Biotech. Adv. Vol. 10, pp. 149-178,1992 0734-9750192 $15.00 P~inted in Great Britain. 1992 Pergamon Press Ltd

EVALUATION OF METHODS FOR DETECTING ECOLOGICAL EFFECTS FROM GENETICALLY

ENGINEERED MICROORGANISMS A N D MICROBIAL PEST CONTROL AGENTS IN TERRESTRIAL SYSTEMS

RAMON J. SEIDLER

U.S. E P A E n ~ r o n m e n ~ l R ~ r c h ~ t o ~ , 2 ~ S.W. 35thS~e~,Corvallis, 0 R 9 ~ 3 ~ ~ S.A.

ABSTRACT

This report summarizes and evaluates research from several

laboratories that deals with the detection of ecological effects

induced through exposure of microbes or plants to genetically

engineered microorganisms (GEMs) and microbial pest control agents

(MPCAs). Some 27 potential endpoints for measuring effects have

been studied. Perturbations induced by GEMs have been detected in

about one-half of these endpoints. Detectable effects have been

recorded for over half of the 16 species of bacteria and fungi

studied. The effects caused by GEMs and MPCAs include inhibition

of beneficial mycorrhizal fungi growing on Douglas fir seedling

roots, depression in plant root and shoot growth, inhibition of

predatory soil protozoa, accumulation of a toxic metabolite during

biodegradation that inhibits soil fungi, increased microbial

community respiration due to rapid lignin breakdown in soil, and

the displacement of a broad group of gram-negative bacteria that

inhabit the root surface of cereal crops. These effects were

usually, but not always, of short duration. However, some of the

changes were irreversible during the observation time of days,

weeks, or in one case, months.

KEYWORDS

risk assessment, genetically engineered microbes, ecological

effects, microcosms, recombinant streptomyces, nutrient dynamics,

mycorrhizal interactions with MPCAs, toxic metabolites

149

150 R.J.SEIDLER

INTRODUCTION

The need for protocols for conducting risk assessments of

genetically engineered microorganisms (GEMs) and microbial pest

control agents (MPCAs) was prompted by the rapidly expanding

biotechnology industry and the parallel need to understand the

ecological consequences of conducting environmental releases. To

date, most products have been for agronomic uses and include an

assortment of viral, bacterial, and fungal pest control agents,

Rhizobium species with enhanced nitrogen-fixation capabilities, and

more recently, bacteria and fungi for biodegrading hazardous

wastes. Various governments and agencies including the United

States Environmental Protection Agency (EPA) and Environment

Canada, have proposed guidelines that necessitate industry to

collect fundamental, ecological information on the recombinant

product (30). These parameters include knowledge of survival time,

potential for gene exchange, dispersal, and potential for effects

on beneficial nontarget organisms and on the ecosystem (18).

Although regulatory agencies do not explicitly require

experimentation, scientists would concur that most of this

information is not available for recombinant organisms or their

corresponding parental forms. Therefore, it is necessary to

conduct experiments in confined or contained systems (microcosms)

to generate the data needed to make adequate risk assessments.

An accurate assessment of risks from GEMs requires

anticipation of their behavior following release into the

environment. Researchers who planned the EPA's approach for

developing test protocols envisioned that relevant experimental

information on microorganisms could be obtained by conducting basic

analyses in contained systems or microcosms (6,18). Microcosms

have been defined by many but the succinct definition of Gillett

and Witt (16) is preferred ("...a controlled reproducible

laboratory system which attempts to simulate a portion of the real

world...".

Two approaches have been used in the microbiological

applications of microcosms to risk assessment. First,

investigations involving nongenetically engineered microbes are

valuable for evaluating general principles of introductions

including knowledge of survival, dispersal, and gene exchange

potential. Second, the use of genetically engineered

METHODSFOR DETECTING GKNE~CENGINEEFJNGEFFECTS 151

microorganisms may be appropriate for evaluating the consequences

of specific metabolic alterations on ecological processes and

effects on nontarget organisms (12). Both approaches have provided

important and relevant technical information for regulatory

purposes.

Microcosms have been suggested as one significant tool in the

assessment of biotechnology products prior to their release to the

environment (12,30). Once GEMs are released, it is not easy to

decontaminate the site or mitigate their effects (9). Thus,

prerelease evaluations of GEMs in contained environments are a way

to facilitate risk assessment information without needless

environmental compromises. Microcosms are varied in design, size,

and sophistication (12,16). Currently the EPA has no standardized

microcosms for GEM risk assessments (30). Therefore, this report

has concentrated on evaluating research that addresses ecological

effects methodologies rather than elaborating on the pros and cons

of the various microcosms that have been used.

The development of methodologies to measure effects from GEMs

is both an intellectual and a practical challenge. One

investigator has likened the job of developing test methods to

finding "a needle in a haystack" (34). The challenge comes from a

lack of scientific precedent and a lack of relevant ecological data

that provide clues as to likely effects. Information on effects

caused by GEMs, and or methodologies to detect such effects were

simultaneously needed due to the rapid commercialization of

recombinant organisms for environmental release. The need for test

methods for conducting risk assessments of biotechnology products

requires ecological adaptation of molecular methodologies. The

application of molecular methods to ecological issues has given

rise to a new discipline, "molecular ecology".

What is known about ecological effects caused by GEMs has been

published within the last two years. These recent studies have

explored in depth some 27 potential endpoints for measuring GEM

induced ecological effects (Table i). Most of these endpoints have

been reviewed and evaluated in the present report. Perturbations

induced from the activities of GEMs have been detected in about

one-half of these endpoints.

Table 2 contains the genera of GEMs that have been evaluated

for producing ecological perturbations. Detectable effects have

been recorded in over half of the genera listed.

152 R.J.SEIDLER

Table i. Experimental endpoints that have been evaluated for

relevancy in measuring ecological effects from GEMs and MPCAs

(adapted from reference 34).

metabolic activity (respiration, kinetics of CO 2 evolution)

numbers of total viable bacteria

numbers of Gram-negative bacteria

bacterial populations in the rhizosphere and on the rhizoplane

total fluorescent pseudomonads

effects on species diversity

biomass of colonized plants

microbial biomass

biomass of plant shoots

total nitrogen content of wheat shoots

total viable fungi

cellulose utilizers

chitin utilizers

denitrifiers

nitrifiers

numbers of protozoa (predators)

nutritional groups (growth in minimal medium +/-growth

factors)

antibiotic resistant biotypes

energy source utilization

activity of soil enzymes

kinetics of nitrogen transformations (ammonia, nitrate,

nitrite)

numbers of nonsymbiotic dinitrogen fixers

competitive ability of GEMs

species diversity measured by colony types

changes in procaryotic and eucaryotic population densities

effects of MPCAs on mycorrhizal colonization of roots

effects of MPCAs on mycorrhizal colonization and plant growth

This report provides an evaluation and overview of methods

and results from research teams that have detected, quantitated,

described ecological effects that occurred following exposure of

various test systems to genetically engineered bacteria or by

exposure to bacterial or fungal microbial pest control agents (2,

3,5,10,13-15,19-21,32,34,36-38).

METHODS FORDETEClqNGGKNE~CENG[NEP2dNGEFFECTS

Table 2. Microorganisms evaluated as potential effectors of

ecosystem effects.

153

BACTERIA

Aarobacterium radiobacter

Alcaliaenes

AzosDirillum lipofe~m

Bacillus cereus

Bacillus subti~s

Escherichia co~i (several strains)

Pseudomonas Dutida

Pseudomonas species

Enterobacter aeroaenes

Enterobacter cloacae

Pseudomonas fluorescens

Serratia Dlvmuthica

StreDtomvces sp.

FUNGI

Gliocladium virens GV-P

Talaromyces flavus TF-I

Trichoderma harz~anum WT-6-6

MYCORRHIZAL INTERACTIONS WITH MPCAS.

Linderman and coworkers developed methodologies and conducted

detailed investigations for detecting possible effects of microbial

pest control agents on the major groups of mycorrhizal fungi

(19-21,26,27). Specifically, they examined the influence of MPCAs

on the development, activities, and functions of mycorrhizal fungi

representing the vesicular arbuscular mycorrhizae (VAM),

ectomycorrhizae (EcM), and ericoid mycorrhizae (ErM).

In general, the test systems consisted of placing the

mycorrhizae in a container of soil in a greenhouse and evaluating

the influence of MPCA exposure on colonization of the roots, growth

of the plant, and inhibition of mycorrhizal mycelial growth or

spore germination. The following experimental systems were

developed:

YAM

evaluation of MPCA effects on spore germination in soil

root colonization of cucumber (% roots colonized)

growth enhancement of onion plants

I~ R.J.SEIDLER

EcM

root colonization and growth enhancement of Douglas-fir

seedlings

inhibition of mycelial growth in pure culture

ErM

effects on root colonization of rooted cranberry cuttings and

tissue-cultured plantlets of rhododendron and mountain laurel

inhibition of mycelial growth of ErM in culture

The VAM fungi used were of the genus Glomus. The inoculum was

produced in a pot culture by growing Sudan Grass (Sorqhum bicolor)

in a sand-soil mix into which a core of G. mosseae-infested sand

was placed before seeding.

Several assays were evaluated involving the VAM fungi. First,

the extent of root colonization of cucumber (Cucumis sativus L.

"Marketer Long" or "Straight Eight") was determined . The VAM

Glomus etunicatum was added to the soil at six levels (19, 9, 5, 2,

i, and 0.6 spores/g soil). The bacterial MPCAs were applied at

about 107 colony forming units per gram (cfu/g) of soil in plastic

tubes. Tubes were watered daily and incubated one week prior to

planting with cucumber. Twelve days after planting, seedlings were

harvested and evaluated for mycorrhizal colonization. In a

separate experiment, G. etunicatum was added to soil at 37, 19, 9,

5, 2, and 1 spores/g soil. Bacterial MPCAs were added as above and

seedlings were harvested 21 days after planting and examined for

VAM colonization. For colonization, roots were washed in water

then cleared and stained using standard methods (28). Percent

mycorrhizal colonization and mean colonization were calculated for

each treatment.

To determine germination of VAM fungi, approximately 300

spores were filtered onto a cellulose triacetate filter. One ml of

bacterial or fungal MPCA at about 108 cfu/ml were also filtered

onto the same membrane. A second membrane was placed on top and

the membrane-spore-membrane sandwich was placed into 40 g of non-

pasteurized soil-sand in a plastic Petri dish. The soil was

moistened and incubated at 27°C. After 4, 5, 6, 7, and 8 days a

pie-shaped wedge of the membrane was cut and removed. The sandwich

was opened, soil was removed, and spores were stained and examined

for fungal germ tubes under 200X microscopy.

Douglas fir seeds were sown in 6 inch pots (i0 seeds per pot)

of REDI-EARTH commercial potting mix. At 8 weeks after

METHODS FORDETECTING GENE~CENGINEERINGF~FECTS 155

germination, seedlings were transplanted into soil containing an

EcM and MPCA inocula.

The EcM used were RhizoDoaon vinicolor (RvA), Laccaria laccata

(S-238A), Hebeloma crustuliniforme (Hecr-2) and Cenococcum

qeophilum (A-145). These ectomycorrhizae were grown on membrane

covered plates of Modified Melin-Norkrans (MMN) agar (22), removed,

and added to sterile vermiculite/peat moss contained in wide-mouth

quart canning jars (19). After 4 weeks at 18°C or 23°C, the

vermiculite substrate was fully colonized.

The colonized vermiculite was washed with tap water and gently

mixed into soil previously inoculated with the test MPCA. In one

series of final experiments, the inoculum level of the mycorrhizal

fungus was varied. The test MPCA was added to soil and mixed

thoroughly to achieve approximately 107 cfu/g. Eight-week Douglas

fir seedlings were transplanted into plastic tubes containing the

inoculated soils. There were i0 replications per treatment

combination per harvest.

In the initial studies, plants were harvested at 2, 4, 6, and

8 weeks after transplanting and, in the final study, harvested at 4

and 8 weeks. At harvest, rhizosphere populations of the MPCAs were

enumerated and percent colonization by the mycorrhizae fungi were

estimated; root and shoot dry weights were taken on oven dried

tissues.

In experiments involving ericoid mycorrhizae, plants of

cranberry, rhododendron, and mountain laurel were utilized. Young

cuttings of cranberry were used following eight weeks of growth in

flats of soil. Eight-week old tissue cultured plants of

rhododendron and mountain laurel were purchased.

In two series of experiments with cranberry, HvmenoscvDhes

ericae was the ErM fungus used. Pure cultures grown on agar plates

were blended, washed, and added to the center portion of small pots

filled with peat moss and the ErM and bacterial MPCA were pipetted

into a center hole in the peat moss. A cranberry seedling was

planted into each pot and, after 12 weeks in a greenhouse, plants

were removed and the root system washed and stained to visualize

mycorrhizal colonization (19). Similar procedures were used with

the mountain laurel and rhododendron plants. After 2 months, i0

plants were removed from each pot, washed, and stained to examine

for mycorrhizal colonization.

The bacterial MPCAs P. fluorescens Pf-5 and 2-79, known

156 R.J.SEIDLER

antibiotic producers, delayed germination of the VAM G_~ etunicatum

spores in unpasteurized soil (19). However, germination observed

at day seven was no different from the controls. Similarly, the

fungal MPCA, Gliocladium, significantly delayed spore germination,

but by day eight no differences from the controls were noted.

None of the other fungal or bacterial MPCAs had an effect on VAM

spore germination.

Table 4. Summary of effects of MPCAs on root colonization by VA

mycorrhizal fungi (adapted from reference 19).

Response

Cucumber Onion

MPCA Exp.l Exp.2 Exp.3 Exp.l Exp.2 Exp.3

A. radiobacter K84 n

Alcaliqenes sp. MFAI +

B. cereus UW-85 n

~. subtilis Quantum 4000 HB n n

E. aeroqenes B-8 +

~. cloacae EcCT-501 n

P. fluorescens Pf-5 (JL3832)-

P. fluorescens Pf-5 Rif n

S. plvmuthica 6109DO1 +

Streptomyces sp. H68-4 n

+ = biocontrol agent enhanced root colonization by mycorrhizal

fungus

- = biocontrol agent decreased root colonization by mycorrhizal

fungus

n = no effect on mycorrhizal colonization was observed.

The effect of MPCAs on colonization of cucumber roots by G.

etunicatum was examined in three separate experiments (Table 4).

Only StreDtomvces sp. H68-4 decreased root colonization by the VAM

A few MPCAs such as Alcaliaenes sp. MFAI, increased the extent of

root colonization and were considered to have a positive effect on

the YAM.

All the fungal MPCAs tested had a negative effect on percent

METHODSFOR DETECTING GENE~CENGENEERINGEFFECTS is7

colonization in R. vinicolor-inoculated plants when compared to

MPCA uninoculated controls. No mycorrhizae formed in the presence

of ~. virens. Shoot and root dry weights of the B. vinicolor

Douglas fir plants were also reduced. Thus, these results verify

the relevance of this assay and the sensitivity of the technique

for detecting effects from certain fungal MPCAs on the activity of

ectomycorrhizae in colonizing roots of Douglas fir seedlings.

Although the fungal MPCA G. virens inhibited growth of the

mycorrhizal fungus R. vinicolor, in only one combination was there

a reduction in root dry weight over uninoculated MPCA controls.

This means that in the artificial potting soil system that received

intermittent fertilizer, the full beneficial effects of the

mycorrhizae on plant growth was not realized. With nutrient

depleted soils, it is more likely that the inhibition of the

mycorrhizal fungus would be accompanied by a reduction in Douglas

fir seedling growth.

Depending upon the mycorrhizal species exposed, bacterial

MPCAs influenced root dry weight, and shoot dry weight

differentially (Table 5). In most cases, the bacterial MPCAs

either had no ecological effect on mycorrhizal colonization of fir

seedling roots or, in certain cases, the bacterial MPCAs actually

stimulated mycorrhizal formation as found with P. fluorescens

colonizing fir roots containing R. vinicolor. It is interesting

that in this case of stimulated mycorrhizal growth, there was

reduced shoot development in the seedlings. The bacterial MPCAs

2. aeroaenes, E. cloacae, and S. plymuthica all increased shoot

and/or root development.

Investigators were able to increase the sensitivity of the

assay procedure by decreasing the amount of mycorrhizal inoculum

added in the final pairing studies involving Hebeloma and Laccaria.

That is, when lower concentrations of mycorrhizal fungi were added

to the soil, an effect of the MPCA was detectable with certain

MPCA/mycorrhizal combinations (Table 5, lower entries). Thus,

colonization by the mycorrhizal fungus Hebeloma was inhibited by 2-

cloacae, and accompanied by a reduction in shoot and root growth of

the Douglas fir seedling. In the earlier assays, run at one

mycorrhizal fungus inoculum level, no inhibition of Hebeloma by E.

cloacae was recorded, similar observations were recorded for P.

fluorescens, in which at lower levels of mycorrhizal fungi the

bacterial MPCA was associated with reduced shoot and root growth of

158

the fir seedling.

R. J. SEIDLER

Table 5. Summary of effects of MPCAs on ectomycorrhizae formation

and growth of Douglas fir (adapted from reference 19).

RvA a Hecr

Rdw b Sdw %EcM c Rdw Sdw %EcM

Bacterial MPCAs

A. radiobacter K-84 0 0 0 + + 0

Alcaliaenes sp. MFAI 0 0 0 0 0 0

B. subtilis EBW-4 0 0 0 0 0 0

E. aerogenes B-8 0 0 0 + + 0

E. cloacae EcCt-501 0 0 0 + + 0

P. fluorescens Pf-5 0 - + 0 0 0

S. plymuthica 6109DO1 0 0 0 + 0 0

Fungal MPCAs

G. virens Gv-P - 0 - 0 0 0

T. flavus 0 0 - 0 0 0

T. harzianum Wt-6 0 + - 0 + 0

Bacterial MPCAs in a

Dose Response Test*

P. fluorescens Pf-5 - - 0

E. cloacae EcCt-501 - - -

S. Divmuthica 6109DO1 0 0 0

Response codes:

+ = significantly greater than uninoculated control (p<0.05)

- = significantly less than uninoculated control (p<0.05)

0 = not significantly different from uninoculated controls

(p<0.05). a RvA is Rhizopoqon vinicolor, Hecr is Heboloma

crustuliniforme

b Rdw is root dry weight; Sdw is shoot dry weight.

c %EcM indicates the percent of the total number of root segments

viewed that were infected.

* = In these experiments the inoculum level of the mycorrhizal

fungus Hebeloma crustuliniforme (Hecr) was reduced to increase the

potential for an effect of the MPCA on the plant.

In the first experiment using cranberries, P. fluorescens Pf-5

significantly increased colonization by the mycorrhizal fungus H.

METHODSFOR DETECqlNGGENE~CENGINEE~NGEFFECTS 159

ericae (Table 6). The response of the mycorrhizal fungus induced

by the MPCA ~. cloacae-treated plants did not differ from the

control. In the second experiment, six MPCAs were tested. The

rate of mycorrhizal colonization in the presence of the MPCAs G__~.

virens and E. aerouenes-treated plants was significantly lower than

the untreated controls. The mycorrhizal relationship induced by

the two other bacterial and two fungal MPCAs did not differ from

that of the control treatments.

Although the effect of MPCAs on root colonization by ErM fungi

differed between rhododendron and mountain laurel, neither plant

had dramatic variations in the incidence of ErM fungi. Thus,

colonization of mountain laurel roots was unaffected by the MPCAs

Table 6. Summary of effects of MPCAs on ericoid mycorrhizal

formation with cranberry, rhododendron, and mountain laurel

(adapted from reference 19).

mountain

Cranberry Rhododendron laurel

Bacterial MPCAs exp 1 exp 2

~. radiobacter K84 0

Alcaliaenes 0 0

B. subtilis Bact-I + 0

~. aeroqenes B-8 - 0

E. cloacae EcCt-501 0 0

P. fluorescens Pf-5 + 0

~. nlvmuthica 0 + 0

Fungal MPCAs

2- virens Gv-P - + 0

~. flavus Tf-i 0 0

T. harzianum Wt-6 0 0

Response codes:

+ = percent colonization significantly greater than uninoculated

controls (p<O.05)

- = percent colonization significantly less than uninoculated

controls (p<0.05)

0 = not significantly different from uninoculated controls

blank = not tested

I~ R.J.SEIDLER

tested. Four of the MPCAs significantly influenced colonization of

rhododendron roots by increasing the percentage of the root length

colonized. Therefore it appears that MPCAs have little, if any,

significant effect on the interactions of ErM fungi with these

plants. It does not appear that this assay would be useful in

future risk assessments of MPCAs unless the sensitivity of the

assay can be demonstrated.

Experimental results of these bioassays demonstrate their

value in detecting effects from certain MPCAs on the colonization

of plant roots by mycorrhizal fungi. In certain cases, the

presence of the MPCA was accompanied by a reduction in Douglas fir

seedling biomass. It should be stressed that very different kinds

of observations were recorded depending upon the combination of

MPCA and mycorrhizal fungal species. Furthermore, concentrations

of the MPCA as well as the mycorrhizae are critical. The

investigators generally only used one concentration of each paired

component. The final concentrations of MPCA ranged from 104 to 106

cfu/g soil after 8 weeks. The current quantitative results on

percent colonization as well as root and shoot dry weight were

recorded after 8 weeks. It is not known how long an effect would

persist nor do we know the magnitude of the effect at various

concentrations of the added MPCA. It is probably just as critical

to vary the concentration of both the MPCA as well as the

mycorrhizal agent in such tests. Much needs to be learned about

the biological reasons for the differential effects. Accordingly,

at this time it is recommended that several species of mycorrhizae

be used to assess the risk of releasing root inhabiting bacterial

or fungal MPCAs.

EFFECTS OF RECOMBINANT PSEUDOMONAS SOLANACEARUM ON

PREDATORY PROTOZOA

Austin and coworkers evaluated the effects of various strains

of Pseudomonas solanacearum (mucoid and nonmucoid, recombinant and

nonrecombinant) on populations of soil protozoa (2). The objective

was to determine if the addition of a transposon or a recombinant

plasmid in P. solanacearum had any effect on bacterial feeding by

protozoa.

Spontaneous mutants were selected from all strains that were

resistant to 1,000 ug streptomycin /ml to facilitate enumeration

from soil. Five stains of bacteria were used. One was a standard

Pseudomonas sp. previously used to feed protozoa; four P.

METHODS FORDETECTING GENETIC ENGINEERINGEFFECTS 161

solanacearum strains were also used. Mucoid strains were

designated AW whereas nonmucoid strains were designated AR. One

strain (PS-6) contained a transposon (Tn 5) and the other

genetically altered strain AW(pHE-3) contained a recombinant

plasmid comprised of a common cosmid vector (pLAFR3, 20kb size) and

a 30kb fragment from P. solanacearum encoding for beta-l,4

endoglucanase. These strains are described in detail by Roberts et

al. (29). Protozoa representing flagellates, amoebae, and ciliates

were enumerated.

The control bottles that did not contain pseudomonads added as

a food source maintained stable protozoan counts over the five day

study period at approximately 102 protozoa/g soil. In the presence

of the Pseudomonas sp. added food source, all protozoan populations

increased 100-fold. When the wild-type mucoid P. solanacearum AW

was provided as a food source, flagellate numbers increased

significantly to about 105/g soil whereas ciliate and amoebae

numbers showed no increase. A similar pattern was recorded with

the mucoid PS-6 strain. However, for the mucoid recombinant P.

solanacearum strain AW(pHE-3), the flagellate population at 2 days

was significantly lower compared to the nonrecombinant parental

strain AW. After 5 days in the presence of the nonmucoid variant

of P. solanacearum (strain AR), all protozoan counts were similar

to those with the standard food strain of Pseudomonas sp. The

authors indicated that the indigenous soil bacteria had no effect

on protozoan predation because they amounted to less than 0.1% of

the added pseudomonad population.

The presence of the recombinant plasmid resulted in a

significantly lower flagellate population. The data were recorded

over a five day period. There was no indication whether protozoan

counts would remain depressed over longer periods of time. Because

in this case a genetically engineered bacterium was not actively

consumed by protozoa, there is an indication that the survival of

released GEMs in the environment might be prolonged. These

observations also provide a justification for conducting predatory

protozoan assays to confirm the potential for GEM effects on higher

trophic levels.

The procedures utilized in this study leave several important

issues unresolved. Thus, the issue of time period in which

protozoan counts are depressed cannot be assessed. It would be

important to know whether protozoan counts return to normal levels

I~ R.J. SEIDLER

following prolonged incubations. One does not know how general

this phenomenon is and whether protozoa in other ecosystems might

be similarly affected. However, the assay is relatively simple to

perform and appropriate replications can easily be conducted to

validate the concerns over the general nature of bioassay

applicability.

RECOMBINANT STREPTOMYCES EXPRESSING LIGNIN PEROXIDASE AND

EFFECTS ON SOIL ECOLOGICAL PROCESSES

Crawford and colleagues are interested in developing strains

of StreDtomyces species with enhanced capabilities to degrade the

natural substrate lignin (7,8,36). These investigators evaluated

the survival, growth, genetic stability, and effects of recombinant

Streptomyces on the rate of lignin mineralization in a soil

ecosystem. In separate publications, Wang et al., (36-38)

reported the cloning of the lignin peroxidase gene into S.

lividans. A list of the strains and their key phenotype are listed

in Table 3. In the first series of experiments, the molecular

construct S. lividans TK23pSEI was unstable and lignin peroxidase

activity diminished following growth of the host bacterium in soil

or in laboratory culture. This TK23pSEI was constructed from a 5.5

kb DNA fragment from the chromosome of S. viridosporus in plasmid

pSPl0. Different cloning vectors and strains were used in a

second study in which stable recombinant cultures were constructed

and evaluated (38). Thus S. lividans recombinant strains TK23.1,

TK24.1, and TK64.1 were constructed by inserting the lignin

peroxidase gene ALip-P3 from S. viridosporus T7A into plasmid

pIJ702 forming pIJ702.LP. The latter recombinant plasmid was

transformed into S. lividans forming strains TK23.1, TK24.1, and

TK64.1

Both recombinant and parental nonrecombinant strains of S.

lividans were used to study carbon turnover kinetics by measuring

the kinetics of carbon dioxide (C02) evolution from lignin added to

sterile and nonsterile soil. Spore suspensions of the S. lividans

cultures were added to 5 g of soil in flasks to yield about 108

cfu/flask. The soil was adjusted to 40% w/w water content and

incubated at 25°C. A high organic carbon content soil (5.2%) was

used. An analysis of this soil was published (36). Flasks were

constructed with sterile gassing ports to allow for continuous

passage of sterile, CO2-free , humidified air into each flask, and

all exit gases from each flask were continuously passed through CO 2

METHODS FORDETEC~NG G E N E ~ C E N G I N E E ~ N G EFFECTS 1~

traps (7,8). Carbon dioxide was quantified by acid titration.

In all cases, CO 2 evolution from nonsterile soil with the S.

lividans recombinant strain was significantly higher than from

soil inoculated with the corresponding wild type. In sterile soil,

there were no significant differences between CO 2 evolution curves

for recombinant strains TK23.1 and TK24.1 and their corresponding

wild types. In proline-amended sterile soil, the curves for TK64

and TK64.1 were essentially identical. These strains are

auxotrophic for proline. In nonsterile soil, strains TK64 and

TK64.1 mineralized much more carbon than they did in soil not

supplemented with proline. Overall, the amount of CO 2 released

over the 12-day period was significantly higher for each

recombinant compared to its corresponding wild type. Enhanced

rates of CO 2 evolution were observed only for recombinant strains

inoculated into nonsterile soil.

The results illustrate that the release of recombinant S.

lividans that overproduce lignin peroxidase in soil had a

significant short-term impact on carbon turnover rates. The

kinetics of CO 2 evolution peaked and leveled off by day 12.

Because recombinant strains did not cause an elevation in CO 2

release in sterile soil, the investigators hypothesized that it was

the native soil microflora and the released recombinant acting

synergistically that enhanced the mineralization of carbon. This

is a significant observation as it illustrates how a released

recombinant microorganism can interact metabolically with the

indigenous native microflora to impact the biogeochemical

processing of carbon. In this interaction, Wang et al. (38)

hypothesized that the action of the recombinant peroxidase

increased the rate of oxidation of relatively unavailable, lignin-

derived soil organic matter. This oxidation, in turn, partially

opened up the lignin molecule and made lignified carbon more

available to the soil microflora that were then able to more

rapidly respire the carbon. This study is the first report of a

GEM having a measurable effect on a biogeochemical cycle. The

intimate metabolic linkage between a GEM and the indigenous

microbes dramatically illustrates how ecological effects may not

necessarily derive directly from a GEM, but may be caused

indirectly through a synergistic relationship with unrelated

species as in the present example.

These studies are important for several reasons. First, the

I~ R.J. SEIDLER

measurement of CO 2 evolution was shown to be highly relevant in

determining an effect from a released GEM in soil. This validates

the measurement of CO 2 evolution and at the same time has provided

an indication of the time course of the effects. The investigators

described the effect as being of "short duration". Indeed

measurements were recorded over a 12-day period and peak evolution

generally occurred by 6-9 days following addition of the GEM. The

issue of the time-effect however, should not be used to downplay

the significance of the observations. Thus, if all the readily-

disrupted lignin was metabolized by 6 days, one would not expect

the CO 2 evolution to continue indefinitely. Little is known

concerning the potential subsequent effects (if any) of rapid

lignin degradation on other soil processes including general soil

fertility, moisture holding capacity, etc. No one has determined

whether structural or functional ecological effects occur as a

result of a burst in lignin degradation. Also, nothing is known

concerning what organisms consume the substrates made available by

the over production of lignin peroxidase. It would be of concern,

for example, if the release of substrates elevated populations of

plant pathogens or other undesirable components of the soil

ecosystem. These questions remain for future studies.

MEASURING NUTRIENT DYNAMICS INFLUENCED BY GEMS IN INTACT

SOIL-CORE MICROCOSMS

Fredrickson and his colleagues have used the intact soil core

microcosms to develop and evaluate risk assessment methodologies

for GEMs (3-5,11,13-15). The advantages of the intact soil core

microcosm include its adaptability for studying nutrients in

leachates as a means for evaluating effects of GEMs on

biogeochemical cycling processes in soil. The core consists of a

physically intact cylinder of soil with natural pores and spatial

distributions of life forms present in the field. As such, the

intact core can better simulate penetration of water and the

removal of leached nutrients then can other microcosm systems. The

intact soil-core has been studied extensively and standard

protocols exist for its use in chemical toxicology (39).

In one series of experiments, investigators examined the

influence of the bacterium Azospirillum liDoferum as a rhizosphere

inhabitant on wheat and corn. The objective of the study was to

determine the impact of A. lipoferum on the efflux of nutrients

from agricultural soil-core microcosms through leaching and uptake

METHODS FOR DETECTING GENE~CENG1NEE~NGEFFECTS 165

by wheat and corn (13).

Intact soil cores were either 17.8 cm or 25.5 cm diameter by

60 cm in depth. The smaller cores contained a Palouse silt loam

soil whereas the larger soil cores were classified as a Burbank

sandy loam type commonly used for irrigated agriculture. Cores

were removed and set up over funnels as previously described

(11,13).

Microcosms were maintained at a water potential of -0.03 MPa

and a temperature of 22 ° C. To generate leachates, i00 ml aliquots

of deionized water were added to each core, allowed to soak in, and

further aliquots were added until breakthrough of water from the

bottom of the core was noted. Leachates were collected 33 and 67

days after planting.

Leachates were analyzed for phosphate, sulfate, nitrate,

nitrite, and ammonium ions. In addition, analyses were conducted

for total carbon (TC), dissolved organic carbon (DOC), and

dissolved inorganic carbon (DIC). Ground plant material was ashed

on day 33 in concentrated nitric acid then dried. The ashed tissue

was brought to 20 ml with deionized water and analyzed for

phosphorus (P), sulfur (S), zinc (Zn), iron (Fe), manganese (Mn),

boron (B), copper (Cu), potassium (K), magnesium (Mg), and total

nitrogen content of tissue.

Table 7 summarizes the data for leachate analyses collected on

day 33 for the two soil types inoculated with heat-killed or living

A. lipoferum. There was a statistically significant difference

between the two soil types with regards to leachate TC, DOC, DIC,

and mineral contents except for ammonium and nitrate (column 5,

Table 7). The concentrations of ammonium and nitrate were very

low, close to the limits of analytical detection, in both soils.

This probably accounts for the lack of differences in these two

elements in the leachates. The control microcosm cores received

heat-killed A. lipoferum. The microcosms inoculated with living A.

lipoferum exhibited no significant effects on the level of soluble

nutrients in leachates compared to the control except for TC and

DOC at day 33. The concentrations of TC and DOC were significantly

higher in controls than in microcosms that received the live

inoculum. These differences were not considered ecologically

significant because the results could be attributed to the addition

of dead microbial biomass, autoclaved Azospirillum, a portion of

which leached out by the treatment on day 33.

I~ R.].SEIDLER

Table 7. Analyses of leachates from intact soil cores collected on

day 33 (adapted from reference 13).

Burbank soil Palouse soil

corn Spring wheat

Azospirillum Azospirillum Prob>F

living control living control Soil a

Nutrient

sulfate 41.02 52.20 18.02 18.89

phosphate 1.90 2.34 0.83 0.96

ammonium 0.04 0.02 0.09 0.03

nitrite 0.04 0.02 0.04 0.05

nitrate 89.94 130.03 279.73 287.31

TC b 59.31 68.70 12.51 17.60

DOC c 16.36 21.42 5.37 6.91

DIC c 42.95 47.28 7.14 10.69

0 00

0 00

0 49

0 43

0 00

0 00

0 00

0 00

Inoculum

0.45

0.12

0.14

0.14

0.61

0.04

0.05

0.ii

a The soil column reports statistically significant differences in

the composition of the two soil types. The inoculum column reports

significant differences between Azospirillum treatments, i.e.,

microcosms receiving live or heat-killed cells.

b total carbon

c dissolved organic carbon

d dissolved inorganic carbon

TC and DOC leachate concentrations were also significantly

different between the soil types at the 67 day analysis (data not

shown). However, none of the minerals analyzed were significantly

different in effluent concentration. There was also no difference

in leached minerals between the microcosms receiving living or

heat-killed Azospirillum.

Mineral concentration in corn and wheat tissues were not

affected by the AzosDirillum cultures (13). The coefficient of

variations were typically lower in the leaf tissue extracts

compared to microcosm leachates. Nutrient analysis of plant tissue

extracts may be a more sensitive indicator of GEM effects on plant

nutrition than nutrient leaching of soil cores (13).

The studies of nutrient analyses of leachates and plant

tissues did not provide assurances of their relevance and

sensitivity for detecting effects from GEMs because no significant

METHODS FORDETECTING G E N E ~ C E N G I N E E ~ N G E F F E C T S 167

effects or alterations were noted in the AZOSDirillum treated

microcosms. However, the lack of effects detected seem to be

attributable to the choice of test bacterium and not a lack of

value of the experimental test system per s e. Other workers have

indeed found value in this holistic measure of nutrient leaching

because it reflects the community action of numerous microbial

groups (24). Nutrient leaching has also been found to be relevant

for detecting effects on microorganisms following addition of toxic

chemicals, including heavy metals, to a microcosm system (37).

However, investigators found that core-to-core variation in

leachate nutrient concentrations was high and limited sensitivity

of nutrient leaching as an indicator for measuring ecological

effects from GEMs (13). These methodologies appear useful in

detecting broad scale metabolic effects on biogeochemical

processes. However, it would be valuable to confirm these

expectations by experimentally validating these systems with a GEM

that induced perturbations.

ECOLOGICAL STRUCTURE AND FUNCTION COMPARISONS WITH

PSEUDOMONAS RC-I IN SOIL-CORE MICROCOSMS

Bolton et al. (5) examined the influence of a rhizosphere

bacterium Pseudomonas sp. RCI on ecologic structure and function

using soil-core microcosms. The approach was to utilize wheat as a

source of rhizosphere for colonization by RCI and compare

properties of the ecosystem in field plots, field lysimeters, and

intact soil-core microcosms enclosed in a growth chamber and in the

laboratory.

Details of the soil-core microcosm are described above and in

more detail in the literature (11,13,39). Briefly, the intact

soil-cores and lysimeters were 17.5 cm wide and 60 cm deep. The

field plots were 17.5 cm wide and 15 cm deep with open-ended

rings. Four experimental treatments consisted of: i) RCl plus

soil amendment consisting of 1% w/w alfalfa; 2) RCl and unamended;

3) uninoculated and amended; and 4) uninoculated, unamended. The

growth chamber microcosms had temperature fluctuations that

simulated the high and low field averages; the ambient microcosms

were maintained at 22 ° C. Inoculation of RCI achieved 1.7 x 108

cfu/g soil with the alfalfa and 5.5 x i0 z without alfalfa. A

solution of 15N-labeled ammonium sulfate was added to achieve a 15

atom percent enrichment. All treatments were brought to a final

soil moisture of 16% with tap water. All microcosms, field

I~ R.J. SEIDLER

lysimeters, and field plots were seeded with winter wheat seeds.

Two samplings of winter wheat were made, one at the three-leaf

stage and a final destructive sampling at the boot stage of growth.

The three-leaf stage was reached at different times in the various

microcosms (18, 45, and 144 days after planting for ambient,

chamber microcosms, and field lysimeters, respectively). A final

sample was collected at 67, ii0, and 205 days after planting,

respectively. At the final sampling the soil-cores were divided

into three sections from top to bottom (0-15 cm; 15-35 cm; 35-55

cm) for various analyses.

Relative numbers and diversity of microbial species were used

as indices of possible changes in ecosystem structural

measurements. Soil adhering to wheat roots from seedlings at the

three-leaf stage and from roots at three soil depths from the boot

stage of wheat were rinsed off in sterile water. Microbial

populations remaining on the root surface (rhizoplane) were

determined by plating onto appropriate media. Indices of species

diversity (i) were calculated for heterotrophic bacteria present on

the rhizoplane. For ecosystem functional parameters, measurements

of wheat shoot biomass, atom % 15N contents of shoots, and soil

dehydrogenase at the soil surface (0-15 cm) were ascertained (5).

The results of ecosystem structural analyses are summarized in

Table 8 for the early sampling taken at the 3-1ear stage of wheat

growth. Statistically significant changes were noted in some of

the experimental microcosms inoculated with the RCI strain. For

example, the total population of pseudomonads on the rhizoplane of

wheat was significantly higher in treatments receiving RCI. There

was no significant change in pseudomonads in microcosms at any of

the four locations (ambient, chamber, field, field lysimeter),

i.e., there was no significant location effect. Pseudomonads were

stimulated, as expected, by the alfalfa amendment. However, the

percentage of total fluorescent pseudomonads was lower in

treatments that received RCl. Thus, RCI out-competed a significant

proportion of the native soil pseudomonads for colonization of the

root surface. This colonization altered the bacterial composition

of the rhizoplane by decreasing the percentage of fluorescent

pseudomonads (24% as fluorescent pseudomonads without RCI, 1% with

RCl; Table 8). The population of fluorescent pseudomonads

continued to be significantly depressed, although not to the same

extent, at the boot stage of wheat growth (data not shown).

METHODS FOR DETECT~qGGENE~CENG~[£ERINGEFFECTS I~

Table 8. Ecosystem structural measurements on wheat rhizoplane at

the three-leaf stage of growth (adapted from reference 12).

%fluor-

Pseudomonads Bacteria RClas% RClas% escent

log CFU/ log CFU/ of of pseudo-

g dry g dry pseudo- bacteria monads

root root monads

Location

ambient 8.3a a 9.0a 86a 48a 10b

chamber 8.8a 9.4a 82a 36a 18a

field 8.4a 9.2a 85a 20a 10b

field lysim. 8.7a 9.1a 83a 58a 8b

Alfalfa amendment

with 8.8a 9.5a 76a 27a 15a

without 8.3b 8.9b 91a 53b 9b

Inoculation with Pseudomonas sp. RCI

with 8.8a 9.3a 84a 41a ib

without 8.3b 9.1a 0b 0b 24a

a Means in the same column that are followed by the same letter are

not significantly different (P<=0.05).

The initial inoculation of RCI at about 108 cfu/g soil provided an

ecological advantage to facilitate competition with other

pseudomonads in this habitat. Interestingly, the relative

percentage of RCI compared to the total bacteria decreased

significantly upon the addition of the alfalfa amendment. This was

likely caused by the stimulation of the indigenous microbes

allowing them to compete more effectively with RCl, due to the

addition of organic matter. The stimulation of RCI by alfalfa

amendment was not to the same extent as with the indigenous

microorganisms.

Others have suggested that because there is a large redundancy

of function in microbial communities, the displacement of a single

species as a result of competition with an introduced species would

probably not be deleterious (35). However, the exclusion of a

broad group of bacteria such as the fluorescent pseudomonads from

the rhizoplane of wheat, as in the present case, could be

detrimental to plant growth. A number of fluorescent pseudomonads

can function as biological control agents to prevent plant diseases

I~ R.].SE]DLER

and some strains can stimulate plant growth (25,40). This shift in

a significant proportion of the rhizoplane microbial population

validated monitoring for structural changes in microbial

components. This methodology is a sensitive indicator of ecosystem

change.

The ecosystem function comparisons are presented in Table 9.

Table 9. Soil and plant ecosystem functional measurements

influenced by Pseudomonas RCl and recorded at the three-leaf and

boot stage of wheat growth (adapted from reference 9).

Soil dehy- Shoot Boot stage

drogenase biomass b shoot N

activity a .............

3 leaf 3 leaf boot % total Atom %

stage stage stage N

Location

ambient 10.0ab c 0.02b 5.7c 3.9a 5.36ab

chamber ll.7a 0.07a 15.3a 1.7b 5.25bc

field 6.9c 0.06a ll.7b 1.2c 4.99c

field lysim. 8.2bc 0.05ab 13.7ab 1.7b 5.57a

Alfalfa amendment

with 14.0a 0.04b 14.0a 3.2a 1.87b

without 4.4b 0.06a 9.8b 1.4b 7.96a

Inoculation with Pseudomonas sp. RCI

with 9.2a 0.06a 12.2a 2.2a 4.85a

without 9.2a 0.04a 10.9a 2.1a 5.85a

aug triphenylformazan produced/g dry soii/24 hr.

b expressed in g of plant tissue

c Means in the same column that are followed by the same letter are

not significantly different (P<=0.05).

Inoculation with RCI had no significant influence on any of the

functional parameters evaluated. Significant variations were seen

by location on the total soil dehydrogenase activity and similarly

in the boot stage plant biomass. As expected, alfalfa amendment

also stimulated dehydrogenase activity as it also stimulated growth

of microbial populations. The alfalfa amendment also significantly

increased shoot biomass.

The addition of alfalfa to soil increased the N content of

METHODS FOR DETECTING GENE~CENGB%[EERINGF~FECT5 I~

plants, as anticipated, and decreased the % 15N enrichment of wheat

shoot. Inoculation with the pseudomonad RCl did not influence

shoot N or atom % 15N contents of plant tissue.

The value of these ecosystem functional parameters (soil

dehydrogenase, plant biomass, plant nitrogen content) was not

demonstrated by their response to the addition of RCl. However,

support for the potential usefulness of these parameters is clearly

seen in the statistically significant changes in data based on

location and alfalfa amendment. Thus, one would anticipate that if

a recombinant bacterium could induce changes in soil enzymes or

plant size, the techniques employed in these studies would be of

sufficient sensitivity to detect those changes.

A METABOLITE OF 2,4-DICHLOROPHENOXYACETIC ACID METABOLISM

IMPACTS SOIL ECOLOGICAL PROCESSES

The use of biotechnology for the remediation of toxic

chemicals holds significant promise. In one example, the

decontamination of agricultural soils containing 2,4-

dichlorophenoxyacetate (2,4-D) was demonstrated to proceed more

rapidly with a GEM than with the indigenous microflora (31). The

parental strain could not degrade 2,4-D. In related studies, the

degradation of 2,4-D in an arid soil occurred only with the added

GEM, Pseudomonas putida PPO301(pR0103), indicating that the

indigenous microorganisms of this soil were not capable of

degrading 2,4-D. Concurrent with the degradation, a metabolite

accumulated that had measurable effects on components of the

ecosystem (32). The soil was collected from a limited use area in

central Oregon, stored in greenhouse flats, and was periodically

wetted to maintain the indigenous microbiota. Soil was sieved

through a 2-mm mesh screen, adjusted to -33 kPa water potential,

and amended with 500 ug of 2,4-D per g of soil. Soil was

inoculated with approximately 107 cfu/g of parental P. putida

PPO301 or the recombinant strain PPO301(pROI03) that expresses

constitutively the ability to degrade 2,4-D to chloromaleyl acetic

acid (32). Soil was placed into individual I00 cm 3 vials and

incubated in a master-jar system consisting of l-gal (3.8L) glass

containers(33).

Two approaches were used to evaluate toxicity of 2,4-

dichlorophenol (2,4-DCP) to fungi. Growth of five morphotypes of

soil fungi was quantitatively measured after spread-plating

dispersed fungi onto Martin's agar containing 2,4-DCP. In the

In R.J.SEIDLER

second study, a replica-plating technique was used to evaluate the

extent of fungal spread from a central inoculation point of soil in

a petri dish that contained 2,4-DCP in soil. Spread of fungal

growth into the soil was evaluated after II days by replica plating

from the soil onto Martin's agar. The spread of each fungal

isolate at each 2,4-DCP concentration was expressed as a percentage

of the spread of each isolate in soil without 2,4-DCP.

Activities of the soil dehydrogenases and acid and alkaline

phosphatases and arylsulfatases, were measured by incubating soil

and substrates followed by extraction and spectrophotometric

measurement of enzyme products (i0). Methods for determining

numbers of total bacteria, spore-forming bacteria, and chitin

utilizers are previously summarized (I0).

The kinetics of 2,4-D removal from soil revealed that over 80%

of the 2,4-D was metabolized approximately 14 days after addition

of the GEM P. putida PPO301(pROI03). Within i0 days after

inoculation, the concentration of the first metabolic product of

2,4-D breakdown, 2,4-dichlorophenol (2,4-DCP) accumulated to 70-90

ug/g soil. No 2,4-D breakdown occurred in uninoculated soil that

received the nonrecombinant parental strain of P. putida PPO301.

Soil that received 2,4-D and glucose, plus the GEM P. putida

PPO301(pROI03), exhibited significantly reduced and delayed

respiration during the first 32 days of incubation. None of the

controls exhibited this effect, suggesting that the accumulation of

2,4-DCP depressed gross metabolic activity.

In soil amended only with P. putida PPO301(pROI03), fungal

propagules (CFU/g soil) of indigenous soil fungi either declined

slowly or exhibited little change. In 2,4-D amended soil

inoculated with the GEM, fungal propagules decreased dramatically

from over 3 x 106 on day 6 to undetectable levels by day 18.

Toxicity of 2,4-DCP was demonstrated in plate assays where

relative fungal viable counts were determined in the presence of

increasing concentrations of 2,4-DCP (Table i0). Growth of all

fungal isolates was detectably influenced by as little as i0 ug/ml

of 2,4-DCP. In the soil replica plate assays, 50 ug/g soil of 2,4-

DCP reduced the spread of fungi in sterile soil by 90 to 99%.

The accumulation of 2,4-DCP did not depress the numbers of

total heterotrophic, chitin-degrading, or spore-forming bacteria.

Thus, by day i0, when 2,4-DCP had accumulated to over 80 ug/g soil,

numbers of total heterotrophic, chitin-degrading, and sporeforming

METHODS FORDETEC~NG GENE~CENGINEERINGEFFECTS 1~

Table i0. Assay for relative amount of growth in the presence of

2,4-DCP or 2,4-D for five fungal isolates on Martin's agar (adapted

from reference 26).

% Relative Colony Formation for fungal isolate:

Martin's agar

1 2 3 4 5

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

unsupplemented i00 i00 i00 i00 i00

+ 10ug/ml 2,4-DCP <i 61 88 79 74

+ 25ug/ml 2,4-DCP <i 25 <i 2 9

+ 50ug/ml 2,4-DCP <i <I <i <i <i

+200ug/ml 2,4-D 83 62 73 69 52

bacteria were approximately the same as in the untreated control.

The accumulation of 2,4-DCP did not appear to affect the activity

of any of the enzymes monitored.

These results represent the first example of an ecological

effect induced through the accumulation of a metabolic by-product

from a GEM intended for bioremediation of contaminated soil. The

effects on soil fungi was dramatic and, under the conditions

studied, nonreversible. The toxicity was also apparent through the

delayed rate of glucose respiration. The toxicity of the 2,4-DCP

was generally greater to soil ecosystems than the parent 2,4-D.

However, it must be emphasized that the 2,4-DCP metabolite only

accumulated following the breakdown of 2,4-D by this GEM. The 2,4-

DCP only accumulates in soil where there are no microbes capable of

further metabolizing this by-product. Because 2,4-DCP is a

volatile compound it is not clear to what level it would accumulate

in soil under field conditions. The application of ecosystem

structural measurements (changes in microbial populations) as

endpoints was demonstrated to be valuable for detecting GEM effects

in these studies. Measuring change in the microbial profile or

relative numbers is easy to perform, sensitive to perturbations,

and amenable to replications to validate results. None of the soil

enzymes responded to accumulations of 2,4-DCP and thus the

sensitivity of using such assays to detect GEM effects needs to be

demonstrated.

I~ R.J. SEIDLER

ACKNOWLEDGEMENTS

The author expresses appreciation to the following individual

who provided suggestions for this manuscript: Jim Fredrickson,

Peter Hartel, William Holben, Mary Hood, Marcia Bollman, and

Deborah Coffey. Mention of trade names or commercial products doe

not constitute endorsement or recommendation for use. The

information in this document has been subject to the agency's peer

and administrative review, and it has been approved for

publication.

METHODSFORDETECTING GENE~CENGINEERINGEFFECTS

LITERATURE CITED

175

i. Atlas, R. M.. and R. Bartha. 1987. Microbial ecology, 2nd ed.

Benjamin/Cummings Publ. Co. Menlo Park, CA.

2. Austin, H. K., P. G. Nartel, and D. C. Coleman. 1990. Effect

of genetically-altered Pseudomonas solanacearum on predatory

protozoa. Soil Biol. Biochem. 22:115-117.

3. Bolton, Jr., H., J. K. Fredrickson, S. A. Bentjen, D. J.

Workman, S. W. Li, and J. M. Thomas. 1991. Field calibration of

soil-core microcosms for evaluating fate and effects of genetically

engineered microorganisms in terrestrial ecosystems. Report PNL-

7667/UC-403. National Technical Information Service, U.S.

Department Commerce, 5285 Port Royal Rd., Springfield, VA. 22161

4. Bolton, H. Jr., J. K. Fredrickson, S. A. Bentjen, D. J.

Workman, S. W. Li, and J. M. Thomas. 1991. Field calibration of

soil-core microcosms: fate of a genetically altered rhizobacterium

Microb. Ecol. 21:163-173.

5. Bolton, H. Jr., J. K. Fredrickson, J. M. Thomas, S. W. Li, D.

J. Workman, S. A. Bentjen, and J. L. Smith. 1991. Field

calibration of soil-core microcosms: ecosystem structural and

functional comparisons. Microb. Ecol. 21:175-189.

6. Bourquin, A., and R. J. Seidler. 1986. Research plan for test

methods development for risk assessment of novel microbes released

into terrestrial and aquatic ecosystems. In Biotechnology and the

Environment: Research Needs. G. S. Omen and A. H. Teich, eds.,

Noyes Data Corp. pp 18-61.

7. Crawford, D. L. 1978. Lignocellulose decomposition by

selected StreDtomvces strains. Appl. Environ. Microbiol. 35:1041-

1045.

8. Crawford, D. L., and R. L. Crawford. 1976. Microbial

degradation of lignocellulose: the lignin component. Appl.

Environ. Microbiol. 31:714-717.

9. Donegan, K., R. J. Seidler, and C. Matyac. 1991. Physical and

chemical control of released microorganisms at field sites. Can.

J. Microbiol. 37:708-712.

I0. Doyle, J. D., K. A. Short, G. Stotzky, R. J. King, R. J.

Seidler, and R. H. Olsen. 1991. Ecologically significant effects

of Pseudomonas putida PPO301(pROI03), genetically engineered to

degrade 2,4-dichlorophenoxyacetate, on microbial populations and

I~ R.J.SEIDLER

processes in soil. Can. J. Microbiol. 37:682-691.

ii. Fredrickson, J. K., S. A. Bentjen, H. Bolton, Jr., S. W. Li,

and P. Van Voris. 1989. Fate of Tn5 mutants of root growth-

inhibition Pseudomonas sp. in intact soil-core microcosms. Can. J.

Microbiol. 35:867-873.

12. Fredrickson, J., and R. J. Seidler. 1989. Evaluation of

terrestrial microcosms for detection, fate, and survival, analysis

of genetically engineered microorganisms and their recombinant

genetic material. US EPA/600/3-89/043.

13. Fredrickson, J. K., H. Bolton Jr., S. A. Bentjen, K. M.

McFadden, S. W. Li, and P. Van Voris. 1990. Evaluation of intact

soil-core microcosms for determining potential impacts of nutrient

dynamics by genetically engineered microorganisms. Environ. Tox.

Chem. 9:551-558.

14. Fredrickson, J. K., P. Van Voris, S. A. Bentjen, and H.

Bolton, Jr. 1990. Terrestrial microcosms for evaluating the

environmental fate and risks associated with the release of

chemicals or genetically engineered microorganisms to the

environment. Haz. Assess. Chem. 7:157-202.

15. Fredrickson, J. K., and C. Hagedorn. 1991. Overview:

Identifying ecological effects from the releases of genetically

engineered microorganisms and microbial pet control agents. In:

Microbial Ecology: Principles, Methods, and Applications. p. 559-

577. M. Levin, R. J. Seidler, and M. Rogul eds. McGraw-Hill, New

York. In Press.

16. Gillett, J. W., and J. M. Witt. 1979. Terrestrial

Microcosms. NSF-79-0024, National Science Foundation, Washington,

D. C.

17. Jackson, D. R., B. S. Ausmus, and M. Levin. 1979. Effects of

arsenic on nutrient dynamics of grassland microcosms and field

plots. Water Air Soil Pollut. 11:13-21.

18. Levin, M. A., R. J. Seidler, A. W. Bourquin, J. R. Fowle, and

T. Barkay. 1987. EPA developing methods to assess environmental

release. Biotechnology 5:38-45.

19. Linderman, R. G., J. E. Loper, M. D. Henkels, N. J. Mosier, T.

C. Paulitz, R. P. Griffiths, and B. A. Caldwell. 1991. Evaluating

effects of microbial pesticides on mycorrhizae. Final report.

USEPA, Environmental Research Laboratory, Corvallis, Or. In Press

20. Linderman, R. G., T. C. Paulitz, N. J. Mosier, R. P. Griffiths,

J. E. Loper, B. A. Caldwell, and M. E. Henkels. 1991. Evaluation

METHODS FOR DETEC~NG GENE~CENGINEE~NGEFFECTS 1~

of the effects of biocontrol agents on mycorrhizal fungi. In: D.

L. Keister and P. B. Cregan (eds.) The rhizosphere and plant

growth, p. 379. Kluwer Academic Publ. The Netherlands.

21. Linderman, R. G. 1991. Mycorrhizal interactions in the

rhizosphere. In: D. L. Keister and P. B. Cregan (eds.). The

rhizosphere and plant growth, pp. 343-348. Kluwer Academic Publ.

The Netherlands.

22. Marx, D. 1969. The influence of ectotropic mycorrhizal fungi

on the resistance of pine roots to pathogenic infection. I.

Antagonism of mycorrhizal fungi to root pathogenic fungi and soil

bacteria. Phytopathol. 59:153-163.

23. Omenn, G. S. 1986. Controlled testing and monitoring methods

for microorganisms. I__nn Biotechnoloqy Risk Assessment. J. Fiksel

land V. T. Covell, eds. pp. 144-163. Pergamon Press, N.Y.

24. O'Neill, R. V., B. S. Ausmus, D. R. Jackson, R. I. Van Hook,

P. Van Voris, C. Washburne, and A. P. Watson. 1977. Monitoring

terrestrial ecosystems by analysis of nutrient export. Water Air

Soil Pollut. 8:2710277.

25. Murdoch, C. W., R. J. Campana, and J. Hoch. 1984. The

biological control of Ceratocystis ulmi with Pseudomonas

fluorescens. Phytopathology 74:805.

26. Paulitz, T. C., and R. G. Linderman. 1989. Interactions

between fluorescent pseudomonads and VA mycorrhizal fungi. New

Phytol. 113:37-45.

27. Paulitz, T. C., and R. G. Linderman. 1991. Lack of

antagonism between the biocontrol agent Gliocladium virens and

vesicular arbuscular mycorrhizal fungi. New. Phytol 117:303-308.

28. Phillips, J. M., and D. S. Hayman. 1970. Improved procedure

for clearing roots and staining parasitic and vesicular-arbuscular

mycorrhizal fungi for rapid assessment of infection. Trans. Brit.

Mycolog. Soc. 55::158-161.

29. Roberts, D. P., T. P. Denny, and M. A. Schell. 1988. Cloning

the egl gene of Pseudomoonas solanacearum and analysis of its role

in phytopathogenicity. J. Bacteriol 170:1445-1451.

30. Seidler, R. J., and J. Settel, (eds.). 1991. The use and

development of environmentally controlled chambers (mesocosms) for

evaluating biotechnology products. US EPA/600/9-91/013.

31. Short, K.A., R. J. Seidler, and R. H. Olsen. 1990. Survival

and degradative capacity of Pseudomonas putida induced or

constitutively expressing plasmid-mediated degradation of 2,4-

I~ R.J. SEIDLER

dichlorophenoxyacetate (TFD) in soil. Can. J. Microbiol. 36:821-

826.

32. Short, K. A., J. D. Doyle, R. J. King, R. J. Seidler, G.

Stotzky, and R. H. Olsen. 1991. Effects of 2,4-dichlorophenol, a

metabolite of a genetically engineered bacterium, and 2,4-

dichlorophenoxyacetate on some microorganism-mediated ecological

processes in soil. Appl. Environ. Microbiol. 75:412-418.

33. Stotzky, G. 1965. Microbial respiration, pp.1500-1572. I_Dn

C. A. Black, D. D. Evans, L. E. Ensminger, J. L. White, and F. E.

Clark (ed.). Methods of soil analysis. American society of

Agronomy. Madison, Wisconsin.

34. Stotzky, G. 1990. Methods to measure the influence of

genetically engineered bacteria on ecological processes in soil.

US EPA\600\3-90\011.

35. Tiedje, J. M., R. R. Colwell, Y. L. Grossman, R. E. Hodson, R.

E. Lenski, R. N. Mack, and P. J. Regal. 1989. The planned

introduction of genetically engineered organisms: ecological

considerations and recommendations. Ecology 70:298-315.

36. Wang, Z., D. L. Crawford, A. L. Pometto III, and F. Rafii.

1989. Survival and effects of wild-type, mutant, and recombinant

Streptomyces in a soil ecosystem. Can. J. Microbiol. 35:535-543.

37. Wang, W., B. H. Bleakley, D. L. Crawford, G. Hertel, and F.

Rafii. 1990. Cloning and expression of a lignin peroxidase gene

from Streptomyces viridosporus in Streptomyces lividans. J.

Biotechnol. 13:131-144.

38. Wang, Z., D. L. Crawford, T. S. Magnuson, B. H. Bleakley, and

G. Hertel. 1991. Effects of bacterial lignin peroxidase on

organic carbon mineralization in soil, using recombinant

Streptomyces strains. Can J. Microbiol. 37:287-294.

39. Van Voris P. 1988. Standard guide for conducting a

terrestrial soil-core microcosm test. Standard EI197. In Annual

Book of ASTM Standards. Vol 11.04, pp. 743-755. American Society

for Testing and Materials, Philadelphia.

40. Weller, D. M. 1983. Colonization of wheat roots by a

fluorescent pseudomonad suppressive to take-all. Phytopathology

73:1548-1533.