penicillin.
If the virus is occluded, separate inoccula of occluded and non-
occlued virus should be made, and used in separate infectivity studies.
Quantification of the virus inoculum can be based either on tissue
culture infective doses (TCID5o), plaque assay, or lethal dose in the
target host (Groner e£ _al_., 1984).
Protozoan: If the protozoan can be cultured, the inoculum should be
prepared in such a manner. If, however, it must be prepared from infected
host, it should be prepared in such a manner as to eliminate contaminating
microorganisms. This may be accomplished by repeated centrifugation
followed by resuspension and incubation of the inoculum with antibiotics
to suppress or eliminate contaminating microorganisms.
6
-------
Quantification of inoculum can be by direct microscopic count in a
hemocytometer, or by LD50 determination in a susceptible host (Kudo,
1966).
F. Intravenous Injection: The ventral wing vein of the bird is the most
convenient for injection. The vein is exposed by plucking the feathers
from the underside of the wing. The vein lies in a depression between the
biceps brachial and tricepts humerials muscles. The vein becomes more
visable if the skin surface is wetted with 70% alcohol. Both wings are
extended dorsally and gripped together firmly with one hand in the area of
the wing web. Surface of the skin over the vein is sterilized with
tincture of iodine (20 grams iodine and 24 grams potassium iodide per 1000
mis of distilled water). A needle (3/4" 20 ga) is inserted into the vein
in opposite direction of the blood flow. The plunger of the syringe is
slowly depressed to expel 1 the inoculum and the needle withdrawn. A
cotton ball or gauze square soaked with tincture of iodine is held on the
puncture side until bleeding stops (Zandler, 1983).
G. Post Inoculation Observation:
1. Time: The inoculated birds should be observed daily for signs of
illness and any mortalities recorded. The length of observation
period should be no less than 30 days.
2. Food and Water: Food and water should be provided ad lib. A daily
measurement of the amount of food and water consumed during the
observation period should be made. Sign of illness are reflected in
loss of appetite and thirst. Recovery from the illness will be seen
in a recovery of appetite and drop in water consumption.
7
-------
3. Signs: The inoculated birds should be observed daily for signs of
illness. Among general symptoms of disease are the following:
labored breathing, ruffled feathers, drooping wings, listlessness,
head lowered, eyes closed, increased water consumption, mucous
discharge from mouth, diarrhea, loss of weight, coughing, gasping,
respiratory difficulty, and weakness. The control group should also
be observed and the two groups compared.
H. Necropsy: At the end of the observation period all of the birds should be
sacrificed and a necropsy performed. The internal organs should be
examined for any overt lesions. Samples of tissue should be removed for
attempts to isolate the MPCA and for histopathological preparation and
examination.
I. Bioassay of Inoculum: It is recommended that the experimental inoculum be
assayed in the target host at the same time as it is inoculated into the
birds. This will assure that the inoculum has not lost any virulence due
to preparation.
J. Reporting: The sponsor should submit to the USEPA all data developed by
the test that are suggestive or predictive of the infectivity of the MPCA
for avian species. Included are to any behavioral changes during the
observation period, gross lesions on any internal organs, results of
attempts to reisolate the MPCA from sampled organs, and results of the
histopathologic examination of tissues. The reporting should follow the
requirements set out in Part 792 of the "Good Laboratory Practice
Standards" and should also include the following:
8
-------
1. The name of the test, sponsor, testing laboratory, study director,
principle investigator and dates of testing.
2. A detailed description of the test MPCA, such as type (virus,
bacteria, etc.), source of inoculum (i.e., pure culture, infected
host, etc.), preparation of inoculum, and standardization of the
dose. Description should also include the type of resuspension fluid
and its composition.
3. Detailed information about species of birds used in the test, their
scientific name, source of test birds (supplier or in-house colony),
and history of any indigenous disease. The sponsor should also
include age of test subjects, ambient conditions, i.e., temperature,
room lighting level, and photoperiod.
4. Description of the test cages, dimensions, number of test birds per
cage, number of replicates per dilution of MPCA.
5. Number of MPCAs injected into each bird and method of verification,
microscopic count, plate count, or tissue culture infective doses
must be indicated.
6. The percentage of test birds affected by treatment at each observa-
tion period must be recorded.
7. Results of bioassay of inoculum in the target host, time of first
mortalities, time of last mortalities, and 1050 determination with
statistical treatment must be provided.
8. Any deviation from this test guidelines and anything unusual about
the test, e.g., temperature fluctuations, disease problems in flock,
etc. must be reported.
-------
References
Surges, H. D., G. Croizer, and J. Huber. 1980. A review of safety tests on
baculoviruses. Entomophaga £5, 329-340.
Case, R. M., and R. J. Robel. 1974. Bioenergetics of the bobwhite. J. Wild.
Manage. 38, 368-652.
Faust, R. M., and R. S. Travers. 1981. Occurrence of resistance to neomycin
and kanamycin in Bacillus popilliae and certain senotypes of Bacillus
thuringiensis: Mutation potential in sensitive strains. J. Invert. Path.
34, 113-116.
Groner, A., R. R. Granados, and J. P. Burand. 1984. Interaction of Autographa
californica nuclear polyhedrosis virus with two nonpermissive cell lines.
Invervirol. _21_t 203-209.
Kudo, R. R. 1966. Protozoology. Chapter 43. pp. 1057-1088. Charles C.
Thomas, Publisher. Springfield, Illinois. 5th ed.
Lewis, G. M., M. E. Hopper, J. W. Wilson, and 0. A. Plunkett. 1958. Introduc-
tion to medical mycology. Year Book Publishers, Inc. Chicago, Illinois.
pp. 382-418. 4th ed.
Zandler, D. V. 1983. Diseases of Poultry. Ed by M. S. Hofstad, B. W. Calnek,
C. F. Humbolt, W. M. Reid, and H. W. Yoder, Jr. 8th ed. Iowa State
University Press, Ames, IA. p. 30.
10
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ASSESSMENT OF METHODS FOR THE
DETECTION, IDENTIFICATION, AND
ENUMERATION OF GENETICALLY-ENGINEERED
BACTERIA IN SOIL
ITEM 6940A
FEBRUARY, 1986
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Assessment of Methods for the Detection, Identification, and
Enumeration of Genetically-Engineered Bacteria in Soil
PETER G. HARTEL
Department of Agronomy, University of Georgia, Athens, Georgia 30602*
INTRODUCTION 2
The nature of soil and rhizosphere 3
Preliminary considerations to soil and rhizosphere methods 3
METHODS OF ENUMERATION 5
Fluorescent antibody 5
Plate counts with selective agents 8
Most-probable-number 10
DNA probes 11
COMPARISON OF METHODS OF ENUMERATION 14
METHODS OF DETECTION AND IDENTIFICATION 17
Phage typing 17
Intrinsic antibiotic resistance 17
Other serological methods 18
CONCLUSION 18
LITERATURE CITED 20
* This report represents the opinion of the author. It carries no official
endorsement by the University of Georgia.
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INTRODUCTION
Recent interest in the deliberate or inadvertent release of genetically-
engineered microorganisms to the environment (2) raises questions about the
ability to detect, identify, and enumerate these microorganisms in order to
ascertain their growth, survival, and potential ecological effects. This
report is an assessment of the methods for the detection, identification, and
enumeration of genetically-engineered bacteria in soil. Because only a few
papers involve genetically-engineered bacteria in soil, general methods for
the study of soil bacteria are included here as well. Methods of enumeration
are considered first because these methods are the most developed and likely
to prove the most useful. Methods of detection and identification are
considered in a later section. Detection is distinguished here from
identification because in some of the discussed methods, bacteria can be
detected without identification. Methods involving total microbial community
studies, such as biomass and activity measurements, which do not differentiate
one bacterium from another are not considered. This is a realization that the
primary need in the release of genetically-engineered bacteria into soil is
to study a particular bacterial strain and not the microbial community at
large.
Methods of enumeration in soil generally fall into two categories:
direct and indirect. Direct methods identify bacteria by some form of
microscopy (visible light, scanning electron, transmission electron,
epifluorescence, fluorescent antibody), whereas indirect methods employ some
form of viable counting procedure (dilution plate counts with or without
selective agents, most-probable-number analysis). DNA probe methods are not
yet sufficiently evolved and currently both direct and indirect methods are in
use. Of the direct methods, only the fluorescent antibody and DNA probes can
-------
differentiate one bacterium from another, so these are the only direct methods
considered. This paper will seek to identify advantages and disadvantages of
•
each method before finally comparing all the methods.
The nature of soil and rhizosphere r~
Soil is probably the most complex environment in which bacteria can
exist. This is because soil is dominated by a solid phase of different-sized
particles surrounded by liquid and gas phases which fluctuate greatly in time
and space (85). Consequently the diversity, activity, and numbers of bacteria
in soil also fluctuate greatly in time and space. Thus, it must be understood
that any method of detection, identification, and enumeration of bacteria in
soil has the capacity to represent but a small fraction of the soil's
heterogeneity.
The rhizosphere, the zone of microbial stimulation under the influence of
plant roots, is a natural subset of the soil habitat. It is important because
1) numbers of bacteria are higher here than in the surrounding soil (34) as a
consequence of chemically heterogeneous organic materials released in root
exudates (28), and 2) the rhizosphere presents some distinct problems for the
detection, identification, and enumeration of bacteria. For example, bacteria
may be present under a mucilaginous sheath on the root surface (48, 75) or
invade and colonize root cortical tissue (24). It is still unknown whether
rhizosphere populations of bacteria are best quantified in terms of surface
area or on a root weight basis. These and other problems have led some authors
to state that a precise determination of bacteria in the rhizosphere is not
yet possible (35).
Preliminary considerations to soil and rhizosphere methods
Regardless of the method use to detect, identify, or enumerate soil
bacteria, many important preliminary steps in the proper handling of the soil
-------
must be considered. First, in the case of sieved soil, a 2-mm sieve should be
used because it conforms with the arbitrary upper size limit used in soil
•
analyses (13). It is important to mix this soil thoroughly. James and
Sutherland (47) found significant differences between aliquot samples from a
supposedly well-mixed sieved soil. If a soil is not sieved, the soil samples
should be as homogenous as possible with as much replication as possible to
reduce sampling error (90). Second, it is impossible to store soil samples
longer than a few hours without quantitative and qualitative changes occurring
within the microflora (50), so soil samples should be processed as quickly as
possible. This is particularly important in avoiding changes of soil water
potential. Third, when soil is suspended in a diluent, it is important to
standardize this procedure, because different cell aggregates are not dispersed
or destroyed with equal ease (49). Finally, in the case of inoculated non-
sterile soils, inoculation densities for determining bacterial growth or
survival should be reasonable. The author strongly supports the concept of a
soil threshold for bacterial numbers (20). For example, protozoan predation
o
may be significant at bacterial inoculation densities of 10 cells per g of
soil, but not at densities of 10 cells per g of soil (40). Thus, an environ-
mental effect is observed at high bacterial inoculation densities that would
not normally occur if the densities were more reasonable.
In the case of rhizosphere, what is or is not rhizosphere soil must be
carefully defined because definitions of what constitutes rhizosphere soil
vary. Generally rhizosphere soil is defined as soil still adhering to the
roots after removal of the plant from the soil (60). Although it is still
difficult to determine the amount of this rhizosphere soil, the dry weight of
the soil can be estimated (41).
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METHODS OF ENUMERATION
Fluorescent Antibody
*
The fluorescent antibody (FA) technique is a method in which fluorescent
markers are attached to the antibody proteins for visualizing a antigen-antibody
reaction. Excellent extensive reviews of the theory and principles of this
technique already exist (37, 54). In the direct FA technique, the specific
antibody against the bacterium of interest is labelled, whereas in the indirect
method, the specific antibody against the bacterium of interest is unlabelled
and a second labelled antibody against the first antibody is then required to
visualize the antigen-antibody reaction. The direct technique has an advantage
of simplicity because only one antibody reagent is involved, whereas the
indirect technique has an advantage of a brighter fluorescent label (54).
The FA technique has three primary advantages:
1) The technique can identify a bacterium that is otherwise difficult to work
with or to isolate. An example is the extensive work in soil (32) and in
rhizosphere (73) with the chemoautotrophic nitrifier Nitrobacter which until
the advent of immunofluorescent methods was usually enumerated by a most-
probable-number method (3).
2) The technique is useful for showing spatial relationships, particularly in
rhizosphere where little is known about the soil's micro- and macrostructure
(51).
3) Assuming antisera are available, the technique is fast.
The disadvantages of the FA technique are extensively reviewed elsewhere
(12, 80) and are summarized below:
1) The specificity of the FA reaction must be studied for each bacterium of
interest. The number of strains or species necessary to satisfy this requirement
varies with each experiment, depending on the experimental objectives, the
-------
specificity pattern that emerges, and the information already known about the
serology of the organism. Monoclonal antibodies will be useful in offering
greater specifity for the FA technique in the future, but as yet ttiey have
not been extensively employed for soil.
2) Although the use of rhodamine gelatin as a primary stain (10) lias effectively
eliminated non-specific staining of soil particles and organic matter, and the
use of a combination irgalan black (45) or india ink and rhodamine gelatin
prestaining has effectively eliminated problems with membrane filter auto-,
fluorescence, plant autofluorescence continues to be a problem for some
rhizosphere studies. For example, Diem et al. (22) were unable to eliminate
the autofluorescence of rice roots, whereas Schank et al. (79) were more
fortunate in that autofluorescence was prominent in the stelar portion of a
grass root, but not in the mucigel area where most of the bacteria were located.
3) The FA technique cannot differentiate between living and dead cells as both
fluoresce brightly and specifically. One alternative is to simultaneously
measure metabolically active bacteria by autoradiography (32) or to use a
tetrazolium dye which reduces to optically-dense, dark red spots in actively
respiring bacteria (93). The length of time that a nonviable cell fluoresces
varies with the cell and the environment. Nonviable cells generally fragment
and clear within a 1-2 week period (11), but this would not be the case in
soils with low water potential.
4) The practical lower limit of detectability of the FA technique is only
O /
fair, about 10 -10 cells per g of soil (Table 1). In an effort to improve
the original Breed slide method (10 |JL of a 1:10 soil dilution spread on 1.0
2 6
cm slide surface) which requires a bacterial population of 10 cells per g of
soil to see one antigen cell per microscope field and where only a limited
amount of soil or soil dilution could be observed before the antigen of interest
-------
Table 1. Lowest bacterial density observed in some selected papers using the FA
technique.
Organism Soil (S) or Lowest density reported, Reference
Rhizosphere (R) log no. cells/g of soil
Rhizobium japonicum
Rhizobium japonicum
Rhizobium japonicum
Rhizobium japonicum
Rhizobium japonicum
Beijerinckia sp.
chickpea Rhizobium
Nitrobacter spp.
Nitrobacter winogradskyi
Nitrobacter sp.
Escherichia coli
S
S
S
R
R
S
S
S
S
R
S
-3.54
4.71
-3.54
3.61
3.15
5.08
-3.00
3.52
4.38
3.95
4.57
20
29
88
80
74
21
55
33
72
73
81
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was obscured, Schmidt (81) developed a method to desorb the bacteria from the
soil particles with a surfactant, separate the bacteria from soil colloid, and
*
collect the bacteria on membrane filters. Although subsequent development of
simple soil blending-centrifugation procedures (5, 6, 30, 91) and use of
different diluents (6, 55, 70) have improved bacterial recovery from soil, the
results still vary greatly with soil texture and bacterial strains.
Plate counts with selective agents
Before considering dilution plate counts with selective agents, it is
important to consider the plate count method in general because the latter
provides the foundation on which the former exists. The method in general has
the advantages of being inexpensive and easy to perform. But despite appear-
ances, the method is subject to large experimental errors and it is not easy
to evaluate the results unless the technique is given the greatest care (50).
For example, certain correctable errors need to be taken into consideration.
These errors are 1) accounting for the autoclave loss of the amount of diluent,
and 2) avoiding long settling times between dilutions that can significantly
decrease counts (46).
The disadvantages of this method are extensively reviewed elsewhere (46,
49) and are summarized below:
1) Plate counts do not mimic the natural environment or show spatial relation-
ships of the soil and bacteria in vivo. Although plate counts were initially
criticized because most soil bacteria are short coccoid rods, a morphology
that was not necessarily shared by the bacteria observed on soil dilution
plates (18), it now seems reasonable that this is a response to starvation
(56). For example, Rhizobium japonicum are known to lose their bacillus-like
shape and acquire a coccoid-like form in soil (20).
-------
2) The method needs greater replication to achieve the same precision as the
fluorescent antibody technique.
3) A proportional decrease in the number of colonies on a plate is never
obtained in a soil dilution series (63). In a soil diluted 1:10, the number
of colonies on a plate usually decreases only in a ratio of 1:5. rThis is
primarily because of adsorption of bacteria to the inner walls of the pipettes.
Furthermore, this proportional decrease is not linear because of increasing
dispersion of the suspension with increasing dilution (49).
4) For spread plates, bacteria can adhere to the spreader when spreading the
inoculum over the agar surface; for pour plates, colonies may coalesce if too
close together making counting difficult, or the heat from the molten agar
during pouring could cause lower counts.
5) Plate counts are difficult to standardize. Unless plate counts are performed
on the same soil at the same time under the same strict standardized conditions,
the results will show significant differences among different laboratories
(27).
6) As mentioned in the Introduction, the presence of mucigel layers in the
rhizosphere or bacterial invasion of the endorhizosphere may cause under-
estimation of bacterial counts in these type of studies. For example, vigorous
washing of Paspalum notatum removed little nitrogenase activity associated
with Azotobacter paspali of the roots (23).
The degree of selectivity of the plate counts is seldom sufficient to allow
enumeration of any bacterium of interest in the presence of closely related
types (71). To overcome this, inhibitors such as antibiotics, dyes, or other
selective compounds are added to the plate count medium and natural mutants of
the wild-type bacterium are selected that are resistant to that selective
compound. Thus, the method allows greater selectivity with a concomitant
-------
10
increase in the ability to detect lower numbers of the bacterium of interest.
Another minor advantage is that nothing needs to be sterilized (17). This
method has widespread use and more recently has been proposed as a "technique
for isolating bacteria of potential use in genetic engineering (59, 62). Two
preconditions are assumed for this method: 1) development of a dependence to
the selective compound is avoided by keeping the stock cultures of resistant
mutants on the appropriate medium without the compound, and 2) the resistance
of the bacterium to two compounds is preferable to one, not only to avoid
naturally occurring resistant bacteria in the soil, but also to lower the
potential for genetic exchange of the resistance markers to other bacteria in
the soil. Otherwise, the technique has the following specific disadvantages:
1) The mutant and wild type organism may not behave similarly. For example,
high level antibiotic resistance may confer symbiotic changes in Rhizobium
(15), result in the loss of pathogenicity for some plant pathogens (9, 77), or
cause plasmid loss (42).
2) In the case of antibiotics as a selective agent, not all antibiotics are
suitable as markers, thus limiting the number of strains that can be labelled
with different markers (82).
3) For some slow-growing bacteria that exist in low densities in the soil,
fungal contamination is difficult to suppress. The more recent use of a
variety of different fungicides (17, 39) may help to eliminate this.
Most-Probable-Number
The most-probable-number (MPN) technique is basically an extinction
dilution method (1) that estimates the density of organisms based on the
highest probability of the observed results (19). Enrichment is accomplished
by withholding or adding a specific nutrient or growth factor, adding a toxic
material, or altering some other chemical or physical factor to the advantage
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11
of the desired microorganism. Thus, the desired bacterium grows at a faster
rate than the unwanted bacteria (18). In some cases, a bacterial species can
•
be enumerated by a plant infection MPN (89). The MPN technique makes three
assumptions: 1) the bacteria are randomly distributed in the diluent, 2) the
presence of one or more bacteria in a tube always gives a positive response,
and 3) the organism possesses a unique and detectable metabolic reaction in
order to be selected. The MPN method shares the same advantages and
disadvantages of the selective plate count as well as several unique advantages
and disadvantages. Compared to a plate count method, an MPN method has advantages
of allowing enumeration of bacteria for which a suitable solid medium does not
exist and allowing enumeration of bacteria that exist at too low a density for
counting on a plate. The disadvantages of the MPN technique are:
1) If the specificity is not great enough, false positives are a problem.
2) The MPN technique is always less precise than a colony count (86). Also,
the standard deviations and confidence limits are too large for accurate
determinations of small but potentially important population fluctuations
(72). To improve the precision of the technique, smaller dilution ratios or
more tubes per dilution can be done, but the number of manipulations and time
involved to do this limit its value.
DNA Probes
A DNA probe is useful for screening a large number of bacterial colonies
for a specified DNA sequence or genes (38) and can be used for any bacterial
species in which a DNA sequence unique to the organism of interest can be
isolated (31). This method has recently been used for enumeration of bacteria
in food (43, 44), feces (47), and pond sediment (78). Briefly, the method
involves 1) isolating the bacteria, 2) lysing the bacteria to obtain their
DNA, 3) denaturing the DNA, 4) fixing the DNA to a nitrocellulose filter, 5)
-------
12
hybridizing a radioisotope-labelled homologous section of DNA containing the
sequence of genes of interest to the denatured DNA, and 6) assaying the amount
•
of hybridization by autoradiography. The steps are given here because depending
on the method, some of the steps are interchangeable. If the original bacteria
are required, colonies must be grown and copies made by replica plating.
The methodology of DNA probes for enumeration is still in its infancy, so
it is difficult to ascertain the advantages and disadvantages because both
direct and indirect methods of enumeration are currently in use. The indirect
method shares many of the advantages and disadvantages of selective plate
counts, whereas the direct method shares many of the advantages and disadvantages
of fluorescent antibody. Before these methods are considered, all DNA probe
methods share several common preconditions and disadvantages. These are:
1) The probes require removal of any nonspecific DNA sequences from the gene
of interest to minimize background hybridization (7).
2) The range of probe specificity on the general microbial population must be
assessed. It is possible that a number of species might possess DNA homologous
with the DNA probe (26, 31). Partial homology has been observed between the
probe for heat-labile, toxin-producing Escherichia coli and Vibrio cholerae
(53, 68).
The disadvantages in common are:
32
1) All the current papers on DNA probes use P as the radioisotope of choice.
32
Unfortunately, the short half-life of P necessitates frequent probe relabelling
and close monitoring of specific activity (57). Stable, nonisotopic DNA
probes are expected to solve this problem in the future (31), but they have
yet to be tested.
2) Although the likelihood is small, hybridization with other sequences in the
bacterial genome cannot be ruled out (7).
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13
An indirect method of using DNA probes was adopted by Sayler et al. (78)
and Echevarria et al. (26). In the first paper, the general bacterial population
•
in a pond sediment was targeted for the presence of toluene- and naphthalene-
degrading genes. The^method was indirect because bacterial colonies were
isolated from sediment samples that were diluted and plated on a minimal
medium amended with toluene and naphthalene. In the second paper, E. coli in
water was targeted for the presence of three enterotoxin genes. Water samples
were filtered and the filters placed on MacConkey medium from which bacterial
isolates were obtained. The method worked in both papers, but the papers
clearly demonstrated the technique was still in its developmental stages.
These and other related papers include many recommendations for increasing the
sensitivity of the method: the use of high copy number plasmids and greater
gene specificity (78), higher specific activity of the probe (31), the use of
a simple steaming step to increase DNA binding to the nitrocellulose filters
(61), and the use of a more highly selective medium (44). The last recommenda-
tion is difficult to ascertain because the limit of sensitivity of the method
is determined in part by the number of cells that can be placed on a filter to
allow sufficient growth so that positive cells may be detected (43). Thus,
Sayler et al. (78) were able to achieve a high probe sensitivity of 1 colony
per 10 nonhomologous background colonies, whereas Hill et al. (44) were able
to recover only 13% of Yersinia enterocolitica from a ratio of 1,820 nonvirulent
cells to 1 virulent cell. Furthermore, all these experiments have used bacteria
with relatively fast growth rates. These limits of detectability may not be
as good for bacteria with slower growth rates because of overgrowth. Thus,
the use of selective media may or may not be warranted depending on the
experimental conditions.
In a direct method of using DNA probes, Kuritza and Salyers (57) enumerated
Bacteroides vulgatus in feces using a chromosomal fragment of that bacterium
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14
as a gene probe. No bacterial colonies were isolated. This method clearly
identifies the major advantage of the direct method: the difficult methodological
*
problems of isolating and enumerating obligate anaerobes were avoided. In
addition, another advantage was that the samples could be frozen and assayed
when convenient. The disadvantages were: 1) the method could not distinguish
between viable and nonviable cells, although this is not such a problem with
feces, 2) the interference of fecal material necessitates additional
centrifugation steps and the need for an internal standard to correct for the
remaining interference, and 3) the method requires that the bacteria be well-
represented in the DNA mixture (>2%). It was suggested this could be improved
by solution hybridization but this was not tested.
This direct technique has yet to be tested on soil. Amplification of the
sensitivity to obtain a lower limit of detectibility will be a major problem
because B. vulgatus numbers exceed 10 cells per g dry weight of feces whereas
such numbers of a single bacterial species in soil do not exist. The direct
method will certainly share the same problem of immunofluorescence in trying
to separate bacteria or even the DNA (87) from soil.
COMPARISON OF METHODS OF ENUMERATION
Before considering the advantages and disadvantages of the methods of
enumeration, it is important to realize the fundamental differences between
direct and indirect methods. Under normal circumstances, direct microscopic
bacterial counts are 10- to 100-fold higher than plate counts (30, 75). These
differences have not been specifically elucidated, but are probably because 1)
microscopy includes dead as well as live bacterial cells, 2) some bacterial
cells in soil are either alive but sufficiently debilitated and unable to
multiply and form colonies on the plating medium (50) or alive but do not grow
on artificial media because of unknown nutritional and physiological needs,
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15
and 3) problems of obtaining complete dispersal of the bacteria in the soil
suspension for counting. The latter is important because bacteria are not
•
randomly dispersed in soil (18) and in rhizosphere (69) but are aggregated in
clumps and patches interpersed with areas where bacteria are sparse or absent.
Direct counting techniques can use harsher and more effective desbrption and
disruption techniques than indirect counting methods because bacterial cell
integrity and not viability is important (72). Differences can become even
larger if the bacterial population is sufficiently debilitated. Xu et al.
(92) noted a dramatic 6-log difference in cell density between fluorescent
antibody and culturable counts for Vibrio cholerae in a marine habitat. It is
also interesting to note that in cases of good agreement between a direct and
indirect method, this often occurs in sterile soil or in the beginning stages
of an experiment before the bacteria can adapt to the physical, chemical, and
biological characteristics of soil. For example, a close agreement between
fluorescent antibody counts and viable plate counts occurred for Rhizobium
japonicum (81) and a chickpea Rhizobium strain (55) in sterile soil. Differ-
ences may even appear between indirect methods if the degree of stress is
different. Bushby (17) observed no difference between a plant infection MPN
and antibiotic resistance plate counts in the beginning of an experiment but
considerable difference after 24 days. It is clear that not enough is known
about bacterial stresses in soil, particularly starvation, to explain why
differences between direct and indirect methods should persist.
Indirect procedures (plate counts, MPN) offer advantages of low cost,
both in terms of startup and routine laboratory testing, and the ability to
distinguish between live and dead cells. The indirect method of DNA probes
offers the latter but not the former. Direct procedures (FA, direct DNA
probes) offer advantages of mimicking the natural environment, and the ability
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16
to work with organisms which are difficult to isolate. In terms of statistics,
plate counts are statistically superior to the MPN procedure (86). Direct
microscopy is statistically superior to plate counts because the latter method
has a three-fold larger coefficient of variation for the same replication
(75). Yet the standard error of plate counts can be reduced by one-third to
equal the error of direct microscopy but this entails a nine-fold increase in
plate count replication and a concomitant increase in time.
One of the most important differences between direct and indirect methods
is the ability to detect low bacterial cell densities. For the FA technique,
3 4
the lowest practical detectable limit is 10 to 10 cells per g of soil,
whereas MPN and plate counts with selective agents can detect as low as 10
cells per g of soil. The limit for the indirect method of DNA probes would be
the same as selective plate counts whereas the direct method has not been
tried on soil. Thus, the method of choice here would be any indirect method.
All of the methods of enumeration share one more major disadvantage in
common: all the methods are time-consuming and tedious. Fortunately, all the
methods can be made less time-consuming and tedious. In the case of the FA
technique, a fully automated device already exists for clinical specimens
(66). For the MPN method, the use of microtiter plates (76) offers greater
statistical precision by using more tubes per dilution and a smaller dilution
rate, while saving time and media. For plate counts, the use of spiral bacterial
plating and laser colony counting (14) is possible. DNA probes are obviously
still under intensive development. Finally, other future advances will enhance
methodological sensitivity. For example, further developments in digital
microfluorimetric imaging, such as the availability of fast arithmetic video
processors and mass storage devices (4), and improvements in low light level
microscopy, such as the development of phycofluor probes (36), will enhance
the FA technique.
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17
METHODS OF DETECTION AND IDENTIFICATION
Besides the methods of enumeration, a wide variety of immunological and
•
biochemical tests exist for the detection and identification of specific
bacteria (67). However, far fewer of these tests have actually been used for
bacteria in soil and even fewer are practical because of expensive instrumenta-
tion. Among the most common of the remaining tests are phage typing, intrinsic
antibiotic resistance, and various other serological methods.
Phage typing
Phage typing, the susceptibility of a specific bacterium to lysis by a
bacteriophage, has wide application in the identification of bacteria (64),
although soil bacteria have not been so extensively studied (83). The major
advantage of phage typing is the ability to distinguish between bacterial
strains which cannot be distinguished in other ways. This advantage is offset
to some degree by variability in host range specificity: some phages are very
specific and attack only one or a few host species whereas other phages can
attack several species (84). Thus, it is important to check phage specificity
before using phage typing. The major disadvantage of phage typing is that
many factors influence the phage-bacterium interaction and the resistance of a
bacterium may occur for even trivial reasons. For example, if a bacterium is
grown at too high a temperature for flagella to form, then a phage that
recognizes a flagellar antigen as a receptor site cannot attack (65).
Intrinsic Antibiotic Resistance
Intrinsic antibiotic resistance of some soil bacteria to low levels of
antibiotics can be used for bacterial identification. The method requires
control strains to standardize the test and careful attention to inoculum size
and time of incubation to give optimal reproducibility (52). The method has
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18
advantages similar to that of selective plate counts with the added advantage
that changes are not conferred on the organism. The method has a disadvantage
•
of not working on all bacterial species, particularly slow-growing bacteria.
In addition, the method only permits distinction among a limited number of
strains (16).
Other Serological Methods
A variety of other serological methods can be used for the generic identi-
fication of bacteria. These methods include gel immunodiffusion, enzyme
linked immunosorbent assay (ELISA), and radioimmune assays. These methods
have advantages and disadvantages similar to that of fluorescent antibody,
however they have additional disadvantages in that the number of bacteria that
can be tested is limited before the tests become unwieldy. In addition, the
production of so many strain-specific antibodies is a time-consuming process.
The tests are routinely used to differentiate typical soil bacteria like
Rhizobium (25), Azospirillum (58), and Pseudomonas (8).
CONCLUSION
In the case of identification, DNA probes offer the greatest sensitivity
and are the method of choice, particularly for genetically-engineered bacteria
where the genetic basis of differentiation is known, but the method still
needs to be developed and refined for soil. In the case of detection and
enumeration, a critical assessment of the superiority of one method over
another is much more difficult. The ideal method would allow detection and
enumeration of a specific bacterial population and would estimate -the propor-
tion which is viable and metabolically active. Rarely is this achieved and
some compromise must be accepted. The current trend in soil microbiology is
for direct methods with greater speed, so in this regard FA and DNA probes
-------
will be the future methods of choice. The direct methods of DNA probes and FA
both lack the ability to detect low bacterial numbers. This is important
because bacterial numbers of a single species in soil are usually lt>w. For
3 5
example, Rhizobium numbers of 10 to 10 cells per g of soil are considered
"abundant" (89). Yet the alternative of plate counts with selective agents
ignores the failure of this method to provide conditions under which every
viable organism in the population can grow (51). The indirect method of DNA
probes remains promising but is still in its developmental stage. In short,
no method of detection or enumeration is perfect. It seems reasonable to
continue to recommend a variety of methods depending on the experimental
conditions and then to assess only relative differences between bacterial
strains rather than comparing a direct and indirect method. Thus, for condi-
tions involving bacterial numbers of > 10 cells per g of soil in the
rhizosphere, fluorescent antibody might be more appropriate, whereas under
4
conditions of < 10 bacterial cells per g of soil, plate counts with select
agents followed by DNA probes for identification are appropriate measures.
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20
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CONJUGAL DNA TRANSFER AMONG BACTERIA:
TECHNIQUES, ISSUES, AND FINDINGS RELEVANT TO THE
RELEASE OF GENETICALLY ENGINEERED BACTERIA
ITEM #6941A
PREPARED BY:
RAMON J. SEIDLER
TEAM LEADER
TERRESTRIAL BIOTECHNOLOGY PROGRAM
U.S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS ENVIRONMENTAL RESEARCH LABORATORY
CORVALLIS, OREGON 97333
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TABLE OF CONTENTS
PAGE
Purpose of this report 3
General Introduction 4
Characteristics of plasmids in bacteria 6
Enhanced frequency of mating 11
Plasmid transfer in habitat simulating
environments 13
Author's conclusions of issues presented
and concerns raised 27
Literature cited 30
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PURPOSE OF THIS REPORT
The purpose of this special report is to familiarize the
reader with some techniques, issues, and findings in the
scientific literature regarding the formation and detection of
genetic recombinants produced from the conjugal transfer of
plasmid DNA from one bacterium into another. Discussions are
oriented towards risk assessment issues regarding the release of
genetically-engineered microorganisms (GEMs). Examples are
presented regarding possible consequences resulting from the
"escape" of engineered DNA from a GEM which enters the indigenous
microbial population in a natural environment. Data from
representative publications dealing with quantitative aspects of
plasmid DNA transfer and molecular/ecological parameters
influencing such transfers will also be discussed. No attempt
has been made to provide a broad based literature review since
this has recently been done (19). The literature reviewed and
the data discussed were chosen to illustrate the kinds of
information available on DNA transfers conducted under laboratory
and habitat simulating conditions. Conjugal transmission of
plasmid DNA is the most relevant to discuss since a data base of
information exists on plasmid transfers and the biotechnology
industry depends heavily on the use of plasmids both as cloning
vehicles and as vectors to shuttle DNA between species.
Page 3
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GENERAL INTRODUCTION
Many uses of genetically engineered microorganisms involve
their deliberate release to the environment to perform functions
which include but are not limited to: pollution abatement, pest
control, crop protection from frost, modification of soil
fertility, extraction/concentration of metals from ore, and
enhanced recovery of oil (11,22). There have been a variety
of concerns raised over the possible biohazards and effects from
the released GEMs.
Research needed for assessing possible environmental
effects of GEMs has been summarized (13). Genome
characterization and genetic stability were seen as high
priorities for short term research needs over the next several
years (13). Genetic stability refers to the potential of
genetic material, especially the engineered DNA, to move from a
GEM into indigenous microbes, or the movement of DNA from
indigenous microbes into the GEM.
The potential hazards of intergeneric movement of DNA
would be heightened if engineered DNA in a GEM is transferred
into organisms which are already established in nature. This
could result in the unintentional exposures of indigenous species
as well as ecosystem processes to novel products of genetic
engineering. Following the transfer of DNA into indigenous
species, potential risks could also arise from changes in gene
expression or changes in specificity of action of gene products.
Page 4
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An additional concern with regards to genetic stablity is the
known ability of at least certain plasmids to increase the
"fertility" of recipient strains for accepting plasmid DNA
in subsequent matings. The ability of certain bacteria to
participate in a mating has been shown to increase up to 1 million
times through prior acquisition of a specific plasmid (21).
These and other issues make it important to be familiar with
research findings on genetic stability issues.
Page 5
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CHARACTERISTICS OF PLASMIDS IN BACTERIA
Genetic exchange and recombination events in prokaryotes can
occur through three mechanisms. These are transformation,
transduction, and conjugation. It is a consensus that transfer
of DNA from cell to cell by intimate contact (conjugation) is the
most common mechanism of DNA transfer between bacterial species.
(6). The most common mechanism for conjugal DNA transfer
involves the participation of conjugative plasmid DNA.
A plasmid is a closed circular DNA molecule which is stably
inherited without being genetically linked to the bacterial
chromosome. Plasmid molecules are of the order of 1-200
Megadaltons in molecular weight, encode for several to over 200
genes, and amount to some 0.04% to 10% or more of a typical
bacterial chromosome (6). Genes carried on plasmids can code
for a variety of functions ranging from the expression of
resistance to antibiotics and heavy metals, virulence factors in
pathogens,and metabolic activities including dinitrogen fixation
and aromatic compound biodegradation (6). Because of these
novel genetic features and their rather modest size, plasmids are
very useful molecules in the commercial exploitation of genetic
engineering.
A single bacterium may contain several different plasmids
which co-exist and are compatible. Incompatible plasmids cannot
coexist in the same cell. The molecular basis of incompatilility
is not known. However, plasmids which bind to the same cell
Page 6
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membrane site might be incompatible because the site is either
essential for the start of replication or because attachment to
the site may be essential to accomplish segregation of plasmids
at the time of cell division (6). Plasmids are designated as
belonging to one of some 30 incompatible groups which are
designated by a capital letter (eg. Inc C plasmids).
Plasmids are conjugative or nonconjugative. Conjugative
plasmids are self-transmissable to other bacteria. Many
conjugative plasmids can also transfer pieces of chromosomal DNA
although this typically occurs at a much lower frequency than
plasmid transfer (6).
Conjugative plasmids have a cluster of genes which allow
them to self-transfer by conjugation. The vast majority of known
conjugative plasmids are found in gram-negative bacteria. To
date, there are no known instances of conjugal DNA transfer
between gram-positive and gram-negative bacteria. Where
information is available, the ability of a plasmid to transfer
itself is known to be encoded by a large set of at least 20
genes. This region is known as the transfer or Tra locus of the
plasmid molecule (6).
Insertion sequences and transposons are genetic elements
occurring on plasmids and chromosomal DNA. Insertion sequences
and transposons are able to make copies of themselves and insert
into plasmid DNA or chromosomal DNA. Insertion sequences are
small transposable elements of the order of 1,000 nucleotide base
pairs (6). Transposons are larger elements and code for many
Page 7
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of the traits specified by plasmids such as antibiotic resistance.
Transposons contain an insertion sequence at each end of the
molecule and a series of genes between. Because of the insertion
sequence at each end, transposons can move readily between
chromosomes and plasmids carrying their genes with them.
The insertion sequence has an identical nucleotide sequence
that is repeated at each end of the DNA molecule. Transposons,
like insertion sequences, also have identical base
sequences that are repeated at each end of the DNA molecule.
These terminal nucleotide sequences establish the bases for
enzymatic insertion into other DNA molecules.
There appears to be substantial transfer of genes between
the same and different bacterial genera and species in naturally
occurring populations (18). Thus the issue of genetic stability
of GEMs is NOT one as to whether gene transfer occurs in nature;
virtually all experts would agree that transfer does indeed
occur. In one situation for example, the plasmid RP1 has been
identified unchanged in 17 different bacterial genera (18).
It is the opinion of Slater (18) that the natural evolution of
interacting bacterial communities may be much more
significant (interacting through genetic transfer of DNA) in the
evolution of new metabolic capabilities of bacteria, than a
series of (mutational) events occurring in one organism. Thus,
the transmission of DNA among microbial populations via DNA
plasmids is a major contributing factor in bacterial evolution.
Significant questions regarding risk assessment arise
Page 8
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therefore over the frequencies by which plasmids can transfer in
natural microbial communites and whether engineered DNA
fragments will be moved into unintended species. This issue has
been poorly investigated (18). It is impossible at present to
predict on the basis of known properties of a given plasmid-host
combination and on the basis of known properties of a given
ecosystem, whether a plasmid will persist, or spread to other
species in that habitat (4).
Aggregation of bacteria on a surface stimulates plasmid
transfer (4). Bacterial aggregates in nature occur widely on
the surfaces of inanimate objects such as stones, twigs, soil
particles, as well as on biological substrates such as roots and
leaves (12). Bacteria in nature occur in highest densities on
such surfaces. Microscopy of plant root surfaces, for example,
11
reveals over 1 X 10 bacteria residing in a cubic centimeter
within the rhizoplane of 0-1 micrometer from plant root
surfaces (12). The microbial populations within this rhizoplane
include some of the most common candidates for genetic
engineering such as Rhizobium, Pseudomonas, Achromobacter, and
Xanthomonas. It is well known by many geneticists that use of
membrane filters (which promote cell aggregates) promotes more
efficient conjugal transfer of plasmid DNA when compared to
broth cell suspensions (12). Indeed, the relative amounts of
cells which participate in conjugal DNA transfer can, depending
on the incompatability group, be rather strongly dependent upon a
solid substrate (Table 1).
Page 9
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Table 1. Dependency of certain conjugal matings on contact with
a physical substrate (adapted from 12).
Plasmid Broth/Solid Ratio Relative Amount of
Incompatability Surface Mating DNA Transfer
Group (solid:broth)
*
I,K,FII,H,J,V No preference .3-5
C,D,T,X Surface preferred 50-100
M,N,P,U,W Surface highly over 2,000
preferred
* Ratio transfer frequency on solid surface/frequency in broth
Thus it can be seen that the more commonly studied plasmids
of Inc Groups W and P are more frequently transferred into
suitable recipient cells by an order of 2,000-fold, when matings
are carried out on physical substrates such as membrane filters
or agar, compared to broth matings. It is important therefore,
when investigating the genetic stability (conjugal
transmissability) of plasmid DNA, to investigate the experimental
transfer capablities under a variety of mating conditions.
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ENHANCED FREQUENCY OF MATING
It is known that plasmids may undergo a variety of
physical changes within recipient cells. Indeed, certain
plasmids are rather unstable in certain species and are lost upon
cultivation in the absence of selective pressure to maintain
traits carried by plasmids.
Pseudomonas species, especially Pseudomonas aeruginosa, is
known to be a poor recipient for many plasmids transferred from
Enterobacteriaceae (21). A novel case was reported following
the transfer of an Inc N plasmid into P. aeruginosa from E. coli.
The transconjugant strain, designated as GT24 was found to have
an enhanced recipient ability for plasmids in subsequent mating
trials (21). Strain GT24 was isolated following the transfer of
Inc N plasmid R45 into strain GT1 of P. aeruginosa. GT24
was found to allow increased entry of plasmids. This increased
"fertility" as a recipient even extended to plasmids of other
Inc Groups as well. Many of the plasmid transfers into GT24 had
never previously been demonstrated in P. aeruginosa. Table 2
summarizes some of these observations.
Page 11
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Table 2. Transfer frequencies of various plasmids from E. coli
to a P. aeruginosa recipient (adapted from 21).
Plasmid Plasmid Frequencies of transfer to P. aeruginosa
in E. coli Inc Group GT1 GT24
donor
R40a
R100-1
pIP69
R45
RP1
-7
C 9 X 10
-8
FII <10
-8
M 1 X 10
-7
N 1 X 10
-1
P 9 X 10
6 X
2 X
4 X
3 X
2 X
-1
10
-4
10
-8
10
-2
10
-1
10
* Transconjugants produced per recipient cell.
Plasmids of Inc Groups C and N were transferred into strain
5 6
GT24 about 10 to 10 times more frequently then into parental
strain GT1. Further studies revealed that strain GT24 is a
special transconjugant in which at least a portion of the plasmid
R45 has become integrated into the chromosome (21). The
antibiotic resistance markers associated with R45 are stable and
do not transfer during conjugation. Also, no plasmid DNA can
be isolated from strain GT24. Concerns over this type of
enhanced fertility in accepting plasmids and how or whether
enhanced fertility might occur in environmental situations, may
become important issues in future risk assessment discussions
on genetic stability.
Page 12
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PLASMID TRANSFER IN HABITAT-SIMULATING ENVIRONMENTS
There are a limited number of studies which address the
transfer of plasmids among bacteria contained under
environmentally "simulated" conditions. Simulated environmental
conditions refer to those studies which make an effort to
maintain and monitor laboratory grown bacteria in chambers
placed into water or sewage, or to release tagged strains into
soil or onto plants.
Mach and Grimes (1982) studied the conjugal transfer of
plasmid DNA encoding resistance to antibiotics between enteric
bacteria confined to diffusion chambers. The chambers contained
sterile domestic wastewater along with a suitable donor and
recipient. The chambers were incubated in primary or secondary
settling tanks at a wastewater treatment plant. Soluble
nutrients from the wastewater were able to diffuse across the
membrane surrounding the immersed chambers. Rates of DNA
conjugal transfer from donor to recipient were then compared to
those observed in laboratory cultures grown in either nutrient
broth or in sterile unchlorinated primary wastewater influent
contained in test tubes. Species of bacteria studied included
Proteus mirabilis, E. coli, and a human pathogen, Shigella
sonnei. None of these bacteria are candidates for release to
the environment by the biotechnology industry. However, methods
of study and general observations may be applicable to plasmid
transfers occurring in eutrophic fresh water environments
polluted with domestic wastewaters. Table 3 summarizes a portion
Page 13
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of the results presented in that study.
Results revealed overall plasmid transfer rates under
-3
laboratory conditions of about 2 X 10 transconjugants per donor
for both nutrient broth and sterile unchlorinated primary
wastewater. Rates in the chambers immersed into primary and
-5
secondary wastewater averaged 2-logs less, 2-7 X 10
transconjugants per donor. Transfer was evaluated at 20C in
laboratory incubation while in situ temperatures at the
wastewater plant are below the known optimum for plasmid transfer
in species of enteric bacteria. The lower temperatures probably
account for the 100-fold reduction in transfer rates compared to
the laboratory observations.
In these studies there were no viable indigenous cells
within the chambers to compete for nutrients or to serve as other
fortuitous participants in the mating process. Furthermore, the
species diversity in the wastewater is significantly different
from the pure culture studies used in the chamber mating
experiments. Plasmid transfers are much more likely between
species of E. coli than in intergeneric transfers involving the
diversity of species present in natural wastewater. Therefore
one must view the absolute numerical results of this study with
caution when attempting to extrapolate to the natural
environment. The transconjugation rates reported are most likely
higher then those occurring in wastewater.
Page 14
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Table 3. Frequencies of antibiotic resistance transfer between
donor and recipient enteric bacteria (adapted
from 10).
_4 *
Donor X Recipient R-plasmid transfer (X 10 )
nutrient sterile sterile 1 sterile 2
broth sewage wastewater wastewater
E.
E.
P.
P.
P.
coli HI X S.sonnei S
coli 2 X S. sonnei S
mirabilis H X E.
mirabilis S X E.
mirabilis H X S.
coli
coli
H3
H3
sonnei S
10
60
23
20
7
19
45
27
0.0
0.0
0.
0.
0.
0.
0.
5
7
4
4
0
0
0
0
0
0
.2
.5
.4
.0
.0
* Transconjugants per donor
An interesting feature involving the dynamic nature of
bacterial numbers and the volume of wastewater treated daily,
deserves mention in attempting an overview of these transfer
frequencies. Mach and Grimes reported an average rate of
-5
5 X 10 transconjugants per donor formed during a 3-hr mating
5
period. If one conservatively assumes there are 5 X 10 E. coli
donor cells in a wastewater containing a total population of 1 X
8
10 cells/ml (0.5% are E. coli donor cells), then there should be
1 transconjugant produced during a 3-hr period. The wastewater
6
plant in this study processes 6.4 X 10 liters of sewage per day
and is a relatively small operation. The total daily number of
transconjugants produced at this treatment plant would be:
9
1 transconjugant/ml/3-hrs X 24 hrs X 6.4 X 10 ml of raw
9
sewage processed per day or about 50 X 10 transconjugants
produced per day.
Page 15
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Naturally the waste treatment plant reduces significantly
the number of bacteria which leave the plant in the final
effluent. Even if the final effluent is chlorinated, it will not
be sterile. If one assumes there is a 7-log (10-million fold)
reduction in total bacterial numbers following wastewater
treatment, there may still be some 5,000 recombinant bacteria
which leave this wastewater plant daily as a result of the single
hypothetical example of conjugation that has been monitored in
the study. Such bacteria would be multiply antibiotic resistant
since the transmission of plasmids specifying resistance to
several antibiotics are being transferred. These transconjugants
may in turn function as new donor strains in the receiving
stream environment. There will be untold additional types of
mating pair combinations among other bacterial species depending
upon the incidence of conjugative plasmids in the bacterial
wastewater population.
A very similar study on conjugation using diffusion
chambers immersed in wastewater was carried out by Altherr and
-5
Kasweck (1982). Rates of plasmid transfer in wastewater were 10
-6
to 10 transconjugants per donor compared with rates which were
10 to 100-fold higher under laboratory incubation conditions.
The similar 10 to 100-fold difference in transfer rates for
laboratory vs wastewater conditions is similar to the range of
differences recorded in the Mach and Grimes study.
Page 16
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Another, quite recent study revealed that so-called "safe"
or nonconjugative plasmids can be mobilized and transferred
into bacteria indigenous to the wastewater environment by a
mechanism called cointegrate formation (5). This process
requires an initial transfer of a conjugative plasmid such as
the RlOO-1 into a second cell which contains a nonconjugative
plasmid, such as pBR325. A conintegrate may form between the two
plasmids making pBR325 take on the ability to transfer into a
third cell. In the report of Gealt et al (1985) plasmid RlOO-1
was transferred from E. coli strain 1784 into E. coli strain
KA1661 which contains pBR325. Thus coincubation of KA1661 and
1784 in L broth resulted in high molecular weight DNA acquisition
in KA1661. The high molecular weight plasmid was the cointegrate
formed between plasmid RlOO-1 and pBR325. Transfer of pBR325
during a triparental mating was subsequently confirmed by
isolation of transconjugants from a mating between KA1661 X 1784
X E. coli 1997. The following list of strains are examples of
the participants in a triparental mating involving the formation
of a cointegrate DNA plasmid molecule.
A. E. coli 1784(R100-1) mobilizer strain
B.I E. coli KAl66l(pBR325) pBR nonconjugative
B.2 E. coli 2656(pBR322) pBR nonconjugative
C. E. coli 1997(recipient cell) contains no plasmids
Biparental matings require organism types A and B or A and
C. Triparental matings require types A, B, and C. Biparental
4 8
matings conducted in L broth generated 10 to 10
Page 17
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transconjugants/ml after 25 hrs of incubation (5). The L broth
matings are obviously not habitat simulating experiments but were
conducted for purposes of comparison with triparental
recombinants found in other experiments. Results of certain
triparental matings from incubations in rich L broth medium
produced results as summarized in Table 4.
Table 4. Transconjugants produced from triparental matings
involving laboratory strains of E. coli incubated
in L broth (adapted from 5).
E. coli strains Initial cell No. transconjugants
used in triparental density of each per ml
matings E. coli strain
9 5
1784(R100-1) 2 X 10 3 X 10
8 from the triparental
2656(pBR322) 2 X 10 mating
9
1997 2 X 10
8 8
1784(R100-1) 1 X 10 4 X 10
7 from the triparental
KA166l(pBR325) 8 X 10 mating
7
1997 5 X 10
Several replications of these crosses were conducted with
7 9
variations in cell densities of the participants from 10 to 10
cells/ml. There was no strong correlation between the number of
transconjugants produced and the cell densities of the
triparental participants over the concentration range
investigated. Nevertheless, in those crosses involving E. coli,
Page 18
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pBR nonconjugative plasmids were mobilized in triparental matings
at rates which did not significantly differ from the transfer
rates recorded for the conjugative plasmid RlOO-1 in biparental
matings.
Through the use of triparental matings, it was also possible
to follow the mobilization of pBR325 into two species of enteric
bacteria recently isolated from wastewater (Table 5). The rates
of transconjugants produced were less than those involving
laboratory recipient strains such as 1997 by a factor of some
100 to one-hundred thousand-fold (5).
Matings of cells were also conducted in wastewater for
detecting possible triparental matings. These matings were
conducted in 300 to 600 microliter volumes of autoclaved
wastewater placed on top of a Millipore filter/saturated filter
pad and incubated at 37C. Transconjugants were formed on the pads
2 5
as a result of triparental matings at the rates of 10 to 10 per
ml. One should be cautioned however, that the filter surface,
the temperature, the potential carryover of nutrients from the
rich broth medium, and the autoclaved wastewater all combine to
make this a highly artifical replication of the "natural"
environment.
Page 19
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Table 5. Transconjugants produced from triparental matings
involving wastewater isolates incubated in L broth
(adapted from 5).
E. coli strains Initial cell No. transconjugants/ml
in triparental density of each
mating E. coli strain
1784(R100-1)
KAl66l(pBR325)
E. cloacae 191*
1784(R100-1)
KAl66l(pBR325)
E. coli 343
7
10
7
10
7
10
8
10
7 8
10 -10
7 8
10 -10
1 3
5 X 10 to 1 X 10
from the triparental
mating
2 3
10 to 10
from the triparental
mating
* Strains which were isolated from wastewater
The general mechanism for the transfer of nonconjugative
plasmids such as pBR325 and pBR322 involves their cointegration
into a helper plasmid, such as the R100-1 used in the studies of
Gealt, et al. Thus the size (molecular weight) of the original
plasmids are likely to change in the transconjugants produced in
a triparental mating. In some cases only a portion of the
original pBR325 plasmid became integrated into R100-1.
Apparently, the sizes of the nonconjugative plasmid will vary
considerably.
No one knows whether triparental matings occur in nature at
rates that are detectable or at rates which may raise a risk
assessment "issue". However, Levine et al (9) have shown that
triparental matings involving recombinant strains of E. coli in
Page 20
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the human intestine are extremely rare. In those studies,
recombinants produced from triparental matings involving the
mobilization of pBR325 were only detected when the "ecosystem"
was disturbed following the oral administration of antibiotics
that selectively favored transconjugants. Those results are
distinctly different from the report of Gealt et al (5).
However, the latter studies were carried out under aerobic
incubation and in the absence of competing natural flora. On the
other hand, it has been indicated that some 50% of the natural
coliform population contains conjugative ("helper") plasmids (6).
Therefore there may appear to be ample opportunities in certain
natural environments for cointegrate plasmid formation to occur.
As mentioned earlier in this report, some plant-associated
bacterial species occur in high cell densities and such species
are of interest to the biotechnology industry. Published studies
have shown that recombinants may form from the conjugal
transmission of plasmid DNA among bacteria associated with plant
crops and other plant matter (7,8, 20).
In one study, rates of transconjugants produced from broth
matings in the laboratory were compared with rates which were
produced during growth of Klebsiella strains on the roots of
radish plants and in moistened sawdust (20). Radish seeds were
3 4
soaked in broth containing 10 to 10 bacteria/ml, resulting in
some 10 to 100 recipient cells adsorbed to the surface of each
seed. Seeds were planted in nonsterile sandy-loam soil
containing indigenous bacteria. Prospective donor bacteria were
Page 21
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2 3
pipetted over the seeds to provide about 10 to 10 viable donors
in the vicinity of each seed. Radish plants were removed weekly
and the rhizosphere analyzed for transconjugants of the Klebsiella
recipient originally applied to the seeds. Table 6 summarizes
the results of that study.
Table 6. Transfer of antibiotic resistance in broth and on the
radish rhizosphere present in natural soil
(adapted from 20).
-5
Donor X Recipient R-plasmid transfer (X 10 )
Penassay radish
broth rhizosphere*
Klebsiella SL5 X Klebsiella UG13 1.3 0.02
" X Klebsiella MH29 5.2 0.2
11 X Klebsiella U010994 500 0.02
11 X Klebsiella PC2 0.3 0.02
UG28 X Klebsiella DS1 530 0.02
* Calculated as transconjugants/donor/gram radish.
The number of transconjugants produced per donor illustrates
that 10% to much less than 0.1% recombinants were formed in the
soil on the radish rhizosphere as compared to those formed in the
rich broth medium. Both donor and recipient bacteria used had
been recently isolated from their natural habitats. Nothing was
known about their genetic compatability or lack thereof. Unlike
previous studies, the production of these transconjugants took
place under field simulated conditions in the presence of
indigenous microflora and may have come close to mimicking the
Page 22
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interactions of GEMs with indigenous microbial flora.
The actual number of recombinant bacteria formed per gram of
radish plant is not an impressive number. One would anticipate
there to be less than 100 such bacteria per plant rhizosphere.
However, in a single row of plants 100 feet in length there would
5 8
be approximately 10 recombinants or some 10 per acre.
The studies conducted by Lacy's research group have been
concerned with the possible transmission of plasmid DNA among
bacteria which cause plant diseases (7,8). The transmission of
antibiotic resistance among bacteria which cause plant diseases
which are controlled by antibiotic therapy has great economic
implications. In the 1975 study, Lacy and Leary demonstrated
that plasmid RP1 was transferred in lima bean pods injected with
O.lml suspensions of donor and recipient cells. Transmission of
the plasmid, both in the pods and in leaves inoculated by
congestion with water containing bacteria, occurred between
Pseudomonas glycinea and Pseudomonas phaseolicola (Table 7).
Page 23
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Table 7. Transmission of plasmid RPl in vitro(in laboratory
media) and in planta (adapted from 7).
-2
Donor X Recipient R-plasmid transfer (X 10 )*
in vitro in planta
P. glycinea X P. phaseolicola 5.8,1.1, 55 (pods)
2.7,.49,2.2 8.2 (leaves)
* counts expressed as transconjugants/recipient
It was conservatively concluded that plasmid transmission as
studied, occurred at least as well in planta as with broth-
cultured laboratory procedures. The authors concluded that this
transfer, along with an ability of E. coli to also transfer the
plasmid into Pseudomonas species in the bean pods, suggests that
plasmid transfer in planta is not just limited to related species
of plant pathogenic bacteria.
In a more recent study ( 8 ) Lacy et al, followed the
transfer of plasmid RPl among species of Erwinia and Pseudomonas
syringae pathovar syringae in planta in pear blossoms. The
investigators mentioned that streptomycin chemotherapy had
previously been effective in the treatment of fire blight
in pears caused by Erwinia amylovora. However, there is an
increasing emergence of streptomycin resistant strains of E.
amylovora. Oxytetracycline has been suggested as an alternative
treatment. Thus, the purpose of the current study was to
ascertain whether plasmid-borne resistance to oxytetracycline
Page 24
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may also occur due to plasmid transmission in planta.
In interpreting the outcome of the research, it seems
relevant to detail how the in planta matings were conducted.
Cultures were pregrown on agar, washed in sterile distilled
water, and resuspended. Approximately 0.03 ml recipient was
added onto the surface of the receptacle of a detached pear
blossom. Then 0.005 ml of the donor was added to the droplet
containing the recipient cells. The detached blossoms were
maintained (with pedicels immersed in water) in moist chambers at
25C for 4 to 7 hrs. Bacteria were washed from the blossoms,
concentrated by centrifugation, and plated onto appropriate media
selective for donor or recipient alone, or for the
transconjugants. Putative transconjugants were verified first on
media selective for phenotypic expression of the recipient and
then by plasmid extraction and purification. Table 8 summarizes
these mating experiments.
Page 25
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Table 8. Frequencies of antibiotic resistance transfer between
Erwinia herbicola or P. syringae donors and
E. amylovora recipients, both in vitro and in planta
(adapted from 8).
Donor X Recipient R-plasmid transfer frequency*
in vitro in planta
-3 -7 -2 -6
P. syringae X E. amylovora 10 to 10 10 to 10
-5 -6 -1 -8
E. herbicola X E. amylovora 10 to 10 10 to 10
* Transconjugants per donor
It is apparent that the in planta transfer of pRPl to E.
amylovora was often greater than the in vitro transfer rate.
The authors indicated however, that there was a great range in
the transfer rates. No explanation was offered for the higher
transfer rate in the in planta studies. The great range in the
rates was attributed to differences in the ratio of donor to
recipient. These data serve to underscore the earlier articles
reviewed where plasmid transfer in habitat simulating
environments was shown to occur, often at rates which approach or
occasionally exceed those found in laboratory media.
Page 26
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AUTHOR'S CONCLUSIONS OF ISSUES PRESENTED AND CONCERNS RAISED
1. Discussions over the risk assessment issues regarding
genetic stability of GEMs should not be directed at WHETHER DNA
exchange occurs among bacteria in the environment.
2. The major issue regarding the intergeneric movement of
DNA deals with the transfer of genetically-engineered DNA from a
GEM into organisms already established in nature. Concerns arise
from unintentional exposures of indigenous species and ecosystem
processes to novel products of genetic engineering. Changes in
expression of engineered genes in new genetic backgrounds may
result in an increase or shut off of transcriptional and
expression activities.
3. Escape of the engineered DNA from the original GEM into
the indigenous population may prolong the persistence of the new
genetic element and make many conventional detection/enumeration
techniques ineffective (selective media, fluorescent antibodies).
4. Transmission of DNA among microbial populations via DNA
plasmids is thought to be a major factor in bacterial evolution.
It follows therefore, that the escape of engineered DNA into the
natural population, may influence the evolution of indigenous
bacteria.
5. Transfer of DNA involving cell contact (conjugation) may
be strongly influenced, depending on the plasmid Inc group, by
contact with a substrate. Substrate contact is common in
symbiotic associations in terrestrial ecosystems (plant roots,
intestinal tract insects, animals, etc).
6. Published studies reveal that DNA transfer has been
demonstrated in a variety of artificial habitats intended to
mimic the "natural" environment. Transfer rates are dependent
upon plasmid Inc groups, temperature, cell densities of the
interacting species, and other physical/chemical conditions of
the mating environs. Estimates of trillions of transconjugants
form daily within wastewater, plant root and leaf surfaces.
7. Standard assay systems are needed to methodically
evaluate, on a case-by-case basis, the genetic stability of
various plasmid groups in an array of bacterial species and
habitats which mimic actual or anticipated GEM release
situations.
Page 27
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LITERATURE CITED
1. Altherr, M. R., and K. L. Kasweck. 1982. In situ studies
with membrane diffusion chambers of antibiotic resistance
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