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5
Plant Diseases and
Insect Pests
The damage to plants caused by competition from weeds
and by other pests including viruses, bacteria, fungi,
and insects greatly impairs their productivity and in
some instances can totally destroy a crop. Today,
dependable crop yields are obtained by using disease-
resistant varieties, biological control practices, and by
applying pesticides to control plant diseases, insects,
weeds, and other pests. In 1983, $1.3 billion was spent
on pesticides--excluding herbicides--to protect and limit
the damage to crops from plant diseases, nematodes, and
insects. me potential crop losses in the absence of
pesticide use greatly exceeds that value.
For about 100 years, breeding for disease resistance
has been an important component of agricultural produc-
tivity worldwide. But the successes achieved by plant
breeding are largely empirical and can be ephemeral.
mat is, because of a lack of basic information about the
function of genes for resistance, studies are often ran-
dom rather than specifically targeted explorations. In
addition, any results can be short-lived because of the
changing nature of pathogens and other pests as new
genetic information is introduced into complex agro-ecol
ogical systems.
An excellent example of the effect of genetic change
is the sterile pollen trait bred into most major corn
varieties to aid in the production of hybrid seed.
Plants containing Texas (T) cytoplasm transfer this male
sterile trait via the cytoplasm; it is associated with a
particular type of mitochondrion. Unknown to breeders,
these mitochondria also carried vulnerability to a toxin
produced by the pathogenic fungus Helminthosporium
81
-
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82
maydis. The result was the corn leaf blight epidemic in
North America in the summer of 1970.
The methods used in the discovery of pesticide chem-
icals also have largely been empirical. With little or
no prior information on mode of action, chemicals are
tested to select those that kill the target insect, fun-
gus, or weed but do not harm the crop plant or the
environment.
Empirical approaches have produced enormous successes
in controlling some pests, particularly weeds, fungal
diseases, and insects, but the struggle is continuous,
since genetic changes in these pests can often restore
their virulence over a resistant plant variety or render
the pest resistant to a pesticide. What is missing from
this apparently endless cycle of susceptibility and
resistance is a clear understanding of both the organisms
and the plants they attack. As knowledge of pests--their
genetics, biochemistry, and physiology, their hosts and
the interactions between them--increases, better-directed
and more effective pest control measures will be devised.
This chapter identifies several research approaches
to a better understanding of the fundamental biological
mechanisms that might be exploited to control plant path-
ogens and insects. Molecular biology offers new tech-
niques for isolating and studying the action of genes.
me existence of susceptible and resistant host plants
and virulent and avirulent pathogens can be exploited to
identify and isolate the genes that control the inter-
actions between host and pathogen. Studies of the fine
structure of these genes can lead to clues about the
biochemical interactions that occur between the two
organisms and to the regulation of these genes in the
pathogen and in the tissues of the plant. It should be
possible in the future to improve the methods and
opportunities for the transfer of desirable traits for
resistance into crop plants and, conversely, to create
pathogens that will be virulent against selected weeds or
arthropod pests. An increased understanding of insect
neurobiology and the chemistry and action of modulating
substances, such as the endocrine hormones that regulate
metamorphosis, diapause, and reproduction, will open new
avenues for controlling insect pests by disrupting their
physiology and behavior at critical stages in the life
cycle.
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83
Molecular Bases of Plant-Pathogen Interactions
The existence of susceptible and resistant cultivars
implies specificity in plant diseases. One explanation
for this high specificity is a "recognition" mechanism
between pathogen and host. Understanding the molecular
bases that determine this specificity in recognition or
in the pathogen's ability to alter the host's metabolism
should yield new, definitive, and more efficient ways to
prevent attacks on crop plants or to mitigate disease
symptoms.
Based on our current, limited understanding of the
types of interactions that occur between host plants and
pathogens, the mechanisms involved are varied and
complex. Theoretically, a minimum of two criteria are
involved. The first is recognition. There may be
preformed molecules in both host and parasite that can
interact. Second, there must be metabolic changes in the
host or pathogen or both that are triggered by the
initial interaction step. Genetic mutations in either
host or pathogen can change the specificity of molecular
interactions or their ability to trigger metabolic change.
The following presents discussions on research
directed toward possible mechanisms involved in recog-
nition between host and pathogen and the metabolic
changes that cause disease symptoms.
Molecular Determinants of Resistance and Susceptibility
It is widely held that some forms of resistance to
fungal and bacterial pathogens are the result of a host
plant's ability to synthesize chemicals that inhibit the
growth and development of the pathogen. During infection
by a pathogen, metabolic pathways in the plant are acti-
vated, leading to the detectable biosynthesis of the
inhibitors. A major class of inhibitors, called phyto-
alexins, are primarily low-molecular-weight, secondary
plant metabolites that possess wide-ranging activity
against fungi and, to a lesser extent, bacteria. In the
last two decades, more than 100 phytoalexins have been
identified. The induction of the biosynthesis of
phytoalexins, however, does not follow the specificity
that most pathogens have for a specific cultivar. For
example, phytoalexin synthesis can be induced by abiotic
agents, such as wounding or other stress conditions, in
both resistant and susceptible plants. Phytoalexins can
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84
be toxic to both virulent and avirulent pathogens. It
appears that phytoalexin synthesis might be a general,
nonspecific type of active resistance.
An alternate approach, the study of susceptibility,
has revealed mechanisms that show a high degree of
specificity. _ ~ ~ ~
Many Pathogens Possess specific agents for
virulence, sucn as toxins or enzymes, that determine the
course of events in susceptible plants. In the last five
years, six host-specific or host-selective toxins have
been chemically characterized. These toxins affect only
susceptible cultivars and are produced only by specific
pathogens that can attack these same susceptible culti-
vars. One well-studied example is the toxin produced by
the fungus Helminthosporium maydis, mentioned earlier.
The H. mavdis toxin disrupts enerav Generation in susceD—
tible mitochondria that characterize the T cytoplasm of
corn. Normal mitochondria are resistant and are
unaffected by the toxin because they apparently lack a
receptor site for it.
Genetic specificity also exists for resistance and
susceptibility to plant viruses, but there is no infor
mation on how such genes act. With respect to plant
viruses the term resistance is used rather loosely.
Quite often only the appearance of disease symptoms is
considered. Thus, a plant that supports virus repli-
cation but shows no symptoms is considered to be
-
resistant because it superf~cially appears to be so.
More correctly, that plant should be called tolerant.
Recent observations suggest that one type of
resistance may involve the ability of viruses to spread
from cell to cell in their hosts. The continuum of
responses ranges from rapid and complete invasion of the
whole plant by the virus to slow invasion to circum-
stances where the virus is unable to spread from an
infected cell, even though it might replicate well
there. Accumulating evidence indicates that viruses
induce the synthesis of proteins that are necessary for
the movement of viruses from cell to cell. The host,
however, depending on its genotype, can in some way
interfere with this protein. Although the process is
poorly understood, it may be, in part, the basis of
resistance of plants to viruses.
In a sense, viruses might be thought of as packages of
genes; they are composed primarily of RNA or DNA, and
they can replicate only in a favorable host cell
environment. Studies of the interactions between viral
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85
RNA or DNA and genes in the host cell can lead not only
to an understanding of how viruses function but also to
the development of viruses as gene-carrying vectors for
genetic engineering.
An improved understanding of the basic concepts con-
trolling resistance and susceptibility will result from
research based on interrelated approaches to the analysis
of the genetics of these traits, the gene products, the
structure of the genes, and the methods that will permit
their transfer between organisms.
Genetics Continued breeding studies and genetic
analysis of resistance traits in host plants and viru-
lence traits in pathogens provide the experimental
systems needed to isolate and determine the properties of
recognition molecules involved in susceptibility or
active resistance, such as phytoalexin biosynthesis.
Single-gene changes that confer resistance against a
pathogen exist and are used in crop breeding to develop
improved cultivars. In other cases multiple genes appear
to be involved in resistance, and complicate crop
breeding. m e growing collection of data on the genetics
of host plants and particularly of pathogens needs to be
strengthened. Such data are essential for identification
of the genes that control the specificity of receptor
molecules, which determine resistance or susceptibility
to bacteria, fungi, or viruses. Genetic analysis of some
important fungal pathogens, however, will be difficult
because sexual reproduction does not occur, and the modes
of genetic reassortment and inheritance are unknown.
Many genetic approaches are now being initiated. For
example, single-pathogen genes responsible for disease
reactions in two bacterial leaf-spot diseases, soybean
blight and bacterial spot of tomato, are being isolated
and cloned. mese techniques have potential for wide
application.
Gene Products ~ =~ -_ __ __ ~ .-_
tein. There is little direct evidence for the role of any
{rho end product of most genes is a oro-
specific proteins in controlling interactions between a
host and a pathogen. Many potential candidates, however,
can be hypothesized. By analogy with animal systems,
surface molecules, such as membrane glycoproteins, may
interact with low-molecular-weight messenger molecules,
such as small carbohydrates released from cell walls.
Cell wall extracts from both hosts and pathogens have
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86
been shown to elicit some resistance responses. Both the
hydrolytic enzymes that release carbohydrate fragments
from cell walls and the enzymes involved in the
biosynthesis of toxins or phytoalexins are gene products
that may be selected for study.
Additional basic information is needed about the
cellular interactions between host and pathogen during
the onset of resistance reactions. For example, the
precise mechanisms employed by phytoalexins to exert
their effects on pathogens are unknown and need to be
actively studied. Metabolic pathways for the biosyn-
thesis of phytoalexins must be clarified, and other com-
pounds associated with resistance need to be identified.
The regulation and coordinated synthesis of the enzymes
involved in these pathways must be detailed.
In addition, the phenomenon of acquired resistance in
plants needs further study. Resistance can be localized
or can occur throughout the plant. Systemic resistance,
however, may be of more practical value. m is phenomenon
can appear after a host plant is inoculated with an avir-
ulent strain of the bacterial, fungal, or viral pathogen.
This exposure somehow induces resistance properties so
that when the plant is subsequently challenged by one or
more pathogenic strains, it will resist infection or
exhibit only mild disease symptoms.
Acquired resistance is most actively being studied
using Pseudomonas solanacearum, some strains of which
cause wilt and stem rot in tobacco, ginger, potato,
tomato, and banana.
resistance.
Other avirulent strains only induce
resistance. The experimental approach is to find mutants
of the avirulent strains that fail to induce the acquired
A comparison of the gene libraries of the
active with the inactive mutants could lead to the iden-
tification of the genes and gene products responsible for
triggering the acquired resistance.
Gene Structure Once the genes and gene products are
identified, it is feasible to alter their activity by
changing the structure of the gene itself. The tools of
molecular genetics can be used to study both the struc-
ture and activity of pathogen genes for virulence and
avirulence and host genes for resistance and suscep-
tibility. Some progress has been made recently with
bacterial pathogens, particularly in characterizing some
virulence factors such as pectolytic enzymes. Much of
the basic information on the molecular biology of fungal
pathogens, however, is yet to be acquired.
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The functions of proteins coded for by viral genomes
must also be established to aid in the understanding of
their possible roles in replication and pathogenesis.
It may now be possible to isolate genes for specific
types of resistance, such as that characterized by so-
called hypersensitive lesions. For example, certain
plant species and cultivars respond to infection by a
pathogen by rapidly undergoing cell necrosis at the site
of infection. The hypersensitive lesion can effectively
stop the spread of a virus or confine the bacterial or
fungal pathogen. In the latter two cases, the pathogen
then dies.
m is response is controlled in most cases by a single,
dominant gene in the host plant. One approach to study
of the mechanism controlling development of the hypersen-
sitive lesion would be to first isolate messenger RNA
from infected plants--those induced to give a hypersen-
sitive response and those with a suppressed hypersen-
sitive response. The mRNA from the suppressed plants
could be used to prepare complementary DNA. This
complementary DNA should recognize and hybridize with all
the mRNAs from induced plants, except for those involved
in the hypersensitive response. In principle, the
remaining free mRNAs could then be used to probe a gene
library of the hypersensitive plant for the gene that
they can hybridize with. This gene should be the one
responsible for inducing the hypersensitive lesion.
Gene Transfer
The ultimate goal of research discussed
in this section on genetics, gene products, and gene
structure is the routine transfer and expression of genes
for resistance in agriculturally useful plants. As noted
in the earlier chapter on genetic engineering, some bac-
terial and viral pathogens may be developed as suitable
carriers for the transfer of genes into host plants.
Current and prospective vectors take advantage of natu-
rally occurring, intimate associations between micro-
organisms and plants, both pathogenic and beneficial. An
appreciable effort is needed to identify and obtain suit-
able vectors in addition to the one successful vector,
the Agrobacterium Ti plasmid that can be used in some
dicotyledonous plants.
m e techniques necessary to manipulate vectors are
available and will likely be refined and improved within
the next few years. It is, unfortunately, the lack of
knowledge of the basic biology of plants and of the
function, transfer, and expression of genes that
restricts progress in this area.
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Molecular Basis of Cellular Drainage in Susceptible Bosts
Although it may appear that research on cellular dam-
age and disease symptoms is a subset of the research
discussed previously on resistance and susceptibility,
its intent is distinct, but of equal major importance.
Research emphasis in this area will yield insights into
the biochemical mechanisms that result in cellular dam-
age, or disease, following successful pathogenic inva-
sion. As yet there is no clear explanation of how major
symptoms, such as the yellowing and loss of chlorophyll
in chlorosis or the tumors, galls, and morphological
changes caused by cellular growth distortion, are induced
once a virulent pathogen becomes established in a tissue.
It may be possible to ameliorate symptoms or prevent crop
damage directly by treatment, if the biochemical details
are known. m e little-understood phenomenon of natural
tolerance to disease is evidence that such treatment
should be possible. Indeed, the study of natural toler-
ance may be a valuable guide for developing disease
protection traits for crop improvement.
Easily observable disease symptoms, such as chlorosis,
necrosis, and cellular growth distortions, can have a
number of diverse causes. Therefore, it is not possible
to make progress on such generalized disease symptoms
without some indication of the kinds of pathogens
involved. Some research approaches hold promise for
establishing general scientific principles of host-
pathogen interactions.
Mode Of Antinn Of Taxi no Research in the last decade
on purification and structural characterization has led
to an acceptance of the concept that toxins are the
potent chemical agents of virulence in many important
diseases caused by bacteria and fungi. Only a small
number of toxins have been chemically identified. Even
fewer have a postulated target or receptor site in the
host cell, as was described earlier for Helminthosporium
maydis. But even in these few cases, it is not known how
interference with the target site leads to cell damage.
Much additional research is needed on toxins--on their
genetics, such as chromosomal versus plasmid inheritance;
on their chemical structure; on the pathways of biosyn-
thesis in pathogens; and on their biochemical effects and
role in pathogenesis.
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Nucleic Acid Interactions It is clear that the mere
replication of a virus or viroid within a plant does not
determine whether that plant will be diseased. mere are
many examples of strains that produce a great deal of
virus, but with very little damage to the host. On the
other hand, some of the most serious plant diseases are
caused by viruses that replicate very sparingly.
Viruses, with their small genomes, have too little
genetic information to code for the variety of proteins
necessary to account for the almost infinite number of
symptom types. Thus, it seems likely that interactions
between the nucleic acid of the pathogen and that of the
host initiate the disease process. Viroids, which are
RNA molecules that do not code for a protein product, can
cause symptoms similar to those caused by viruses. This
lends support to the supposition that viruses as well as
viroids interact directly with the genome of the host
plant.
Complete nucleic acid sequences are now available for
several viroids; for satellite RNAs, which modify the
symptoms of their carrier viruses; and for a few plant
viruses. Complete complementary DNA clones have been
made for some of these RNA agents and have been shown to
be infectious. Because DNA is technically easier to
modify than RNA, such DNA clones provide the opportunity
to make site-specific modifications in the sequence of
the nucleic acid by inserting or deleting short stretches
of DNA. me effect of such changes on the agent's
ability to infect and on the symptoms produced can then
be determined.
Using current methods the nucleotide sequences respon-
sible for the disease syndrome should be identified.
Furthermore, these complementary DNA clones could also be
used in hybridization studies to locate regions in the
host genome where the host and the virus, satellite RNA,
or viroid sequences interact. As knowledge of the fine
structure of the host's genes increases, future studies
should enable researchers to determine the specific genes
and processes that are perturbed by the presence of the
pathogen.
If the DNA clones themselves are not infectious, the
cloned viral or viroid DNA can be transcribed back to RNA
using any of several in vitro systems. Thus, site-
specific modifications made in the DNA clone can be tran-
scribed into the RNA to test the effect of such changes
on infectivity and disease symptoms. In this manner,
critical regions of the genome could be identified,
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which would aid in the understanding of their functions
and possibly the functions of viral-coded proteins.
Bacterial Interactions Bacteria that cause diseases in
plants cause symptoms, at least in part, by the produc-
tion of various metabolites. Relatively few of these
substances have been identified. The metabolites in-
clude, but are not limited to, toxins, polysaccharides,
pec tic enzymes, and plant hormones. All the bacterial
toxins identified to date appear to be general toxins
affecting a wide spectrum of plants. Many of these
plants are not considered to be host species for the
bacterial pathogen producing the toxin.
Other bacterial metabolites appear to have specific
effects on host plant species.
charides, which are associated with wilting of plants,
can be released in amounts great enough to clog up
transport between plant cells, and may act by disrupting
plant cell membrane functions.
. , . _ . . .
~ , _ _ _,
Bacterial polysac-
Soft rots, for example J
are the result of bacterial enzymes that degrade the
cementing pectin layer between plant cells. m e pro-
duction of plant hormones by bacteria disrupts the
endogenous hormone balance in the host plant and can be
part of the mechanism leading to crown gall tumors and
other abnormal growths.
The molecular and genetic bases of the synthesis of
these pathogen metabolites and the basis of the symptoms
they cause in the host plant are largely unknown. There
is increasing knowledge, however, about the genetics of
some of the bacterial virulence factors that contribute
to the severity of a disease. For example, in crown
gall, which is caused by Agrobacterium, both bacterial
chromosomal and plasmid genes are known to be required
for pathogenicity. The molecular Genetics of crown nail
is the most thoroughly studied of any plant disease.
In the genetic analyses of virulence in bacteria, two
different approaches are currently being used. One is
the introduction of transposons into virulent strains of
bacteria to create avirulent mutants. The transposon is
used as a probe to locate and isolate the turned-off
virulence gene. DNA clones of virulence genes can be
used for an analysis of gene products. The second
approach is molecular and genetic analyses of known or
suspected determinants of pathogenicity, such as cell
surface components, hormones, toxins, and extracellular
enzymes. Both approaches hold promise for the elucida-
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tion of the biochemical steps in pathogenesis. There is
an essential need to have basic knowledge about the
structure, function, and regulation of virulence factors
in the pathogen to provide a basis for directed plant
breeding and to design effective inducers of plant
resistance.
Research Status
It is important to recognize that considerable exper-
tise and training in molecular biology are necessary for
many of the research approaches discussed in this section
of the report. Progress is facilitated by individuals
working together in groups. Interactions with
researchers in other laboratories are important sources
of intellectual stimulation as well as sources of tech-
nical expertise.
The tools of genetics and molecular biology offer some
new methods for understanding the highly specific inter-
actions between host and pathogen. Studies of the
molecular aspects of plant pathology must receive high
priority and emphasis within the ARS research programs on
plant diseases.
Currently the ARS research centers are undertaking
relatively little basic work in molecular plant path-
ology. The ARS does have a few strong research programs
in virology and in viroids, but very little work at the
molecular level is being conducted with bacteria or
fungi. A single laboratory, at Beltsville, is studying
plant mycoplasmas.
To strengthen programs in the molecular basis of plant
diseases, research investigations should emphasize:
The molecular bases of the factors that determine
whether a host-pathogen pair will result in a resistant
or a susceptible interaction.
· me basic concepts of the interaction between the
host and the invading pathogen that result in disease.
This should lead to novel methods of preventing damage
from disease, including natural plant tolerance.
o The transfer of resistance traits to normally
susceptible plants through the development and subsequent
exploitation of vector systems that allow for gene
transfer between plant species.
It is significant to note that very few laboratories
in the world have undertaken studies to understand
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The use and genetic manipulation of insect pathogenic
bacteria and viruses constitute a promising but compara-
tively underdeveloped approach to insect control. me
potential exists for genetically improving these organ-
isms to increase their pathogenicity, either by enhancing
existing pathogenic traits or by introducing desirable
pathogenic characteristics.
Basic knowledge about potentially useful pathogens
must be acquired. This includes identification of the
pathogen and characterization of the insect host. The
specificity between pathogen and host and the techniques
for production and storage of candidate pathogens must
also be studied. With this information the physiology,
biochemistry, and genetics of the host-pathogen inter-
action can then be investigated. More specific areas of
study include the molecular basis of processes such as
recognition, virulence, and toxicity and the mechanisms
regulating gene function during these interactions.
Progress in this line of research is apparent from the
work of many laboratories worldwide. Candidate micro-
organisms identified by this research include baculo-
viruses and Bacillus thuringiensis. With recent devel-
opments in insect cell culture, some of the fundamental
processes detailed here, in principle, can be directly
probed in vitro with any of these microorganisms.
Control of Nematodes Control of plant parasitic
nematodes has been largely accomplished through the use
of chemical nematocides, many of which have now been
shown to be harmful to the environment and have been
withdrawn from use. Biological control measures using
resistant plant varieties and trap crops have been
effective in some cases. A trap crop can stimulate the
hatching of nematode eggs but does not support nematode
growth, thus reducing nematode populations to harmless
levels.
More information is needed on the basic biology of
nematodes to provide directed approaches to their
control, using less toxic, target-specific substances.
This might include the use of the hatching stimulants
that are apparently produced by plants and trigger nema-
tode eggs to hatch. The growing nematodes then perish in
the absence of a suitable host plant. Studies of nema-
tode pheromones and hormones could lead to methods for
controlling reproduction or development.
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as
Plant Bealth Microorganisms In recent years some
information has been gathered on soil microorganisms,
specifically, certain bacteria, that can improve plant
vigor and contribute to increased yields. me mechanisms
by which such bacteria exert these effects are essen-
tially unknown, nor are their relationships to pathogens
or other microorganisms in the environment well under-
stood. Indeed, candidate organisms suited for particular
crops remain to be identified and characterized. Such
bacteria contribute a desirable and perhaps essential
microflora for optimal plant growth. While a range of
microflora is known to be essential for human health,
virtually nothing on a comparable basis is known for
plants.
Several mechanisms have been suggested that describe
the effects of soil microorganisms on plant health.
Beneficial microbes may produce antibiotics that inhibit
the growth of pathogens, or they may be involved in the
acquired resistance phenomenon. Recent evidence suggests
that some plant growth-promoting bacteria produce sidero-
phores, iron-chelating molecules, that restrict the
availability of this essential element to pathogens and
other members of the microflora.
Biological Degradation of Organic Pesticides Timely
and appropriate disposal of pesticide residues in water
and soils is an important and attainable goal in routine
agricultural production practices. The biological degra-
dation of pesticides is theoretically feasible. For
example, pseudomonads have been identified as being able
to degrade the herbicide 2,4-dichlorophenoxyacetic acid
(2,4-D) to innocuous compounds. Lack of knowledge of the
chemistry, the fate of breakdown products, and the
ecology of the organisms involved, however, is still a
constraint to their use.
Both waste disposal of agricultural by-products and
biomass reduction on an industrial scale are under inten-
sive investigation. The processes are not commercially
feasible as yet, however, because of low yields and
organism management problems. These problems can be
overcome using genetically engineered organisms, espe-
cially bacteria, that are currently more amenable to
manipulation than other microorganisms.
Research Status
The ARS laboratories are among those contributing to
progress in biological control of plant pathogens and
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insect pests. With increased emphasis, the ARS could be
at the forefront of this research. The potential return
for the ARS extends beyond the control of plant pathogens
and insect pests; it would involve the development of
general methodologies for gene transfer, cloning, and
gene expression using microbial and insect systems. Basic
research on the microflora of the rhizosphere is also an
area that ARS can strengthen.
There is enormous potential for the identification,
development, and application of microorganisms that can
degrade pesticide residues and other toxic wastes. The
ARS should expand its efforts--some of which are exemplary
--in these areas. It is high-risk, long-range research
and requires the multidisciplinary base that is already
in place in some locations.
Specifically, the ARS should focus research toward:
· Exploring and identifying microbial agents that
can control plant diseases and insect pests. Further,
the agency should seek conventional genetic or
recombinant-DNA technologies to make these agents more
effective;
· Generating more knowledge of the basic biology of
plant pathogenic nematodes to develop novel, nonpesticide
means of control by perturbing reproduction and devel-
opment; and
· Developing unique microorganisms that will pro-
mote plant health and others that can be used to detoxify
or destroy organic pesticide pollutants.
Molecular Basis of Pesticide Action
Pesticides are major tools in the production of food
and fiber and in the maintenance of high standards of
veterinary, human, and plant health. Better pesticides
are needed, relative to cost effectiveness, potency,
selectivity, persistence, environmental impact, and
safety for domestic animals, humans, and plants. Most of
the early pesticides were discovered in industrial pro-
grams involving the synthesis and screening of thousands
of synthetic chemicals for safe and effective molecules.
The emphasis in current discovery efforts favors research
on the natural chemicals produced by plants and micro-
organisms, such as occurred for the pyrethroids. Equally
important are investigations into the molecular basis of
pesticide action.
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Advances in bioregulation research provide new vistas
in seeking enzyme or receptor targets for pesticide
action. Increasing fundamental knowledge of the function
and regulation of communication systems within living
organisms focuses attention on new targets and greatly
facilitates the molecular design of optimal compounds for
pest control. Greater diversity is needed in the targets
for future pesticides, such as insecticides, herbicides,
nematocides, and fungicides to avoid or minimize the
impact of pesticide resistance and toxicity against non-
target species. Susceptible and tolerant species often
differ only in the sensitivity of their pesticide recep-
tor site or their facility for detoxifying the pesticide.
A clear definition of the mechanisms involved will
provide the background for the next generation of
improved pesticides. New pesticides, in turn, provide
unique probes to explore cellular entities such as
enzymes, receptors, and membranes.
me molecular basis for metabolic activation and detox-
ification must be defined. Using this background know-
ledge genetic engineering can provide opportunities for
modifying receptor sites and detoxification mechanisms
for improved animal and crop safety.
Research Status
Research on the molecular basis of pesticide action is
carried out in many laboratories within industry, univer-
sities, and the ARS. Industrial labs tend to focus on
the modes of action of their proprietary compounds.
Universities more often use pesticides as probes for
physiological and pharmacological investigations. me
ARS has placed considerable emphasis on the mechanism of
pesticide action. The laboratory defining a new target
often reaps the benefit of finding alternative agents
working at the same site or in the same way.
Research on pesticide mode of action requires the
creative teamwork of biochemists, chemists, and geneti-
cists with adequate instrumentation and the appropriate
environment to stimulate communication. This multi-
disciplinary approach and the requisite personnel are now
in place in several ARS laboratories. The ARS should
increase its emphasis on the molecular basis of pesticide
action, using the available expertise in microbial,
plant, and insect physiology, biochemistry, and natural
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products chemistry. Success in this program will serve
as the basis for improving animal health and for reducing
crop losses during production and storage.
More specifically, the ARS should emphasize:
Definition of the molecular basis for metabolic
activation and detoxification of pesticides;
Study of new targets for selective pesticide
action;
Identification of new natural chemicals
important in regulating pest populations;
Investigation of the basic molecular biology of
vectors for gene transfer and elucidation of
gene regulation in insects; and
Continued research on both insect genetics and
on natural products chemistry.
Insect Neurobiology and the Regulation of
Development and Reproduction
The functional responsiveness of an insect is depen-
dent on rapid chemical communications among its own cells
and between the individual and other insects. Intercel-
lular communication is mediated primarily by the nervous
system, through substances such as neurotransmitters,
neurohormones, and neuromodulators as well as by the
endocrine system, through hormones. me endocrine system
is closely coupled to the functioning of the nervous
system. Communication between individuals is achieved
through volatile chemicals called pheromones. Their
production and action is mediated by the nervous system.
Insect Neurobiology
The function of the nervous system makes it a logical
focus for investigations of alternative means of insect
control that could potentially have considerable selec-
tivity. Before investigations can be initiated, however,
basic information about the function of the insect
nervous system must be obtained, specifically, infor-
mation about nervous processes involving chemical
communication. This approach is the only potentially
successful avenue to the solution of applied research
problems. For this reason a research emphasis in
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fundamental insect neurobiology should be developed by
the ARE.
Insect neurobiology is now experiencing a period of
exponential growth. Despite the fact that the insect
nervous system has been used for many years as a model
for studying certain neurophysiological processes, basic
research using modern techniques has only recently begun
on the neurochemistry, neuroendocrinology, neurogenetics,
and neuropharmacology of the insect nervous system.
For example, the number of identified insect peptides
with neurohormonal activity is fewer than 20. Only 4 of
these insect neurohormones have been purified and
sequenced. These include the neurotransmitter/neuro-
modulator proctolin, the two adipokinetic hormones, and
cardiac accelerator peptides. Proctolin is important in
the stimulation of muscle contraction and is co-released
with other neurotransmitters. me adipokinetic hormones
mobilize lipid for its metabolism by muscle in insect
flight, and the cardiac accelerator peptides control the
heartbeat of the insect. It now appears that the struc-
tures of the prothoracicotropic hormones, the primary
effecters of insect metamorphosis and the first hormone
of neural origin described for any animal (1917), are
finally being resolved. In addition, a new brain peptide
that regulates the production of pheromones has been
described and promises to introduce a renaissance in
pheromone research.
Study of these and of yet-undiscovered hormones will
aid in an understanding of the physiology of the insect,
its growth and development. Such studies will also
define the mechanisms by which the central nervous system
integrates and regulates these processes. This under-
standing may allow scientists eventually to selectively
manipulate the neuroendocrine system, and thus control
insects by altering their ability to fly, curtailing
metamorphosis, or disrupting sexual recognition. The
study of neurohormones may not provide an immediate
answer to insect control. The resulting knowledge, how-
ever, will provide scientists with the sound foundation
necessary to propose and pursue new directed and applied
research on the neural regulation of insect growth and
development.
The top scientific priority for neurobiological
research on insects is the elucidation of the mechanisms
by which chemical communication directs and coordinates
the growth, development, homeostasis, and reproduction of
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insects. me basic information still lacking includes
the identification of neural regulators and an eluci-
dation of their chemistry, synthesis, secretion, and
metabolism.
Other opportunities for manipulation of insect pests
include the neurohormones bursicon, diuretic hormone, and
egg development neurotropic hormone. Bursicon causes the
insect skeleton to harden. Inhibition of the secretion
of this hormone would cause death. Manipulation of the
diuretic hormone, which regulates water and salt balance,
might also result in death, through ionic imbalance and
dehydration. Secretion of egg development neurotropic
hormone from the brain of the female mosquito is stim-
ulated following a blood meal. The hormone indirectly
causes the ovary to mature the eggs. Manipulation of
this reproductive hormone would prevent the development
of generations of mosquitoes.
These hormones are examples of the potential in this
field. To realize this potential the hormones must be
studied extensively at the chemical, molecular, and
physiological level.
At this point a major reesarch program encompassing
the physiology, biochemistry, and molecular biology of
these regulators can be initiated. Research should in-
clude the study of mechanisms of communication within the
nervous system, between organs and organ systems, and
between individuals of the same species. Studies of
interorganismal communication should emphasize the
neuroendocrine and neural bases of this process and
relate this communication to behavioral patterns in
nature.
Knowledge gained from such a fundamental research
program in insect neurobiology could be used in con-
junction with genetic engineering methodologies to
investigate the basic molecular biology of vectors for
gene transfer and to elucidate gene regulation in
insects. These new technologies could also aid in
mapping the insect genome, particularly the genes for
regulatory peptides.
Peptides offer researchers an extremely important
direct line of study; they probably are all products of
single genes. An understanding of these gene products or
polyprotein precursors and their posttranslational pro-
cessing to a bioactive peptide is essential for the
potential control of insects. (Posttranslational pro-
cessing, which follows the translation of RNA, is proving
to be a fundamental mechanism that determines the protein
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nature of the neurosecretion from a given cell.) A dis-
ruption of the synthesis or processing of neurohormones
would be lethal.
The long-term goal of this research is modification of
the normal function of the insect nervous system to
affect viability. A research program on insect pathogens
as vectors for gene transfer would clearly be important
in achieving this objective.
Endocr ine Regulation of Metamorphosis, Diapause, and
Reproduction
The postembryonic development of the insect involves a
series of dramatic physiological and biochemical trans-
formations that culminates in its emergence from a pupa
as an adult form with its own unique function. It is
generally accepted that these transformations and their
associated metabolic processes all are directly or
indirectly under endocrine control, including production
of hormones by neural tissue. The full extent of the
role of the endocrine system is not completely known,
mainly because of a lack of knowledge of the hormones
involved, the molecular basis of the developmental and
reproductive processes these hormones control, and their
mechanisms of action. m e progress made in this field in
recent years has largely been at a descriptive level.
Thus, basic research is needed to identify and chemically
characterize insect hormones and to define at the molec-
ular level both their physiological function and their
mechanism of action.
Although some insect hormones, such as the sesquiter-
penoid juvenile hormones and the ecdysteroids, have been
intensively investigated, the extent of their involvement
in regulating insect development and reproduction is only
now being realized. They are known to exist as struc-
tural and functional families of molecules, each acting
at a specific time during the life cycle of the insect.
The multiple functions of these hormones provide multiple
avenues for pursuing control of the insect. Substantial
stantially more research is needed, both in the above-
cited areas as well as on the mechanisms of their inter-
action at the level of the target gland and interendo-
crine feedback control. Research studies must be
designed to show how these hormones regulate one
another's synthesis and secretion to drive development
and growth.
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A virtually unknown family of insect regulators that
control metamorphosis, diapause, and reproduction is the
peptides. Only a few have thus far been identified, and
as has proved to be the case with vertebrates, there are
numerous peptide hormones involved in the control of
embryogenesis, postembryonic development, reproduction,
and homeostasis. These peptides need to be charac-
terized, their physiological functions defined, and
mechanisms of action elucidated.
The regulation of the synthesis, secretion, and
metabolism of these insect hormones, whether peptide,
steroid, or other chemical structure, is another rela-
tively unexplored research area of considerable signif-
icance and potential application to the control of
insects. The secretion of these hormones has consis-
tently been shown to be precisely regulated, frequently
in response to discrete environmental cues such as photo-
period, temperature, and stress. The mechanisms by which
these cues are transduced by the nervous system to elicit
an endocrine response are important areas for basic
research in insect neurobiology.
Knowledge of the regulation of insect development and
reproduction is applicable to the manipulation of these
systems for improved pest control. Some natural and
synthetic chemicals, including insecticidal compounds,
alter growth and development by inhibiting the biosyn-
thesis or action of juvenile hormones or ecdysteroids and
by governing the initiation and termination of diapause.
Certain antibiotics and the highly insecticidal benzoyl-
phenyl ureas interrupt chitin synthesis necessary for the
formation of the insect cuticle or skeleton. Studies on
insect genetics indicate the possibility of breeding
sterile hybrids for use in pest control. Bacteria and
other microorganisms producing insecticidal materials and
the plant itself may also be modified by selection and
genetic engineering to increase the impact of natural
toxicants or feeding deterrents in host-insect pest
interactions. Further development of insect cell
cultures and vectors for gene transfer in insects may
permit the introduction of deleterious effects into pest
populations.
The benefit from research in insect neurobiology is
not the potential control of insect pests alone.
Although the insect is a relatively simple system
structurally, it is functionally complex, much like that
of vertebrates. An understanding of the insect endocrine
system will lead to a further understanding of similar
processes in all eukaryotic organisms.
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Research Status
The ARS is recognized worldwide for developing the
sterile insect release method of control and for investi-
gations on insect genetics and ecdysteroids. The agency
also is internationally recognized for natural products
research, particularly pheromone chemistry, and the
application to insect development and reproduction. This
type of interdisciplinary research requires a coordinated
team of entomologists, physiologists, biochemists, and
chemists.
There are a number of ARS laboratories currently
conducting excellent research on the physiological and
chemical aspects of endocrine control of insect devel-
opment and reproduction. By bolstering these existing
programs with the appropriate additions of scientists
skilled in protein chemistry, basic biochemistry, and the
study of nuclear and membrane proteins as receptors, the
ARS should be able to make substantial contributions to
this research area.
Although the ARS is becoming increasingly more
involved in fundamental insect neurobiological research,
this program is not developing in a focused manner.
While most of the research skills necessary for a major
program in insect neurobiology--chemistry, neuro-
physiology, behavior, biophysics, and physiology--are
already in place within the ARS, additional expertise in
neurochemistry, peptide chemistry, and biochemistry
(mechanistic aspects or chemical regulation), and immu-
nology must be added. Generally, adequate instrumen-
tation for this research exists within the ARS.
Analytical facilities are needed, however, for peptide
and neurotransmitter structural identification.
Of the few laboratories worldwide engaged in insect
neurobiological research, a number are emerging as
centers of excellence. me comparative paucity of such
centers, however, means that relatively few neuro-
biological systems are currently being explored. Thus,
the scientific opportunities in this field are enormous.
Unfortunately, the lack of basic information has created
a situation wherein the most important areas of research
are high risk and will require considerable effort and
resources. Such high-risk research is well suited for
government-supported organizations like the ARS.
To date, a multidisciplinary program in insect neuro-
biology does not exist. me ARS has an opportunity to
establish the first program of this kind. The success of
such a program greatly depends upon the centralization of
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research at a single site, preferably near a university
or another research institute that has a strong program
in neurobiology.
ARS research should specifically focus on the
following:
· Chemistry of the brain factors that control
pheromone production and release, and their mechanisms of
action;
· Neural regulation of the synthesis, processing,
and secretion of cerebral pheromonotropic peptides;
· The endocrine basis of insect reproduction, in
particular, identification of the cerebral neuropeptides
involved and their target glands, and identification of
the mechanisms regulating these glands;
· Mechanisms that regulate the synthesis of
ecdysteroids and juvenile hormones, and the biosynthetic
pathways of these two hormome families; and
· Interhormonal endocrine feedback; regulation of
insect growth, development, and reproduction; and the
roles and molecular mechanisms of the principal devel-
opmental hormones in regulating one another's synthesis.
Representative terms from entire chapter:
molecular basis
