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V. Additional Uses of Genetically Engineered Microorganisms in Weed Management

V. Additional Uses of Genetically Engineered Microorganisms in Weed Management

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Biodegradation, or mineralization, of herbicides in water and soils is almost

always the result of microbial activity. Soil microbes utilize either adaptive

or constitutive enzyme systems which can alter biochemically a pesticide into a utilizable nutrient and energy source (Alexander, 1981; Ghosal et al.,

1985; Kaufman and Kearney, 1976; Torstensson, 1980). Apart from the

physicochemical characteristics of a given soil, the microbial degradation of

a herbicide can be influenced also by the rate and frequency of its application, the cropping system, and the presence of other pesticides applied

simultaneously or sequentially to the same soil (Kaufman et al., 1985). Frequent, repeated applications of a given pesticide to the same soil may lead to

an increase of microbial populations to the point that succeeding application of this or other pesticides are ineffective on target pests because of the

accelerated rates of their biodegradation. Such soils are currently referred

to as “conditioned,” “problem,” or “history” soils (Kaufman et al., 1985;

Roeth, 1986).

Although this phenomenon has been known for some time (Audus, 1951),

the recent documentation of the accelerated degradation of thiocarbamate

herbicides in soils with previous thiocarbamate herbicide exposure has attracted considerable attention (Fox, 1983; Kaufman et al., 1985; Roeth,

1986). Thiocarbamate herbicides such as EPTC are used extensively in corn

fields for the control of selected annual and perennial grass weeds. Soils conditioned to the rapid degradation of thiocarbamate herbicides are found

primarily in the midwestern states where the bulk of the United States’ corn

production is centered. However, the existence of thiocarbamateconditioned

soils has been also documented in southern states and in other countries

(Murdock et al., 1984; Rahman et al., 1979; Roeth, 1986). Accelerated

microbial degradation has been also demonstrated with the phenylacetamide

herbicide diphenamid and the chloracetanilide herbicides alachlor and

metolachlor (Kaufman et al., 1985; White et al., 1987).

The ability of soil and water microorganisms to degrade a wide variety of

pesticides and other industrial chemicals has been known for some time

(Alexander, 1981; Ghosal et al., 1985; Kaufman and Kearney, 1976;

Torstensson, 1980). A number of herbicides are completely mineralized by

selected soil microganisms and specific examples are presented in Table VI.

For simplicity, in Table VI only the names of the genera of these soil

mocroorganisms are given. It should be emphasized, however, that in most

cases only defined strains of these microorganisms are capable of degrading

the herbicides listed in Table VI.

At present, the mechanism@)of the accelerated or natural biodegradation of herbicides in soils are poorly understood. In analogy to mechanisms

involved in the development of bacterial resistance to antibiotics, it has been

postulated that degradative genes for soil-applied herbicides may be carried

on bacterial plasmids which can be traded freely among various



Table VI

Microbial Strains Known to Metabolize Selected Herbicides in Soils



(2,4-D, MPCA, 2,4,5-T)


Pen tachlorophenol












Alcaligenes sp.

Achromobacter sp.

Arthrobacter sp.

Aspergillus sp.

Brevibacterium sp.

Corynebacterium sp.

Nocardia sp.

Pseudomonas sp.

Strepromyces sp.

Aspergillus sp.

Pseudomonas sp.

Arthrobacter sp.

Flavobacterium sp.

Pseudomonas sp.

Phenylobacterium sp.

Pseudomonas sp.

Pseudomonas sp.

Chaetomium globosum

Rhizoctonia solani

Fusarium oxysporum

Unspecified actinomycete

Chaetomium globosum

Bacillus circulans

Bacillus megaterium

Fusarium sp.

Mucor racemosus

Fusarium solani

Baccilus sp.

Pseudomonas sp.

Arthrobacter sp.

Fluorescent Pseudomonads

Mycobacterium sp.

Flavobacterium sp.

Streptomyces sp.

Fusarium sp.


Don and Pemberton (1981)

McCormick (1985)

McCormick (1985)

McCormick (1985)

McCormick (1985)

McCormick (1985)

McCormick (1985)

Kilbane et al. (1982)

McCormick (1985)

McCormick (1985)

Cripps and Roberts (1978)

Staniake and Fink (1982)

Ghosal et al. (1985)

Karns et al. (1983)

Eberspacher and Lingens


Cripps and Roberts (1978)

Moore et al. (1983)

McCormick (1985)

McCormick (1985)

Kaufman and Blake (1973)

Krause et al. (1985)

McGahen and Tiedje (1978)

Saxena et al. (1987)

Saxena et al. (1987)

Saxena et al. (1987)

Saxena et al. (1987)

McCormick (1985)

McCormick (1985)

McCormick (1985)

Tam et al. (1986)

Meredith et al. (1987)

Mueller et al. (1987)

Imai and Kuwatsuka,




microorganisms to speed adaptation to these herbicides (Don and

Pemberton, 1981; Fox, 1983; Pemberton, 1981; Roeth, 1986). However,

only in relatively few cases have the enzymes that attack herbicide molecules

and cause a loss in their phytotoxicity been documented. Studies on the

isolation and cloning of microbial genes encoding for herbicide degradation

are also limited.

The role of plasmids in microbial evolution and their potential involvement in pesticide degradation have been reviewed by Chakrabarty (1982),

Clarke (1984), Reanney (1976), and Reineke (1984). Pemberton and Fisher

(1977) presented evidence that the microbial degradaton of the phenoxyacetic acid herbicides 2,4-D and MCPA is specified by plasmid-borne

genes. The presence of a conjugal plasmid encoding for the ability of a

strain of Alcaligenes paradoxus to degrade 2,4-D and MCPA has been

documented (Don and Pemberton, 1981; Fisher et al., 1978).

Chakrabarty and his co-workers have studied extensively the role of

transmissible and nontransmissible plasmids in the microbial degradation

of 2,4,5-T (Chatterjee and Chakrabarty, 1981; Chatterjee et al., 1982;

Kellog et al., 1981; Kilbane et al., 1982, 1983). Tam et al., (1986) have

reported the isolation of an Arthrobacter spp. (strain A56) from EPTChistory soils which was capable of degrading this herbicide. Meredith et al.

(1987) and Mueller el al. (1987) reported the isolation of several strains of

fluorescent pseudomonads from butylate-history soils which were also

capable of growing on culture media containing EPTC as a carbon source.

In both cases, the enhanced degradation of EPTC by these soil bacteria was

thought to plasmid-mediated, and a 50.5-kb plasmid has been partially

characterized from the A56 strain of Arthrobacter spp. (Tam et al., 1986).

Another indication of plasmid transfer in connection with herbicide

degradation comes from the work of Senior et al. (1976), who showed that a

strain of Pseudomonasputida acquired the ability to grow on the herbicide

dalapon through the evolution of an extant dehalogenase. Dehalogenases

(also known as halidohydrolases) catalyze the hydrolytic removal of halides

from halogenated alkanoic and, particularly, chlorinated acetic and propionic acids such as the herbicide dalapon (Goldman et al., 1968; Little and

Williams, 1971; Slater et al., 1979). Beeching et al. (1983) suggested that

plasmid-mediated transfer of dehalogenase genes is the most likely reason

for explaining the increased occurrence of different bacteria possessing

dehalogenases after challenging the mixed microbial flora with herbicidal

halogenated alkanoic acids. However, in a study utilizing the PP3 strain of

Pseudomonas putida, which possesses no detectable plasmids, Slater et al.

(1985) suggested that the dehalogenase genes were present on chromosomally located transposable elements and that spontaneous mutations involved

excision of these elements. The frequency of the excision events was strongly dependent on environmental conditions. Thus, both mechanisms



(plasmid-mediated transfer and excision of chromosomally located transposable elements) appear to be important in the evolution of bacterial

mutants that can degrade and grow on specific herbicides following the

repeated exposure of soil microflora to these herbicides.

Plasmid-borne genes coding for degradative enzymes can be transferred to

selected bacteria, and such genetically engineered mirobes could find a number

of practical applications. Ongoing studies on the biochemistry and genetics of

herbicide metabolism by soil microflora could definitely maximize the effective

utilization of the biological degradation process for the feasible waste disposal

of persistent pesticides and other industrial chemicals (Chakrabarty, 1985;

Ghosal eta/., 1985; Kearneyet a/., 1987; Steiertand Crawford, 1985; Summers,

1985; Trevors, 1985). In addition, a better understanding of the biochemistry

and genetics of the microbial degradation of thiocarbamate and chloroacetanilide herbicides in history soils could result in the development of chemical inhibitors of soil microorganisms to eliminate this problem. In fact, chemicals

marketed as extenders are already available for use as short-term aids for

coping with the enhanced microbial degradation of thiocarbamate herbicides

(Roeth, 1986). The chemical dietholate is currently used as an additive in the

formulation of the thiocarbamate herbicides EPTC and butylate (e.g.,

ERADICANE! EXTRA@)to act as a microbial inhibitor and extend the persistenceof these herbicidesin history soils (Roeth, 1986). Applied with EPTC in

history soils, dietholate doubled the half-life of EPTC, slowed ’‘C02 evolution from I4C-labeled EPTC, and increased grass control by this herbicide

(Obrigawitch et al., 1982). However, dietholate did not extend the persistence

of EPTC in nonhistory soils nor restored the lag period to EPTC degradation in

history soils (Roeth, 1986). Contradictory results and questions on the efficacy

of dietholate as an extender of thiocarbamate herbicide in history soils have

been reported by a number of investigators (see review by Roeth, 1986).





The term “herbicide safener” is commonly used to describe a chemical

compound which protects crop plants against injury from herbicides

(Hatzios, 1983). Other terms such as antidote, protectant, and antagonist

are also used for the same purpose. At present, all herbicide safeners

marketed commercially are synthetic chemicals developed by means of random or empirical screening (Hatzios, 1983). Recent advances in

biotechnology, however, appear quite promising for the potential development and use of microbial herbicide safeners in the near future.

Microbes carrying plasmids with herbicide degradative genes could be used

as crop protectants to increase the selectivity of soil-applied herbicides.

In principle, the idea is simple. Genes encoding for degradative enzymes

which break down specific herbicides, isolated from plants or microbes,



could be cloned into appropriate plasmids and transferred to bacteria (e.g.,

Pseudornonas fluorescens) that colonize the roots or seeds of susceptible

crop plants. Such a system could then protect these susceptible plants from

soil-applied and root-absorbed herbicides. Although the practical utilization of such a system may seem remote at present, recent success in utilizing

a similar system for the microbial control of a root-feeding insect of corn

demonstrated the possible use of this approach (Kaufman, 1986). This

model system utilized the cloning of the genes coding for the active endotoxin of Bacillus thuringiensis kurstani into isolates of Pseudornonas

fluorescens, which colonize the roots of corn. Data from monitoring studies

of the risks associated with the release of these genetically engineered

bacteria into the environment showed that this approach is safe. Release

and use of this bioinsecticide is associated with low pathogenicity, low

capacity for genetic exchange, and limited environmental persistence (Kaufman, 1986). Additional support for the potential development of microbial

herbicide safeners comes from earlier research conducted in Poland showing that herbicide injury to sugar beets could be reduced with the use of

selected bacterial fertilizers such as azotobacterin (Geller and Nikolaenko,

1972; Wegrzyn, 1975).

An alternative approach for the use of microorganisms as safeners for

the protection of crop plants against herbicide injury could include the

direct insertion of microbial genes encoding for herbicide degradative enzymes into the chromosomes of susceptible plants. The goal of this approach is to manipulate the plant genetically and transform it to a producer

of the desirable enzyme which provides protection against injury from a

given herbicide. Partial success with this approach has been reported by

Meusen and Zabeau (1986), who attempted the insertion of the gene coding

for the endotoxin of Bacillus thurigiensis into the chromosomes of tobacco

plants in order to make them resistant to insects. Coating corn seeds with

the altered P . fluorescens via a propriatary process developed by Monsanto

Chemical Company produced a plant whose roots contained the endotoxin

of Bacillus thurigiensis that inhibits the attack of the root worm (Kaufman,


This approach of microbial herbicide safening could be extended further

to include the use of endophytic bacteria which live internally in the

xylem of higher plants. Genetically engineered endophytic bacteria could

be modified to produce large amounts of specific amino acids whose

biosynthesis is known to be inhibited by selected classes of herbicides such

as the sulfonylureas, imidazolinones, and glyphosate (see Section IV for a

detailed discussion). Thus, it is feasible that genetically engineered endophytic microorganisms could be used as herbicide safeners in the









Apart from their potential use as bioherbicides or as sources of novel

phytotoxins and leads for new herbicide chemistries, microorganisms could

be further exploited as biological catalysts for the synthesis and commercial

production of sophisticated plant protection chemicals (Alder et al., 1985;

Bewick et al. 1986).

The most obvious attraction of biocatalysts is that they make it possible

to utilize the higher stereo- and regiospecificity of enzyme reactions and to

perform transformations which may be difficult or impossible to perform

chemically. Isolation and characterization of microorganisms which can invert the optical configuration of the S-enantiomer present in racemates of

herbicides containing an asymmetric carbon atom in their molecules is of

utmost importance for the commercial production of the optically resolved

and herbicidally active R-enantiomer (Bewick et al., 1986; Fedtke, 1982).

Activity, specificity, and physical properties represent some of the key factors which need to be considered in the screening of microorganisms as


Although research in this area has been limited, the potential of this

biotechnological approach is enormous. Table VII illustrates the successful

application of this approach in the production of the herbicidally active

enantiomers of selected herbicides including members of the phenoxy carboxylic acid, phenoxy-phenoxy carboxylic acid, and pyridinyl-phenoxy carboxylic acid groups. Such herbicides exhibit excellent grass activity and

good crop selectivity and they are very important in modern weed management programs of major crops such as soybeans and winter cereals.

Genetically engineered microorganisms could be also exploited for their

potential to produce biosurfactants, which can enhance the activity of

chemical herbicides under field conditions. Banerjee et al. (1983). reported

recently that a culture specifically developed for the degradation of the

Table VII

Use of Microorganisms as Biocatalysts in the Production of the

Active Enantiomers of Selected Herbicides


Active enantiomer



Chlor fenprop-methyl



Clostridium klujveri

Rhodococcus sp.

Fedtke (1982)


Mecoprop (Duplosan@)


Not given

Koning (1986)

Bewick et al. (1986)



herbicide 2,4,5-T produces a potent biosurfactant (emulsifier) which forms

stable emulsions with 2,4,5-T. Such emulsions may be very useful for the

herbicidal application of 2,4,5-T in the field as a uniform film.



The potential applications of biotechnology in addressing selected issues

related to weed management and herbicide technology are quite promising.

Bioherbicides, naturally produced herbicides, and constructed microbes

with novel degradative ability have been already developed and used commercially. In addition, the concept of using genetic engineering techniques

for the generation of herbicide resistance or tolerance in sensitive crops has

created great excitement.

The commercialization and marketing of the mycoherbicides DeVine@

and College@ illustrates the great potential of the use of phytopathogenic

agents as bioherbicides to control selected weeds. At present, bioherbicides

are viewed as complementary adjuncts to current weed management practices rather than as alternatives to chemical herbicides. In particular, the

future potential of mycoherbicides is seen in areas that are currently served

inadequately by chemical herbicides. According to Templeton et al. (1986)

such areas include (1) control of parasitic weeds; (2) control of weeds closely

related to crops (crop mimics), in which case a high degree of selectivity is

necessary; (3) control of weeds resistant to chemical herbicides; and (4) control of weeds infecting small, specialized areas where development of

chemical herbicides would be too costly.

Advances in our knowledge of the interactions of plants and

phytopathogenic organisms at the molecular level will increase our

understanding of the role of toxins in pathogenesis, toxin production, and

its genetic control by the pathogen. Such knowledge could lead to the commercialization of new bioherbicides or the synthesis of new chemical herbicides developed by means of a biorational design based on the chemistry

of natural toxins. It is expected that in the near future, chemical herbicides

would be developed by new approaches, based on the principles of

biotechnology and chemistry, rather than by the currently dominant process

of empirical synthesis coupled with biological screening. The commercial

exploitation of the microbial toxin, L-phosphinothricin, which is the active

ingredient of the microbial herbicide bialophos and of the chemically synthesized herbicide glufosinate illustrates this point.

Plant cell cultures and fermentation broths of microorganisms are expected to have a major impact in agriculture through the novel production

of agrochemicals such as herbicides used in weed management. Additional



advances are also,expected in the area of biocatalysis benefiting from the use

of plant or microbial cell enzymology, which is presently superior to the skills

of organic chemists in performing complex biotransformations. Learning

more about the plasmid-determined resistance of soil microbes to relevant

herbicides will allow us to employ effectively such resistance in several

microbially related processes such as the decontamination of soil residues of

persistent herbicides or the development of microbial herbicide safeners.

Advances in genetic technology will also facilitate our efforts to understand, manage, circumvent, and exploit the resistance of plants to selected

herbicides. Isolation and characterization of plant or microbial genes

coding for mutations altering the sensitivity of specific target proteins to

herbicides or for herbicide-detoxifying enzymes will improve our

understanding of the biochemical and genetic basis of plant resistance or

tolerance to herbicides. Such information coupled with advances in recombinant DNA technology will enable us to engineer herbicide-resistant determinants and develop herbicide-resistant crop plants. At a theoretical level,

progress in this area has been enormous. However, at the practical level,

success has been only partial. Presently, only one herbicide-resistant crop,

atrazine-resistant canola, developed from classic breeding techniques rather

than sophisticated genetic engineering technology, is marketed in Canada.

The development of tobacco and petunia plants resistant to the herbicides

glyphosate and chlorsulfuron by means of gene transfer and transformation

techniques indicates that genetic engineering of herbicide resistance is feasible. The full utilization of biotechnological procedures as tools for incorporating herbicide resistance to major agronomic crops (e.g., cereals and

legumes) is presently limited by a number of unresolved problems such as a

limited pool of genes of interest, lack of appropriate vector systems for gene

transfer, and inefficient methods for the regeneration of selected crops

from cell or tissue cultures. Future research, hopefully, will address and

solve these problems.

For a more fruitful application of biotechnology in engineering herbicide

resistance several considerations should be examined or reevaluated in

future investigations. According to Gressel(l985) and Widholm (1978) such

considerations include the following:

1. A given herbicide should be considered only when it more cost effectively controls specific problem weeds in a given crop better than other

presently available herbicides.

2. All biochemical mechanisms conferring plant tolerance or resistance to

herbicides should be considered and exploited for practical application.

3. If we are looking for resistance at the mode of herbicide action level,

we need more information on the biochemical mechanisms involved and

their genetic basis.

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