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BIOLOGICAL PEST CONTROL USING PATHOGENS
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-------------------------------------------------------------------------------------------------- Introduction Extensive discussions of the use of pathogens in
biological control in tropical climates was given by Federici (1995, 1999)
and Weiser (1984). Insects are susceptible to a variety of diseases caused by
pathogens, many of which are acute and fatal, and can be important short-term
regulators of insect populations. Therefore, insect pathogens are the subject
of a considerable research effort aimed at developing the most effective
pathogens as biological control agents. There has been interest shown for
more than 100 years in controlling insects with pathogens, but their further
development is now greater than ever due to the continually increasing need
to replace chemical insecticides.
However, as of 2001, there are an ever increasing number of scientific
papers dealing with the development of resistance to the various organisms,
not unlike that witnessed earlier with flurocarbon insecticides. In modern agriculture the mass production of microbial
insecticides was more common than that for parasitoids and predators. This is
because the production of microbes is easier than rearing predators and
parasitoids. The latter are stored, packed and transported with a great risk
of loss and also the methods for the technology available for their
application is underdeveloped. Microbes are easily stored, packed and the
method of application is very similar to that of chemical pesticides. Also
the evaluation of treatment effects is performed in a way similar to
insecticides, except that the period between application and effective
mortality is usually longer. Other key references are Burges (1981), Burges
& Hussey (1971), DeBach (1964), Franz (1980), Huffaker & Messenger
(1976), Kurstak (1982), Poinar & Thomas (1978) and Weiser (1966, 1972,
1977). In the tropics, environmental conditions of high RH,
extreme temperatures and intensive insolation together with alternating heavy
rains are important factors. Year-round temperatures which remain in the
range suitable for the feeding and activity of insects provide suitable
conditions for ingestion of adequate doses of oral materials, such as
bacteria. On the other hand, high temperatures reduce viability of such pathogenic
forms as fungal conidia, viral polyhedral inclusions and protozoan spores.
Thee conditions are critical not only during application but also during
storage and transportation of the materials. Infectivity is dependent on
different strains' response to temperature, the range of a material's
activity. High temperatures accelerate the development of target insects and
also that of infections, especially those caused by bacteria and viruses. In
principle there is no difference in infectivity to different pathogens in the
tropics and in mild climates, all pathogens isolated in mild regions can be
used in the tropics and vice versa (Weiser 1984). Changes in RH also effect the efficiency of application of
microbial insecticides. During storage high RH causes germination of some
spores and ultimately their destruction. Products must be well formulated to
guarantee a minimum of RH in the bulk material. This is the main reason for
the increased demand for local production, which tends to avoid damage in storage
and transportation. Not only the high RH but also extremes between dry
periods before the rains come in savanna regions. During this dry period most
entomophilic nematodes die or are so much reduced in distribution that their
spread must begin again during the rainy season. Heavy rains may wash down
any residue on plants, necessitating reapplication. Another factor causing heavy losses on viability is solar
radiation. In the tropics more than anywhere else it is important to direct
an essential part of a particular microbial application to the lower parts of
plants and under the leaves. Used in combination with treatments early in the
morning or late in the afternoon, this may preclude the need for repeated
treatments. History.--Interest in using pathogens to control insects dates
back over 100 years. Steinhaus (1975), Bassi (1835), Auduoin (1837) and
LeConte (1874) and Metchnikoff (1879) suggested that pathogens might prove
effective agents for controlling crop pests. But none of the early
suggestions led to the successful deployment of pathogens. Renewed interest
in the 20th Century followed the successful use of other natural enemies such
as predaceous beetles and parasitic Hymenoptera. Control of the cottony
cushion scale by natural enemies in the United States led to the
establishment of permanent research programs by governmental agencies in the
U.S. and several other countries aimed at using natural enemies, including
pathogens, as pest control agents (Paillot 1933, Steinhaus 1975). Two early
successes emerged from these programs, the development of Bacillus popilliae Dutky as a control agent for the Japanese
beetle, Popillia japonica Newman, in the U,.S.
(White & Dutky 1942), and use of the nuclear polyhidrosis virus (NPV) of
the European spruce sawfly, Gilpinia
hercyniae (Hartig), as a
classical biological control agent in Canada (Balch & Bird 1944). Shortly after World War II, E. A. Steinhaus was hired by
the University of California (Steinhaus 1963). Trained as a bacteriologist,
Steinhaus was well aware of the success attained with B. popilliae
and the G. hercyniae NPV, and he began an
concentrated effort aimed at using insect pathogens, particularly bacteria
and viruses as control agents. Although other researchers were influential in
the development of insect pathology and microbial control (Cameron 1973), the
modern era of these two closely related disciplines was due largely to the
leadership of E. A. Steinhaus. He reemphasized the use of Bacillus thuringiensis Berliner as a biocontrol agent, and his
studies and those of Hannay (1953) and Angus (1954) were important in the
commercialization and its successful use as a microbial insecticide. Although
earlier studying the NPV of the alfalfa caterpillar, Colias eurytheme
Boisduval, Steinhaus focused attention on the potential of viruses as
microbial insecticides. His studies of viral and bacterial diseases of
insects, and other pioneering efforts in the field of insect pathology,
invigorated studies of the fungi and protozoa as control agents, and were
important to the development of the international fields of insect pathology
and microbial control. Although there is an extensive literature on insect
pathogens and microbial control, only one pathogen, B. thuringiensis
is in routine use in control in industrialized countries, where it has been
variously successful. It has been noted by many specialists that this
pathogen is the one in which current interest regarding further development
and use is the highest. He considered that it is worthwhile to consider why
this is so because such an assessment identifies some of the key features
required for a pathogen to be successful as a biological control agent. To do
this requires a few definitions regarding the different ways in which
pathogens are used or considered for use in insect control programs, and of
the performance expectations by which the potential of insect pathogens and
their degree of success are evaluated. The guiding principle is one of economics; the pathogens
that will be developed and used are those that are the most cost-effective,
either in the short or long term. Classical biological control is the most
cost effective, but the best example in insects is the use of an NPV to
control the European spruce sawfly in Canada. Pathogens may also be used in
seasonal introductions. The protozoan Nosema
locustae Canning has been
used in this way to reduce grasshopper populations over a period of several
weeks. The common strategy is to use pathogens as microbial insecticides, and
because this strategy has proven quite successful with bacteria and viruses,
it will probably see even greater future use. Depending on the target pest,
applications may often be fewer than those required with a chemical
insecticide because the pathogens are quite specific and typically do not
kill predatory and parasitic insects. Also, the reproduction of the pathogen
in the target insect, as is true of viruses, adds to the amount of the
pathogen in the treated environment, and this can extend control and cost
effectiveness. A single application can yield effective seasonal control when
the pests have only one or a few generations. Performance expectations are important to consider when
using pathogens. A successful pathogen reduces the pest to below an economic
or transmission threshold routinely and reliably at a cost that is economical
in proportion to the value of the crop or impact of the disease. Under most
circumstances pathogens are evaluated on the basis of how they compare with
chemical insecticides, leading to a microbial control agent paradox. Being
generally agreed that the two properties of many chemical insecticides that
were originally considered to be their best attributes are a broad spectrum
of activity and a significant residual activity. Such properties are often
responsible for the destruction of natural enemy populations and the
production of insecticide resistance. But most insect pathogens have a narrow
spectrum of activity and relatively poor residual activity. Although such are
now considered desirable properties, until recently they have discouraged
interest by industry in the development of many potentially useful pathogens
because of the relatively high costs of development and registration in
comparison to the likely return on investment. Therefore, when considering the economics of pesticide
development, it is apparent why B.
thuringiensis has been the
most widely developed and used insect pathogen. It compares favorably with
chemical insecticides in many crop and forest systems where lepidopterous insects
are the key or major pests. It is relatively inexpensive, easy to produce and
formulate for use and it acts rapidly. It has a rather narrow spectrum of
activity and possesses a low residual activity. Nevertheless, the range of
insects it controls continues to provide a market large enough to justify
commercial development. As discussed in the section of medically important
arthropods, this bacterium most likely has been responsible for halting
development of many other biological control possibilities, even though the
latter might offer longer range and more thorough success. Microbial pathogens which are used in pest control stem
from four major groups of insect pathogens: viruses, bacteria, fungi and
nematodes. Although nematodes sometimes are considered as parasitoids, the
fact that they include in their activity the action of symbiotic bacteria,
distinguishes them as a special group. Presently protozoa are not utilized on
a large scale in field applications, but they may be useful in some integrated
control situations . Cytoplasmic polyhedrosis
viruses Use
of Viruses as Insect Control Agents Many viruses affect insects which are potentially useful in biological
control. They may be divided into about 10 different groups according to
their localization and their appearance under the optical microscope. Most
are very host specific. Their formation and reproduction is bound on the
genetic substance of the nuclei (DNA viruses) and sometimes connected with
RNA replication (cytoplasmic viruses). In all viruses the infection is
also egg transmitted and in many cases direct feeding of infectious materials
do not immediately result in apparent lethal infections (Weiser 1984).
Inapparent infections may remain in a population and then may appear in the
next generation if suitable stresses are applied. Among the stresses
initiating outbreaks of latent infections are crowding, cold storage, and
secondary infections with other pathogens. Most common are the nuclear polyhedrosis viruses
(NPV) which appear as visible refringent irregular proteinic inclusions in
the nuclei of the fat body, the hypoderm and other tissues. The nuclei are
hypertrophic, which finally burst and the cell is destroyed. The host dies
after a short period of time, and remains hanging on the last pair of larval
legs. Its interior autolyses into a grey liquid with masses of refringent
polyhedra, which do not stain with Giemsa. The polyhedra contain many virus
rods. In the gut of the newly infected host the virus enters host cells and
undergoes new development. Several NPVs are produced as formulated microbial
insecticides. Most widely used is ELCAR against Heliothis, VIRIN NS against the gypsy moth, and VIRIN KS
against the cabbage worm. There is also NPV available for control of
caterpillars of Trichoplusia
ni, the tussock moth, of Spodoptera littoralis and several other pests. It is essential to
apply the virus against early stage larvae. In many cases the virus is only
collected from outbreak areas and stored at 4°C for the next season. One Trichoplusia
ni caterpillar is sufficient
to treat one acre of infested alfalfa. In sawflies the polyhedrosis is localized in the nuclei of
the midgut. Several NPV's were used for control of important pests such as Neodiprion , Gilpinia and Trichiocampus. In these cases
material was produced on collected caterpillars, stored and applied during
the next season. Granuloses are another type of DNA virus with rodshaped virions,
each closed in a proteinic capsule which is visible only in dark field. Among
the broadly used viruses of this group are the granulosis of the fall webworm
and codling moth. Viral rods without a proteinic capsule are those of the baculovirus
such as infects the rhinoceros beetle, Oryctes
rhinoceros. This pest of
coconut palms in southeast Asia and the Pacific, has an infection attacking
mainly the midgut and causing heavy mortality. The virus is produced in
rearing stations on collected grubs of the beetle and used for spraying the
sprouts of palms, or late instar grubs are infected and eclosed adults
released so that they carry the infection into habitats of the beetle via the
feces. In many areas the infective level of the virus in the field is much
reduced and a recolonization of the infection with infected grubs is relied
upon. Less commonly used in field applications are the cytoplasmic
polyhedrosis viruses, which are common in the cytoplasm of midgut cells
of many caterpillars. They are usually not used alone but in conjunction with
other pathogens, such as microsporidia. A specific virus group are the pox
viruses which are localized in the cytoplasm of fat body cells, with large
oval polyhedra with cushion-shaped virus particles. They are not very
infectious and are not tested as microbial pathogens. Some non occluded
viruses are important factors in reduction of very specific pests, as is the densonucleosis
virus of the greater wax moth. The latter is very infectious, but easily
inactivated due to lack of a protective coating. The group of iridoviruses is
a natural control factor of mosquitoes and blackflies, of grubs of some
beetles (e.g., Sericesthis),
craneflies and others. Viruses may be produced locally by collecting infected
individuals in the field and storing them with care to suppress bacterial
growth. Dry material remains viable for at least one year. A second step is
the controlled production on caterpillars collected in nature or reared from
eggs and infected in rearing cages in the laboratory. A third sophisticated
step is the mass production on artificial media. This type of rearing avoids
the usual contamination with other pathogens such as cytoplasmic
polyhedrosis, bacteria or protozoa (Weiser 1984). Viruses are obligate intracellular parasites, and as such
must be grown in living hosts. Insect viruses must be grown either in insects
or in cultured insect cells, as there is no method known for growing viruses
on artificial media. Most viruses that occur in insects belong to one of
seven major taxonomic families, but generally they are divided into two broad
non-taxonomic categories: occluded viruses and non-occluded
viruses. Occluded viruses are occluded with a protein matrix after formation
in infected cells, forming paracrystalline bodies that are referred to as
either inclusion or occlusion bodies. Non-occluded viruses occur freely or
occasionally form paracrystalline arrays of virions that are also called
inclusion bodies. However, the latter have no occlusion body protein
interspersed among the virions. In pest control, the biological properties of the viruses
are more relevant than the physical and biochemical properties. The most
important biological properties for the four most common virus types are as
follows: Iridoviruses are non-occluded DNA viruses that replicate in the
cytoplasm of a wide range of tissues in infected hosts, causing disease that
is usually fatal. Virions can form paracrystalline arrays in infected
tissues, imparting an iridescent aspect to infected hosts, from which the
name of this virus group is derived. Over 30 types are known, and all have
proven extremely difficult to transmit per
os. Host range of each type
is quite narrow based on natural occurrence in host field populations.
Prevalence and mortality rates in natural populations of host insects is
typically <1% (Kelly & Robertson 1973). Cytoplasmic
polyhedrosis viruses are occluded
RNA viruses which replicate and form large (ca. 0.5-2 milimicrons) polyhedral
to spherical occlusion bodies in the cytoplasm of midgut epithelial cells
causing a chronic disease. Infection in early instars retards growth and
development, extending the larval phase by several weeks, which in many cases
is fatal. This is a relatively common type of virus found among lepidopterous
insects, and dipterous insects of the suborder Nematocera (mosquitoes,
blackflies, chironomids). Detailed studies on isolates shown them to be easy
to transmit per os to the original host and
other species of the same order, and therefore the host range of this virus
type is probably the broadest among the insect viruses (Katagiri 1981). Entomopoxviruses are occluded DNA viruses which replicate in the cytoplasm
of a wide range of tissues in most hosts, causing an acute fatal disease.
Occlusion bodies, depending on the isolate, vary from being ovoidal to
spindle-shaped and generally occlude 100 or more virions. They are most
common from coleopterous insects, from which there are over 20 isolates, but
also known from Lepidoptera, Diptera and Orthoptera. This virus type is
easily transmitted per os, though the experimental
host range of individual isolates seems relatively narrow, generally being
restricted to closely related species (Granados 1981, Arif 1984). Occluded
baculoviruses consist of two
types: the granulosis viruses (GVs) and the nuclear polyhidrosis viruses
(NPVs). Both types are highly infectious per
os, and in some insects
these viruses cause widespread epizootics that can result in significant
declines of larval populations. The NPVs occur in a wide range of insect orders as well as
from Crustacea, but they are most common in Lepidoptera (>1,000 isolates).
Many occur as distinct viral species. NPVs are easily transmitted per os and replicate in the nuclei of cells, generally causing
an acute fatal disease. The occlusion bodies are referred to as polyhedra
because of their typical shape. They are large (ca. 0.5-2 milimicrons), and
form in the nuclei, where each occludes as many as several hundred virions.
The NPVs of Lepidoptera infect a range of host tissues, but those of other
orders are typically restricted to the midgut epithelium. Some NPVs have a
narrow host range, and may only replicate efficiently in a single species,
while others such as the AcNPV, e.g., of the alfalfa looper, Autographa californica (Speyer), have a relatively broad host range
and are capable of infecting species from different genera (Granados &
Federici 1986, Blissard & Rohrmann 1990). The GVs (ca. 200 isolates) are closely related to the NPVs
but differ from the latter in several important ways. GVs are only known from
Lepidoptera. Like NPVs, they initially replicate in cell nuclei, but
replication involves early lysis of the nucleus, which in the NPVs only
occurs after most polyhedra have formed. After lyses, GV replication
continues throughout the cell, which consists of a mixture of cytoplasm and
nucleoplasm. When completely assembled, the virions are occluded individually
in small (200 x 600 milimicron) occlusion bodies called granules. Many
GVs primarily infect the fat body, while others have a broader tissue tropism
and replicate throughout the epidermis, tracheal matrix and fat body. One GV
of the grapeleaf skeletonizer, Harrisina
brillians Barnes &
McDunnough, only replicates in the midgut epithelium (Federici & Stern
1990, Tweeten et al. 1981). Use of Viruses as Insect Control Agents The best example of the use of a virus as an insect control
agent is the NPV of the European spruce sawfly, G. hercyniae,
as a classical biological control (Balch & Bird 1944, Cunningham &
Entwistle 1981). The European spruce sawfly was introduced into eastern
Canada from northern Europe around the turn of the century and was a severe
forest pest by the 1930's. Hymenopterous parasitoids were introduced from
Europe in the mid-1930's as part of a biological control effort, and carried
the NPV, which was first detected in 1936. Natural epizootics caused by the virus
began in 1938, by which time the sawfly had spread to >31,000 km2.
Sawfly populations were reduced to below economic thresholds by 1943. More
than 90% of the control is attributed to the NPV. Although viruses, particularly NPVs, are often associated with
rapid declines in the populations of important lepidopterous and
hymenopterous pests, the G. hercyniae NPV is the only true
example of a virus that has proven effective as a classical biological
control agent. Another baculovirus, the non-occluded baculovirus of the palm
rhinoceros beetle, Oryctes rhinoceros (L.) has been a
partial biological control success as introduced into populations it can
yield control for several years, but usually dissipates and must be reapplied
against the pest population (Bedford 1981). Thus, the control potential of
most viruses is best evaluated by assessing their utility as microbial
insecticides. From this position, the iridoviruses are essentially useless
because of their poor infectivity per
os. Cytoplasmic polyhidrosis
viruses are not appreciably better because, although highly infectious per os, the disease they cause is chronic (Aruga & Tanada
1971, Payne 1981). CPVs have, nevertheless, been used in some situations,
such as against the pine caterpillar, Dendrolimus
spectabilis in Japan
(Katagiri 1981). Entomopoxviruses have not yet been developed as control
agents for any insect (Arif 1984, Granados 1981). NPV's as Conventional Microbial
Insecticides.--Because NPVs
are common in and easily isolated from pest populations, production in their
hosts is relatively inexpensive, and the technology for formulation and
application is simple and adaptable to standard pesticide application
methods. Most NPVs, however, are narrow in their host range, infecting only a
few closely related species. Several can be grown in vitro
in small volumes, but no fermentation technology exists for their mass
production commercially. Despite such drawbacks, several NPVs have been
registered as microbial insecticides, though few are marketed. There is a
renewed interest in developing NPVs as insecticides due to the adverse
effects of chemical insecticides and their increasing costs and because
recombinant DNA technology offers potential for improving the efficacy of
these viruses. Production and Formulation.--Viruses are mass produced in larval hosts grown on
artificial diets or natural host plants. Larvae are infected per os at an advanced stage of development, and reared either
in groups or individually for species which are cannibalistic. After virus
ingestion, the occlusion bodies dissolve in the alkaline midgut, releasing
virions. In Lepidoptera, the virus first invades midgut epithelial cells
where during the firs 24 hrs of infection it undergoes an initial colonizing
phase of replication in the nuclei of these cells. No occlusion bodies are
produced in these nuclei, but rather the progeny virions migrate through the
basement membrane, and invade and colonize almost all other tissues. In these
a cycle of replication occurs during which the virions are occluded in
polyhedra. Maximum production of polyhedra occurs in tissues which are the
most nutrient rich and metabolically active such as the fat body, epidermis
and tracheal matrix. This definitive phase of viral disease occurs over a
period of 5-10 days, and represents several cycles of replication as the
virus spreads throughout the tissues and invades most host cells. The actual
length of the disease depends on several factors including the host and viral
species, larval instar at the time of infection, amount of inoculum and
temperature. Near the end of the disease, after most polyhedra have formed,
the nuclei lyse. As more nuclei lyse, the larva eventually dies after which
the body liquifies, releasing billions of polyhedra. In commercial production
larvae may be harvested prior to liquefication to keep bacteria, which
quickly colonize dead larvae, at a lower level in the final product.
Antibiotics also can be added to the diet to combat bacteria. After the
larval production phase is complete, the larvae are collected and formulated.
Formulation varies considerably, and depends on how the virus will be used
(Ignoffo 1973, Shapiro 1986). The production of lepidopteran GVs and sawfly NPVs is
similar to that described lepidopteran NPVs (Cunningham & Entwistle 1981,
Shapiro 1986). However, the sawfly NPVs differ from the lepidopteran NPVs in
that the former only replicate and form polyhedra in midgut epithelial cells.
Polyhedral yields are thus lower than those obtained with lepidopteran NPVs. NPV's and Economics / Efficacy.--The extent to which conventional NPVs can be used as
microbial insecticides depends on several factors which include the relative
importance of the target pest in the pest complex attacking a crop, the amount
of virus that must be used to control the pest in both the short term and
long term, the value of the crop, and the cost and availability of
alternative control measures. NPVs are suited for use where a single
lepidopteran species is the major pest for most of the growing season on a
crop with a high cash value where other available pest controls are not cost
effective. Examples are NPVs that are effective against insecticide-resistant
species of Heliothis and Spodoptera on crops such as
corn, sorghum and cotton, and more importantly on tomatoes, strawberries and
floriculture. The cost-effectiveness of these viruses is determined by the
amount of virus that must be applied and the frequency of application
necessary to maintain the pest below the economic threshold. This will, of
course, vary with the kinds of virus, pest, crop and in different parts of
the world. The mount of virus required is assessed in terms of larval
equivalents (LEs) necessary to achieve effective control (Ignoffo 1973). The
number of LEs required to obtain effective control is a critical component in
the determination of cost-effectiveness. This can range from 150 LEs per ha.
per treatment using H. zea NPV to control Heliothis on cotton to 500 LEs
for the S. exigua NPV on lettuce and
chrysanthemums. Also, the number of LEs required to control a specific pest
can vary with the crop due to differences in plant phenology and chemistry.
For example, whereas 500 LEs/ha. may be required to control S. exigua on lettuce, the value may be as high as 1,000 LEs
on alfalfa or as low as 100 LEs on strawberries. This kind of economic
evaluation is essential for determining whether a specific NPV merits
commercial development as well as use against a specific insect on a crop.
Additional examples may be found in Fuxa (1990), Shapiro (1986, 1992), Payne
(1982, 1988) and Pinnock (1975). Limitations.--viruses are not extensively used in industrialized
countries because chemical pesticides are readily available and effective,
although this may change with increasing residue and resistance problems.
Nevertheless, viruses in comparison to chemicals are relatively slow to kill,
possess a narrow host spectrum of activity, have little residual activity and
lack suitable cost-effective in
vitro production. But these
limitations have not inhibited the development and use of viruses in some
developing countries where NPVs and even a few GVs are used, especially on
field and vegetable crops. Included are India, China and Latin America,
Africa and southeast Asia (McKinley et al. 1989, Moscardi 1989, 1990).
Reasons are that chemicals are too expensive, their widespread and heavy use
has resulted in resistance, labor costs for virus production in vivo are low, production technology is simple, and
registration for their deployment is not required or is easily secured. Improvement of NPVs with
Recombinant DNA.--Because
conventional viral insecticides are relatively slow in killing and have a
narrow host range, it is possible that both of these limitations may be
overcome through the use of recombinant DNA technology (Miller 1988, Maeda
1989, Hawtin & Possee 1992). One approach to improving the efficacy of
viruses is aimed at developing broad spectrum viruses that will cause a
cessation of larval feeding within 24-48 hrs of infection. This is done by
engineering the viruses to express proteins such as enzymes or peptide
hormones that disrupt larval metabolism, or peptide neurotoxins that paralyze
or kill the insect directly. Because it already has a broader host range than
most occluded baculoviruses, the AcNPV virus has been the subject of most of
the engineering studies to 1995. The virus is engineered by placing the
candidate gene under the control of a strong and late viral promoter. With
this strategy, genes for juvenile hormone esterase (Hammock et al. 1990), B. thuringiensis endotoxins (Martens et al. 1990,
Merryweather et al. 1990, Pang et al. 1992) and insecticidal neurotoxins from
the straw itch mite, Pyemotes
tritici, and the scorpions, Androctonus australis Hector, and Buthus epeus, have been engineered into the ACMNPV (McCutchen et
al. 1991, Tomalski & Miller 1991). Of these the most promising has been
the AcNPV expressing the P. tritici toxin, which shortened
the time between infection and paralysis or death to less than 72 hrs in
older larvae. Engineering viruses to express insecticidal proteins in
many cases should also result in an expanded host range, because in many
lepidopteran host species that do not develop a disease when infected by
conventional viruses, there can be limited viral replication. The less
susceptible hosts develop a mild disease and survive infection. But, the same
hosts infected by an engineered virus that expresses a potent insecticidal
protein will probably die because the virus does not need to replicate very
much in order to paralyze or kill the larva. History of Bacillus thuringiensis Bacillus popilliae for Scarab Control Bacillus thuringiensis details --------------------------------------------------------------------------------------------------------------------------------------------- Sporeforming bacteria are the most important in biological
control due to the possession of resistant stages. Among these are Bacillus thuringiensis, Bacillus
pppilliae and Bacillus sphaericus. History of Bacillus thuringiensis.--A detailed history of B.
thuringiensis given by
Beegle & Yamamoto (1992) is paraphrased as follows: The Bacillus
thuringiensis Berliner story
began in the first decade of the 20th Century when the Japanese
bacteriologist S. Ishiwata isolated the bacillus from diseased Bombyx mori (L.) larvae. He named it Sottokin, which means
"sudden death bacillus." He described the pathology it causes in
silkworm larvae and its cultural characteristics (Ishiwata 1905a). He also
noted that many of the larvae that did not die when exposed to the bacillus
were very weak and stunted. In a subsequent report (Ishiwata 1905b) he stated
that "From these experiments the intoxication seems to be caused by some
toxine, not only because of the alimentation of bacillus, the death occurs
before the multiplication of the bacillus..." This showed that from the
very beginning it was realized that a toxin was involved in the pathogenicity
of B. thuringiensis. Ernst Berliner (1911, 1915) isolated a
similar organism from diseased granary populations of Anagasta kuehniella
(Zeller) larvae from Thuringia, Germany, which he named Bacillus thuringiensis,
and because Ishiwata did not formally describe the organism he found,
Berliner is credited with naming it. Aoki & Chigasaki (1916) reported on their studies of
Ishiwata's isolate, noting that its activity was due to a toxin present in
sporulated cultures, but not in young cultures of vegetative cells. The toxin
was not an exotoxin because it was not found in culture filtrates. It is
obvious from their data on inactivation of the toxin by acids, phenol, mercuric
chloride, and heat that they had a protein. Nothing further was accomplished
with B. thuringiensis for over a decade, which was due perhaps to
the fact that in Japan the Sotto disease was not a serious problem in
silkworm culture and in Europe World War I was in progress. Berliner's
isolate was lost, but in 1927 Mattes reisolated the same organism from the
same host as did Berliner (Heimpel & Angus 1960a). Mattes' isolate was
widely distributed to laboratories in various parts of the world, and most of
the early commercial B. thuringiensis-based products
and most of the early microbial control attempts used this isolate (Norris
1970). Both Berliner and Mattes observed in addition to the spore, a second
body, which they called a Restkörper in the developing sporangia. A serious problem with the European corn borer, Ostrinia nubilalis (Hübner) in corn in North America led to the
formation of the International Group for Corn Borer investigation. At a
meeting in Chicago, IL. it was proposed to attempt to use B. thuringiensis as a control agent (Briggs 1986). Under this
program field trials were conducted in Hungary by Husz in the late 1920s and
early 1930s, and Vouk and Metalnikov in Yugoslavia in the early 1930s. The
results ranged from inconclusive to promising. The depressed economic
conditions in the 1930s in North America resulted in an end to funding of the
corn borer work (Heimpel & Angus 1960a, Weisner 1986). But because of the
promising nature of some of the B.
thuringiensis field trials,
commercial production was begun in France by Laboratoire Libec, and the
product Sporeine became available in 1938. World War II stopped further
production. Jacobs (1951) reported on the effectiveness of Sporeine, finding
that he could protect flour from A.
kuehniella by applying
Sporeine to the flour. In the early 1950s Steinhaus at the University of
California at Berkeley published several articles (Steinhaus 1951, 1956a,
1956b) that stimulated interest in the United States in the use and
commercial exploitation of B.
thuringiensis as a microbial
control agent of some lepidopteran pest insects. In 1951 Steinhaus grew B. thuringiensis in large cafeteria trays containing nutrient
agar, washed the spore-crystal complex off after sporulation, and used the
recovered material in a successful field trial against Colias eurytheme
Boisduval larvae on alfalfa (T. Angus 1988, pers. commun. to C. Beegle).
Steinhaus knew a researcher with Cutter Laboratories in Berkeley, where
antibiotics and vitamins were produced in aerated and agitated liquid media
in relatively large fermenters. Cutter Laboratories then produced B. thuringiensis preparations for Steinhaus which he used
successfully against C. eurytheme larvae (Briggs 1986).
In 1956 Steinhaus and R. A. Fisher met with the president of Pacific Yeast
Products, J. M. Sudarsky, to explore the practicality of producing a B. thuringiensis-based product (Heimpel 1972). Pacific Yeast
Products (later Bioferm Corporation) was a yeast and vitamin B-12 producer in
Wasco, CA. The decision was made to produce B. thuringiensis
and by 1957 a product called Thuricide was available for testing. Thuricide
was formulated as liquid concentrates, dusts, and wettable powders. Bioferm
successfully petitioned the U. S. Food and Drug Administration for an
exemption from residue tolerances of B.
thuringiensis products on
agricultural crops, on the presumption of safety toward beneficial insects,
plants, humans and animals based on historical evidence (Briggs 1986). The
Wasco facility in 1992 is owned by Sandoz, and is still producing a variety
of B. thuringiensis products. In 1959 Nutrilite Products entered
the market with their product Biotrol (Ignoffo 1973), which was produced by
semisolid fermentation on a wheat bran medium. Several other U.S. Companies (Merck,
Agritrol; Rohm & Haas, Bakthane; and Grain Producers, Parasporine)
produced B. thuringiensis for short periods
(van der Geest & van der Laan 1971). Besides the production of Sporeine in France in the late
1930s, there was the development of B.
thuringiensis production and
usage in European socialist countries in the 1950s. The 058 strain of B. thuringiensis subsp. thuringiensis
was used by the Research Institute of Antibiotics in Roztoky, Czechoslovakia,
to produce the product Bathurin in 10,000-L fermenters. This material was
priced at 40 korunas per kilogram and was used for control of insect pests on
vegetables and ornamentals, and in forests and orchards (Weiser 1986). In the
former Soviet Union, the All Union Institute for Microbial Products for Plant
Protection produced the product Entobaktrin using a B. thuringiensis
subsp. galleriae isolate
found by Isakova in 1956 (Isakova 1958). In Moscow, the government agency for
the Direction of Microbial Industry started producing the product
Dendrobacillin for use against larvae of the Siberian silkworm, Dendrolimus sibiricus Tshetverikov, a
serious pest of conifers. The B.
thuringiensis subsp. dendrolimus isolate used in the
product was discovered in 1954 by E. V. Talahaev from dead D. sibiricus larvae (Talalaev 1956). The same facility also
produced the product Insektin using a B.
thuringiensis subsp. thuringiensis isolate. Insektin
also was used for forest protection (Weiser 1986). In Yugoslavia the product Baktukal
was produced by Serum Zavod, in Germany Hoechst produced Biospor, and in
France Procidia produced Plantibac. Edward Steinhaus was perplexed that at sporulation in B. thuringiensis the spores were not centrally located, but
they were rather displaced to one end. In 1953 Steinhaus sought the advice of
the Canadian bacterial morphologist C. Hannay regarding this phenomenon (T.
Angus, 1988 pers. commun. to C. Beegle). Upon examining B. thuringiensis
sporulated cells, Hannay noticed a second body in the sporangium, as had
Berliner and Mattes. But Hannay went one step further and speculated that the
parasporal inclusion bodies had some role in the pathogenicity of the
bacterium toward susceptible lepidopterous larvae (Hannay 1953).
Coincidentally in 1951 Steinhaus published a picture of a sporulated and
lysed B. thuringiensis culture showing
bipyramidal crystals, but did not make note of them in the text. T. A. Angus,
a Canadian also was working with B.
thuringiensis at the time,
and Hannay sent Angus a copy of his manuscript prior to publication. Angus
quickly proved that Hannay was correct, that the parasporal crystal was
responsible for the toxicity of B.
thuringiensis (Angus 1954).
Angus showed that spores by themselves had not effect, and that dialyzed
supernatant of alkali-dissolved crystals had the same toxic action as did the
spore-crystal complex when fed to B.
mori larvae. Angus also
noted that toxicity varied with crystal count, and was independent of the
number of spores present. Beegle & Yamamoto (1992) concluded that the early
years were not without problems. For about the first 10 years of B. thuringiensis commercial production, the amount and range
of usage was limited because of low potency strains (nearly all subsp. thuringiensis) and inadequate
standardization techniques. The latter were a result of a decision by Pacific
Yeast Products Co. to optimize spore production in their fermentations. This
eventually led to the acceptance of spore counts by the Pesticide Regulation
Division of the USDA-ARS as a method to standardize B. thuringiensis-based
products (Heimpel 1972). This was disastrous because there proved to be no
reliable relationship between spore counts and insect killing power (Angus
1954, Menn 1960, McEwen et al. 1960, Hall et al. 1961, Mechalas & Dunn
1964, Krieg 1965b, Burgerjon 1965, Vago & Burges 1964, Burges 1967,
Dulmage & Rhodes 1971). It was also an unsettled period in regards to the
identification and classification of B.
thuringiensis. Modern Isolate
Discoveries.--According to
Beegle & Yamamoto (1992), before 1970 the majority of B. thuringiensis products were based on subsp. thuringiensis, and often
contained heat-tolerant Beta-exotoxin. These products were low in activity,
having potencies from a few hundred to ca. 1000 IU per mg. These low potency
products could not compete with chemical insecticides in either efficacy of
cost because of expedient controls demanded by users at the time. Probable
long-range effects on lowering Lepidoptera population vigor and densities as reported
by Legner & Oatman (1962) & Oatman & Legner (1964) were not
considered of sufficient expediency. The presence of Beta-exotoxin in some of
the products resulted in the confusing host range and safety data that exist
in the literature for those subsp. thuringiensis-based
products. In 1962, Edouard Kurstak isolated another subspecies of B. thuringiensis from diseased A. kuehniella
larvae from a flour mill at Bures sur Yvette near Paris, France (Kurstak
1962). He gave the isolates, designated K-17 and K-18 (short for AP.77.BX.17
and AP.77.BX.18), to A. M. Heimpel of the U. S. Dept. of Agriculture in 1962
and again in 1963 (Beegle & Yamamoto 1992). In 1970 Dulmage reported
isolating from diseased Pectinophora
gossypiella (Saunders)
larvae an isolate he named HD-1 (Dulmage 1970). DeBarjac & Lemille (1970)
examined the five isolates (AP.77.BX.17 and AP.77.BX.18 from Kurstak, K-17
and K-18 from Heimpel and HD-1 from Dulmage) and found that on the basis of
flagellar serotyping they were a new subspecies of B. thuringiensis,
which they named kurstaki. One of the new subsp. kurstaki
isolates (HD-1) was responsible for B.
thuringiensis-based products
being able to compete with chemical insecticides on the basis of efficacy and
cost against insects such as Trichoplusia
ni (Hübner) on cruciferous
crops. The kurstaki isolate
proved to be 20- to 200-fold more potent than the isolates in the existing
commercial B. thuringiensis products (Dulmage
1970). In 1970 Abbott Laboratories entered the market with Dipel, which was
the first commercial preparation based on the new kurstaki isolate. It was not long before all companies
producing B. thuringiensis-based produced in
the United States were subsp. kurstaki.
By 1992 several million kilograms of kurstaki-based
products were produced annually in the United States with usage registered
for almost 30 crops and against over 90 pest insect species worldwide. The
single largest market for B.
thuringiensis-based products
by 1992 was against forest insect pests in North America. However, the market for kurstaki based products in agriculture fell to about 20%
of its peak in the mid-1970s, due largely to competition from synthetic
pyrethroids. In the late 1970s a considerable amount of kurstaki-based B.
thuringiensis product was
used on cotton for Heliothis
spp. Because HD-1 was not the most active isolate against Heliothis available, a number
of the most promising isolates were evaluated in the laboratory and field
tested. Of the cultures in the Brownsville, TX. U.S. Dept. Agriculture B. thuringiensis collection (now at Peoria, IL>0, HD-263,
a kurstaki subsp. originally
isolated by G. Ayerst from a dead Ephestia
cautella (Walker) pupa found
by H. Burges in England, proved to be superior against all Heliothis spp. tested. Three
years of field testing against H.
virescens (F.) on cotton
showed HD-263 to be superior to HD-1 (Beegle 1983). But, even at very high
application rates, HD-263-based material did not control H. virescens
larvae as well as the synthetic pyrethroid chemicals. Discovery of isolates
with superior activity against H.
virescens caused Adang et
al. (1983) to clone the crylA(c)
gene that codes for a crystal protein that is highly toxic to Heliothis spp. (Hofte &
Whiteley 1989). In the mid-1980s Ecogen Corporation tried to develop a B. thuringiensis product using genetically manipulated
isolates containing the crylA(c)
gene. Although Ecogen created HD-263-derived isolates much more potent than
the original isolate, the higher laboratory potency was never translated into
similar field performance (I. Gard 1987, pers. comm. to C. Beegle).
Eventually Ecogen abandoned this project. The question of why B. thuringiensis-based products are not effective against Heliothis spp. on cotton is
unclear especially as there are formulations which are certainly potent
enough to Heliothis spp.,
especially H. virescens. Beegle &
Yamamoto (1992) believed the reason is that Heliothis during most of its larval life on cotton is a
covert feeder, spending most of its time inside the squares and bolls.
Because B. thuringiensis has to be
ingested to be effective, all the potency is not going to do any good if the
target pest insect does not consume the applied material. During the period
that Abbott Laboratories was attempting to penetrate the cotton insect pest
control market with Dipel, nearly 100 adjuvants were examined in hopes of
finding one that would result in B.
thuringiensis being
adequately effective against Heliothis
spp. on cotton (T. Couch, 1985, pers. comm. C. Beegle). Beegle & Yamamoto
(1992) thought it might take the development of a highly effective bait for
use with B. thuringiensis crystal toxin
gene(s) and expressing high levels of crystal toxin (Perlak et al. 1990), for
b. thuringiensis to be adequately effective against Heliothis spp. on cotton. Another commercial used kurstaki isolate, NRD-12, was discovered by Normand Dubois
of the U.S. Dept. of Agriculture Forest Service (Dubois 1985). It was
significantly more active against Spodoptera
exigua (Hübner) larvae than
HD-1. But, Moar and associates presented evidence that this was true only
when the HD-1 isolate being compared with NRD-12 is missing the crylA(b) crystal toxin gene
which codes for a toxin that is especially active against S. exigua larvae (Moar et al. 1989, 1990). NRD-12 is a unique
kurstaki isolate because it
produces considerable levels of heat-tolerant exotoxin under conditions
favorable for production of such exotoxin. The other subsp. kurstaki isolate that is known
to produce measurable amounts of heat tolerant exotoxin is 62B-1-(4)
discovered by Michio Ohba in Japan (Ohba et al. 1981). In the mid-1980s
Sandoz registered the product Javelin, based on NRD-12, with the U.S.
Environmental Protection Agency. In late 1985 Sandoz replaced NRD-12 with
another subsp. kurstaki
isolate which did not produce detectable levels of heat-tolerant exotoxin,
but was as active as NRD-12 against S.
exigua larvae, for use in
the product Javelin Current subsp. kurstaki-based
commercial products listed by Beegle & Yamamoto (1992). The worldwide
market for kurstaki-based
products for forestry and agriculture was estimated at $20-25 million in the U.S.
in 1992 (Beegle & Yamamoto 1992). Goldberg & Margalit (1977) discovered B. thuringiensis isolates with mosquito larvicidal activity
after screening ca. 1000 isolates from 10 soil samples taken from known
mosquito larval breeding sites in Israel. Only 1% of the isolates showed
mosquito larvicidal activity, but one (ONR60A) had extremely high activity.
The new isolate was classified as B.
thuringiensis subsp. israelensis (de Barjac 1978),
and had a good level of activity and killing speed. Mortality of Aedes aegypti (L.) larvae could occur as soon as 30 min. after
exposure (Singer 1980) and with an LC-50 as low as 22 ppb (Dame et al. 1981).
Goldberg obtained a U.S. patent on the organism and assigned the patent to
the U.S. government (Goldberg 1979). By 1992 both U.S. and European companies
were producing and marketing subsp. israelensis-based
products for use against mosquito and blackfly larvae. The worldwide market
for israelensis-based
products was ca. $10-US million. Keio Aizawa of Kyushu University in Japan discovered an
isolate in 1962 that Bonnefoi & de Barjac (1963) determined as a new
subspecies, naming it aizawai.
Subsp. aizawai isolates were
particularly effective against Galleria
mellonella (L.) and Spodoptera spp. larvae.
However, no B. thuringiensis isolate is nearly
as active against Spodoptera
spp. larvae as are the best isolates against the larvae of such species at T. ni and H.
virescens. When beekeepers
lost the use of certain effective insecticides for killing G. mellonella larvae in honey combs, Sandoz recognized that
empty niche and developed their product Certan, based on an aizawai isolate, for control of
G. mellonella larvae in honey comb. The market was small at
ca. $50,000 U.S. per year. By 1992 the latest new B.
thuringiensis subspecies to
be found that had commercial promise was tenebrionis,
which was found by Krieg and associates in diseased Tenebrio molitor
L. (Krieg et al. 1983), and is active against other Coleoptera. Herrnstadt et
al. 91986) of Mycogen Corp reported finding a B. thuringiensis
isolate active against coleopterous larvae, naming it B. thuringiensis
subsp. san diego. Krieg et al. (1987)
compared the two isolates, tenebrionis
and san diego, and found them to be identical in terms of
biochemistry; serology; antibiotic and chemotherapeutic inhibition; plasmid
DNA; crystal morphology, solubility, and protein pattern; and host range. As
the two isolates appeared identical, and tenebrionis
was described first, it follows that tenebrionis
is the correct name for the subspecies and the name "san diego"
should only be used to designate the particular isolate of subsp. tenebrionis that Mycogen
utilized. In 1988 Mycogen brought out its product M-One based on its isolate,
primarily for use against larvae of Leptinotarsa
decemlineata (Say) on
potatoes. M-One was reported effective against small L. decemlineata
larvae in warm weather, but was less effective in cool weather or against
large larvae (Ferro & Lyon 1991; Zehnder & Gelernter 1989). Because
of the problem of resistance to chemical insecticides by L. decemlineata
in some potato growing areas, subsp. tenebrionis-based
products have become the preferred L.
decemlineata control
product. In those areas it was noticed that a reduction in the level of
resistance to synthetic pyrethroids in L.
decemlineata occurred. This
accidental discovery suggested the possibility of managing resistance to both
control agents by alternating usage of each (D. Ferro, 1992, pers. commun. C.
Beegle). Ecogen received registration for its product Foil in 1992, which was
a combination f subsp. kurstaki
and subsp. tenebrionis for
use against both lepidopterous and coleopterous pests on potatoes. Beegle
& Yamamoto (1992) listed a number of subsp. tenebrionis-based products). Characteristics.--Bacillus
thuringiensis causes
extended mortality in nature among lepidopterous insect epizootics or in mass
culture. High mortalities appear in dense populations of stored product
pests, such as Ephestia kühniella, Plodia interpunctella
and others, in the rearing of silkworms and in laboratory mass rearings of
insects in general. The rather thick, clear staining rod of this bacillus
produces a diamond-shaped refringent crystal of proteinic material in
addition to the spore itself, which is the seat of its activity. The crystal
dissolves in the midgut of Lepidopteran caterpillars to a toxin which
attacks the membranes of midgut cells and finally kills the host. In other
hosts the Lep-toxin does not find proper conditions for its activation and is
harmless. Therefore, it is safe for all organisms including humans and other
vertebrates. Conditions in different caterpillars differ slightly, and there
are of the >1,000 known isolates, groups of strains which have some specific
affinity to specific hosts. With flagellar antigens the strains are divided
into more than 20 serotypes differing also in some principal details such as
their activity against noctuids and large caterpillars, activity against the
wax moth, etc. Strains are selected for mass production for controlling
caterpillars of agricultural pests. Serological grouping does not dictate the
general virulence of a strain and it can be isolated with minimum or maximum
virulences. Improvement of the virulence is achieved by selection, optimum
medium for cultivation and passing over selected host insects. With increased
virulence the time between administration and mortality is reduced, from a
usual five days to three or less. Bacillus thuringiensis
is produced in large fermentors, dried, blended with inert ingredients,
emulgators and stickers. It must be stored in the dry state. Ready mixtures
have to be used during the same day. It is administered as a food and for
maximum effect must be ingested in adequate quantity. Applications on the
first three instars of a caterpillar are preferable. Spores on leaves are
damaged by ultraviolet light and usually retain their activity for
<1-week. Spraying from below and to the sides of plants is recommended.
Biological activity of a preparation is determined on the basis of comparison
with a standard of Heliothis
or Lymantria. Local target
insects can also be used for comparison with a standard. The activity is
expressed in bio-units (Weiser 1984). Some serotypes are able to produce a soluble exotoxin.
This is released into the liquid fermentation medium and may be removed by
centrifugation. However, it remains in the total spray-dried formulations
because it is a thermostable toxin. This toxin interferes with DNA-dependent
RNA polymerase and it is a general rather than nonspecific poison. Its active
dose for insects has a safe range far below vertebrate activity, but it is
not yet used broadly in pest control. In the Soviet Union this kind of
material is used for the control of housefly maggots and the Colorado potato
beetle and other pests. In low doses treated animals develop teratologies in
their moth parts and legs. Bacillus thuringiensis
preparations are used for the control of most lepidopteran pests of
agriculture (Legner & Oatman (1962) & Oatman & Legner (1964)). Due to their
nontoxicity to the honeybee, they can be used on flowering plants. The usual
dosage is 0.5-1.5 kg/ha of the preparation, according to density of the
treated crop. Material is used in a water suspension. Lepidoptera are divided
into three major groups of susceptibility. Most susceptible are Pieris, Plutella, Tortricidae, bagworms and moths. Here the dosage
is 0.2-0.5 kg/ha. The second group contains most caterpillars, mainly the
Noctuidae, armyworms, budworms, large caterpillars, etc., where the dosage is
1-1.5 kg/ha. In the third group there are hidden caterpillars (e.g., codling
moth) that do not normally access treated leaves, or exceptionally resistant
species such as Spodoptera. Bacillus thuringiensis
may be used in a mixture with insecticides. Generally the bacillus is
introduced to a diluted insecticide to avoid damage to from solvents.
Mixtures are not recommended because they usually are made with reduced
content of insecticide and after expiration of the microbial agent the
sublethal insecticide may produce resistance. There is no resistance to B. thuringiensis beyond normal fluctuation of activity after
30 generations of genetic stress and pressure (Weiser 1984). The strain used
most in agriculture is H-3 kurstaki. For specific treatment of the wax moths
the serotype H-7 aizawai was developed. Of economically important nontarget
insects the silkworm is the only endangered species and where sericulture is
developed proper care must be taken to avoid its introduction into the
cultures. This danger is analogous to that from insecticides. There is good
adaptation of B. thuringiensis preparations to
entomophagous insects. The five day phase before full mortality enables
parasitoids to finish their development and active adult parasitoids are not
encumbered. The shelf life of dry formulated powders is >2 years, the
spores and crystal toxin remain active for more than 15 years. Moisture usually
causes spore germination and decreases the activity of the preparation. Among the many isolates there are some strains with
activity for beetles, but their development as microbial insecticides has not
been accomplished. The serotype H-14 (B.
thuringiensis israelensis) is a very
important strain which infects and kills mosquito and blackfly larvae. All
tests have shown identical parameters of this strain with other strains. Only
the endotoxin is entirely different, being nontoxic for Lepidoptera and highly
toxic to mosquitoes. The crystal is more spherical and ultrastructurally is
composed of two different substances. The larvae respond after feeding for
three hours with a symptom of knock down, hanging motionless on the surface.
Once this symptom appears there is no recovery for the larvae and they die in
24-48 hrs. The active concentration of the bacteria ranges from 500-1,000
cells/ml. Due to the ecological range of distribution of mosquito larvae in
shallow water, the volume factor does not need to be calculated and a dosage
of 1 kg/ha of wettable powder for treatment of mosquito habitats is usual.
The difference in feeding of early and late instar larvae is not very
important and therefore treatments are timed for the period of older larvae,
before pupation. The treatment does not hold longer than one week and must be
repeated when last instar larvae are again present. Bacteria are filtered
away by different aquatic organisms (e.g., rotifers, ciliates, crustaceans,
etc.). If large dosages are used, some chironomid midges are damaged. For the
treatment of running water that contain larvae of blackflies, it is necessary
to provide a concentration of 1.5 mg/l of Tecnar or Vectobac for 10 min., by
continuous release of the concentrate. Other bacteria are less commonly used in field
application. Bacillus sphaericus is specific only for
mosquitoes. Bacillus popilliae was used earlier for
control of such grubs as Popillia
japonica in the United States.
The bacterium has a large parasporal body and infects grubs by feeding but
only very slowly. It was mass produced by injection of pasteurized spores
into normal field collected grubs. Dying or dead infected grubs were
triturated with talcum or bentonite and the mixture was dusted over lawns
infected with grubs. Several local strains occur in populations of different
grubs in Australia and New Zealand and Weiser (1984) believed they must be
present also in other areas. Bacteria are relatively simple unicellular microorganisms
lacking internal organelles such as a nucleus and mitochondria, and reproduce
by binary fission. With few exceptions most bacterial used as microbial
insecticides grow readily on a wide variety of inexpensive substrates, a
characteristic which facilitates their production. Most bacterial currently
in use or under development as microbial control agents for insects are
spore-forming members of the bacterial family Bacillaceae, in the genus Bacillus. Such pathogenic
bacilli occur in healthy and diseased insects, but they also occur and can be
isolated from many other habitats including granaries, plants, frass, soil
and aquatic environments. Biological Properties.--Two major types of bacteria used in insect control are
those which cause fatal infectious diseases and those which kill insects
primarily through the action of insecticidal toxins. Bacillus popilliae
Dutky is an example of the firs type. It is a bacterium that infects and
kills Coleoptera larvae, particularly soil-inhabiting Scarabaeidae. The
second type is Bacillus thuringiensis Berliner, a
species which produces toxins, both protein endotoxins and nucleotide
exotoxins, that are able to kill insects whether or not they are directly
associated with the bacterium. Biologists have made extensive use of the
latter property by inserting the genes encoding various endotoxins into other
microorganisms and plants thereby making them insecticidal (Fischoff et al.
1987, Vaeck et al. 1987. Perlak et al. 1990, Raymond et al. 1990). Bacillus popilliae for Scarab Control Scarab milky disease was first discovered over 50 years
ago (Dutky 1937, 1963). It is caused by B.
popilliae and B. lentimorbus. The term "milky" derives from the
opaque white color characterizing diseased larvae which results from the
accumulation of sporulating bacteria in the hemolymph. The disease in
initiated when grubs feeding on the roots of grasses or other plants ingest
the spores, the latter than germinating in the midgut and vegetative cells.
They invade the midgut epithelium where they grow and reproduce, changing in
form as they progress toward invasion of the hemocoel (Splittstoesser et al.
1978). After passing through the basement membrane of the midgut, the
bacteria colonize the hemolymph over a period of several weeks and sporulate
reaching populations of 108 cells per ml. The disease is fatal if
the larvae ingest a sufficient numbers of spores early in development, and
dead larvae become foci for spores which can serve as a source for infection
for over 30 years (Klein 1981). One drawback of B.
popilliae and its close
relatives is that suitable media for their growth and mass production in vitro are not available. This has encumbered both research
and large scale commercial development of B.
popilliae. Commercial
development is from field-collected scarab larvae. Such larvae are collected
from field populations, injected with spores, and held in environmental
chambers for 1-2 weeks until the bacteria have sporulated and killed most of
the larvae. Powdered formulations of the bacterial spores are then made by
drying and grinding larvae. Such preparations may be applied to soils
infested with grubs at rates of ca. 1 kg. of formulation per ha. Although
treatment can be expensive, a single treatment typically lasts for 10 years. Bacillus thuringiensis This bacterium has been the most widely used insect
pathogen to date. A complex of bacterial subspecies comprises this bacterium,
all of which are typified by the production of a parasporal body during
sporulation. This parasporal body contains one or more proteins, often in
crystalline form, many of which are highly toxic to certain insects. In the
insecticidal isolates, the toxins are known as endotoxins and often
occur in the parasporal body as protoxins which after ingestion are activated
by proteolysis in the gut. The activated toxins destroy midgut epithelial
cells, causing death. Systematics &
Biology.--B. thuringiensis
is very closely related to Bacillus
cereus, which is separated
by the occurrence of the parasporal body in the former (Baumann et al. 1984).
It seeps that in almost all known isolates of B. thuringiensis,
the proteins which make up the parasporal body are encoded on plasmids borne
by the bacteria. These plasmids can be lost during growth and reproduction,
which can change the description to B.
cereus, using conventional
identification and classification. At least several thousand isolates of B. thuringiensis
have been obtained from a variety of sources by 1991. These include soil,
frass, grain dust, water, and living and dead insects. They are divided into
groups, of which there are nearly 30 (De Barjac & Frachon 1990), that are
differentiated on the basis of the H antigen, i.e., flagellar serotype, which
is indicated by a number or a number and letter combination (e.g., H3a3b,
etc.) as well as a serovar name (e.eg., H-1 thuringiensis, H3a3b kurstaki).
A list of the H antigen types and serovar names as of 1990 is provided by
Beegle & Yamamoto (1992). The H antigen and serovar are usually used to
identify a specific isolate, although the serovar name is often referred to
as either a subspecies or variety (e.g., B.
thuringiensis subsp. thuringiensis, or B. thuringiensis var. thuringiensis).
Beegle & Yamamoto (1992) pointed out that B. thuringiensis began as an independent species
distinguishable from other bacilli, such as B. cereus
Frankland & Frankland. It is close to B.
cereus except that it
produces a crystal at sporulation (sometimes more than one), which is usually
bipyramidal in shape (sometimes square, flat or amorphous), and is toxic to
lepidopterous, dipterous or coleopterous insect larvae. This resulted in some
taxonomic problems, and some bacterial taxonomists felt that B. thuringiensis should be a subspecies of B. cereus (Smith et al. 1952, Bordon et al. 1973). This is
based on such observations as certain strains of B. thuringiensis
and B. cereus both killed mice when injected intraperitoneally
(Lamanna & Jones 1963); the spores of both shared common antigens (Yoder
& Nelson 1960; Lamanna & Jones 1961) and showed cross sensitivity to
bacteriophages (Yoder & Nelson 1960); it was not possible to separate B. cereus and B.
thuringiensis by crossed
immunoelectrophoresis of ultrasonic extracts of sporefree-grown vegetative
cells (Hartung & Hellsmann 1987); some B. anthracis
Cohn strains were sensitive to bacteriophage from B. thuringiensis
(Yoder 7 Nelson 1960); it was not possible to separate B. cereus
and B. thuringiensis isolates by fatty acid patterns (Vivoli
& Fabio 1967, Kaneda 1967, 1968); the flagellar antigens of some B. thuringiensis and B.
cereus isolates overlapped
(Krieg 1970); there was a close enzymoserological relationship (casein
precipitating proteinases) (Sandvik 1973); thin-sectioned spores appeared
identical in fine structure (Gerhardt et al. 1976); and of greatest
importance, deoxyribonucleic acid (DNA) homologies of 80-101% (Kaneko et al.
1978) and 54-80% (Seki et al. 1978). Singer (1980) believed that strains of
bacteria having greater than 70% DNA homology belong to the same species. On the other hand, there are a number of reports that
support separate species status for B.
thuringiensis. For example,
Somerville & Jones (1972) believed they could distinguish between B. thuringiensis and B.
cereus by DNA competition
studies; Krieg (1965a) reported that he could differentiate B. thuringiensis isolates from B. cereus
isolates with immunofluorescence and phase-microscopy; and O'Donnell et al.
(1980) using pyrolysis gas-liquid chromatography were able to show that
pyrograms of B. cereus and B. thuringiensis
isolates were consistently different and allowed the separation of a large
number of isolates of each species into distinct, non-overlapping groups.
Additionally, Rogoff & Yousten (1969) listed several biochemical
characteristics by which B. thuringiensis isolates could be
distinguished from those of B.
cereus. As more flagellar
serotypes of thuringiensis
were found, and the importance in some cases of the crystal serotype became
apparent (Krywienczyk et al. 1978), the taxonomic scheme of Smith became
increasing cumbersome. By 1992 most researchers used the taxonomic scheme
where B. thuringiensis was considered a
separate species. With increasing commercial importance, and numerous
obviously different isolates discovered, the need for a method to identify
and classify B. thuringiensis subspecies was
apparent. The first real effort was made by Heimpel & Angus (1958, 1960b)
based on morphology and biochemistry. DeBarjac & Bonnefoi (1962, 1968,
1973) and de Barjac (1981) developed an identification and classification
based on serological analysis of vegetative cell flagellar (H) antigens plus
biochemical characteristics. Norris (1964) advanced an identification and
classification method based on analysis of esterase patterns of vegetative
cells by starch gel electrophoresis. Norris found that although there were
striking similarities between groupings of B. thuringiensis
isolates by esterase and H-antigen analyses, he felt esterase analysis had
several advantages. Esterase analysis could distinguish between subsp. sotto and dendrolimus, which H-antigen analysis could not, and
esterase analysis was much faster than either biochemical or serological
analysis. Bacillus thuringiensis heat-stable
somatic O-antigens (Ohba & Aizawa 1978, Sekijima & Ono 1982) ,
lectins (De Lucca 1984), crystal serology (Krywienczyk & Angus 1960,
Krywienczyk et al. 1978, Smith 1987), and phages (Jones et al. 1983) have
also been examined for usefulness in identifying or classifying, or both, B. thuringiensis isolates (Beegle & Yamamoto 1992).
Flagellar serotyping became the method of choice largely because the Institut
Pasteur provided a flagellar serotyping service to scientists wishing their
newly found isolates serotypes. By 1992 there were 36 generally recognized B. thuringiensis subspecies based on serotype and some
biochemical and host range information, but de Barjac & Frachon (1990)
proposed abandoning the use of biochemical and host range information as well
as the subspecies concept in B.
thuringiensis
classification. They suggested restricting the identification and
classification of B. thuringiensis strains to only
the serology of vegetative cell flagella, and using the term serovar to
designate the different groups, of which they recognized 34. One limitation
of their scheme was that a group such as subsp. tenebrionis, which is very unique in host range, crystal
morphology, crystal toxin gene, and crystal protein chemistry, did not merit
separate status. Effective techniques that have been applied to the
identification and classification of B.
thuringiensis are high
performance liquid chromatography (HPLC), plasmid mapping, and cloning and sequencing
of the crystal toxin genes. Yamamoto (1983) isolated 135-kDa crystal protein
by column chromatography, digested the protein with trypsin, and mapped the
resulting peptides using reverse-phase HPLC. The results indicate that HPLC
can distinguish crystal types within a serotype with great detail and
reproducibility. Bacillus thuringiensis cells contain one
or more plasmids, and it was determined that the crystal toxin genes are
harbored in the plasmids in most strains (Gonzalez & Carlton 1982, 1984;
Whiteley et al. 1982; Schnepf & Whiteley 1981; Ward & Ellar 1983;
Held et al. 1982; Klier et al. 1982, 1983; Kronstad et al. 1983; Gonzalez et
al. 1982). There have been several reports showing that plasmid profile
(number and molecular weights of cell plasmids) can be used to identify B. thuringiensis strains (Iizuka et al. 1981, Lereclus et al.
1982, Jarrett 1983, Gonzalez & Carlton 1980, Gonzalez et al. 1981). The
latest developments have been the identification and sequencing of the genes
that code for the crystal toxins (Beegle & Yamamoto 1992). Hofte &
Whiteley (1989) proposed a nomenclature and classification scheme for crystal
genes (cry) based on their
phenotype, types of crystal proteins produced, and the protein's host range
as insecticidal toxins. Some biochemists and molecular biologists believe
that a taxonomic system for B.
thuringiensis based on
flagellar serotypes is no longer appropriate because the primary interest in
this organism is its ability to kill insects, and we now have the ability to
determine the DNA sequences of the crystal toxin genes that determine the
level and degree of its activity. Principal Pathotypes.--There are three major pathotypes according to whether
they exhibit toxicity to either Diptera, Lepidoptera or Coleoptera. Most
isolates and subspecies are from Lepidoptera, which until the 1970's was the
only pathotype know. The first isolate with substantial toxicity to Diptera,
principally to mosquito and blackfly larvae, was the ONR 60A isolate of B. thuringiensis subsp. israelensis
(H 14) DeBarjac discovered in Israel in 1976 (Goldberg & Margalit 1977,
De Barjac 1978). The first isolate with high toxicity to Coleoptera was B. thuringiensis subsp. morrisoni
(H8a8b), discovered in Germany (Krieg et al. 1983). The most common pathotype
has continued to be from Lepidoptera. Insecticidal Protein Types.--The insecticidal proteins that occur in the parasporal
bodies of B. thuringiensis are referred to
in general as delta-entotoxins, the delta designating a particular class of toxins,
and endotoxin referring to their localization within the bacterial cell after
production as opposed to being secreted. With new recombinant DNA techniques
and the discovery in the early 1980's that delta-endotoxins were encoded by
genes carried on plasmids, a major research effort developed to understand
the genetic and molecular biology of the toxins. This led to cloning and
sequencing of many B. thuringiensis genes and
characterization of the toxicity of individual gene products. Hofte &
Whiteley (1989) summarized this work through 1988. A variety of names and
terminology was used to refer to B.
thuringiensis insecticidal
proteins and genes, and Hofte & Whitely (1989) proposed a simplified
terminology for naming all insecticidal B.
thuringiensis proteins and
the genes encoding them. The terminology is based on the spectrum of activity
of the proteins as well as on their size and apparent relatedness, suggested
from nucleotide and amino acid sequence data. All genes sequenced to 1992,
except a 27-kDa protein from B.
thuringiensis. subsp. israelensis, appear related,
and probably were derived from the same ancestral gene. Hofte & Whiteley
called these cry, for crystal, genes and the proteins they encoded Cry
proteins. This is followed by a numeral which indicates pathotype (I & II
for toxicity to Lepidoptera, III for Coleoptera and IV for Diptera), followed
by an upper case letter indicating the chronological order in which genes
with significant differences in nucleotide sequences were described. The I
and II for Lepidoptera-toxic proteins also indicate size differences, with
the I referring to proteins of ca. 130 kDa, and the II to those of 65-70 kDa.
Sometimes a lower case letter in parentheses is included, indicating minor
differences in the nucleotide sequence within a gene type. Therefore, CryIA
refers to a 130 kDa protein toxic to Lepidoptera for which the first gene
(cryIA) was sequenced, while CryIVD refers to a 70 kDa protein with
mosquitocidal activity for which the encoding gene was the 4th from this
pathotype sequenced. The 27-kDa CytA protein that was first isolated from B. thuringiensis israelensis
differs from other B. thuringiensis proteins not only
in its smaller size, but also in that it is highly cytolytic to a wide range
of cell types in vitro, including those of
vertebrates (Chilcott et al. 1990, Federici et al. 1990). Also, it shares no
apparent relatedness with Cry proteins. Because of these differences and its
broad cytolytic activity, it is referred to as the CytA protein encoded by
the cytA gene (Hofte & Whiteley 1989). Activity Spectrum & Toxicity
Genetics.--Two features
are common to most genes regardless of pathotypes (Hofte & Whitely 1989).
First, most proteins share in common five blocks of highly conserved amino
acids. These blocks are distributed over the molecule from amino acid
position 153 to ca. 680. Secondly, there is a variable region between, in the
C-terminal portion of the activated toxin core. The conserved regions are
believed to comprise the structural domains that account for the toxicity of
most Cry proteins, while the variable region defines the spectrum of activity
or host range. Ge et al. (1989) gave experimental evidence for the latter. Composition of Pathotypes &
Shape of Parasporal Body.--The shape of the parasporal body is a reasonable
indicator of an isolate's pathotype. Most isolates of B. thuringiensis
produce a large bipyramidal parasporal crystal (0.5 x 1 micro-m.) that is
almost always only toxic to Lepidoptera (Heimpel & Angus 1963, Moar et
al. 1989). The bipyramidal crystal may be accompanied by a smaller cuboidal
crystal toxic to mosquitoes, such as occurs in B. thuringiensis
kurstaki (Yamamoto &
McLaughlin 1981). Other subspecies, such as B. thuringiensis
israelensis (H 14) and the
PG-14 isolate of B. thuringiensis morrisoni (H 8a8b) produce
spherical parasporal bodies (0.7- 1 micro-m.) that are toxic primarily to
Nematocera (mosquito and blackfly larvae). The "tenebrionis' strain of B. thuringiensis morrisoni
(H 8a8b) produces a thin, square crystal that is toxic only to certain
species of Coleoptera (Federici et al. 1990, Krieg et al. 1987). Protein complexity within these parasporal bodies varies
considerably. Single crystals might be composed of a single type of protein
molecule or a mixture of as many as three. Also, a single parasporal body may
be composed of 2-4 crystals (Federici et al. 1990, Hofte & Whiteley
1989). A simple crystal is found in the HD-73 isolate of B. thuringiensis
kurstaki, where only the
CryIA(c) protein is encoded and produced. This forms a typical bipyramidal
crystal at sporulation. However, the HD1 isolate of the same subspecies
carries five cry genes [cryIA(a),(b),(c);cryIIA; cryIIB] producing at least
four of these during sporulation. The 3 CryA proteins crystallize together
into a single bipyramidal crystal, while the smaller CryIIA protein forms the
associated cuboidal inclusion. Most complicated of all combinations is found
in B. thuringiensis israelensis,
where the 3 CryIV proteins and CytA proteins crystallize into three inclusion
types, bound together in a fibrous envelope (Federici et al. 1990). Toxins Produced by B. thuringiensis.--There is
surprisingly little known about the biology and role of the insecticidal
parasporal body in nature. Although isolated from a wide variety of habitats,
it does not grow well in many because it is not a major or dominant species.
Unlike many viruses, fungi and protozoa, B.
thuringiensis has never been
reported in large-scale epizootics. B.
thuringiensis is quite
commonly isolated from grain dust, and the original description by Berliner
(1915) was based on an isolate from the Mediterranean flour moth, Anagasta kuehniella (Zeller). The cadavers of insects killed by B. thuringiensis provide very suitable substrates for
reproduction and sporulation (Aly et al. 1985). Therefore, the bacteria which
harbor plasmids encoding insecticidal bacterial proteins have a selective
advantage when the spores and parasporal bodies occur together, and the number
of parasporal bodies ingested by an insect are sufficient to cause death. The parasporal crystals of the isolates of the three
pathotypes as described by Krieg et al. (1983), are very different. The
crystals of pathotype A, active against lepidopterous larvae, are usually
bipyramidal. The crystal is made up of 230-kDa dimers (Holmes & Monro
1965) which, in the reducing high pH environment of a susceptible insect's
midgut, are dissociated into protoxin, whose size is in the vicinity of 135
kDa apparent molecular weight, which is not toxic until it is enzymatically
digested by midgut proteinases into the active fragment. The reported sizes
of the active fragment range from 500 Da to 708 kDa. Faust & Bulla 91982)
have summarized the treatment conditions, methods of separation, and results
of the many reports. Chestukhina et al (1978) found that B. thuringiensis
crystals were contaminated, either on their surface or within the crystal
lattice, with proteinases that could digest the crystal protein, especially
when dissociated. This explains why Bulla et al. (1979) found an active
fragment of 68 kDa, not 135-kDa protoxin, when an alkali-dissociated crystal
solution was incubated for 6 days at 28°C. When Zalunin
et al. (1979) and Chestukhina et al. (1980) took steps to inactivate the
contaminating proteinases, their data on the dissociation of B. thuringiensis crystals were much more consistent. Yamamoto
& Iizuka (1983) investigated the activation process for the toxin under
controlled conditions similar to that in the midgut of T. ni
larvae. They found that the 135-kDa crystal protoxin, prepared by
dissociating the crystal in a reducing alkaline environment, was rapidly
digested by T. ni gut proteinases until the
protein reached a 62-kDa proteinase-resistant core which retained 100%
activity. The B. thuringiensis pathotype A
crystal often contains additional cuboidal or round crystals within them
(Beegle & Yamamoto 1992). Grigorova & Kalucheva (1966) described
bipyramidal crystals of subsp. thuringiensis
with ovoid bodies embedded in the sides of the crystal matrix. Sharpe &
Baker (1979) observed similar embedded bodies in subsp. kurstaki crystals that appeared to be both cuboidal and
round. Yamamoto & McLaughlin (1981) isolated from a commercial kurstaki strain a 65-kDa
protein which was immunologically and biochemically distinguishable from the
135-kDa protein. They termed the protein P2 and found that it was toxic to
both lepidopterous and dipterous larvae. Iizuka & Yamamoto (1983) then
speculated that the embedded bodies were composed of the P2 protein. Pathotype B consisting of subsp. israelensis and a dipterous active subsp. morrisoni isolate (PG-14), are
highly active against mosquito and blackfly larvae. Several irregularly shaped
crystals per sporangium are produced at sporulation. The crystals consist of
three groups of proteins, 128-135 kDa, 72-78 kDa, and 27 kDa. The 27-kDa
protein is unique in that it is cytotoxic to mammalian cells, and although it
has little or no insecticidal activity, it may have synergistic action with
the 72- to 78- and 128- to 135-kDa toxin proteins (Wu & Chang 1985). Pathotype C, active against coleopterous larvae, consists
of subsp. tenebrionis. The
crystal is unique, both in shape and protein characteristics. It is square
and flat, and is composed of 67-kDa protein molecules (McPherson et al.
1988). Unlike any other B. thuringiensis subspecies,
subsp. tenebrionis crystals
dissolve in NaBr solutions and in denaturing agents in the absence of reducing
agents, suggesting that disulfide bonds are not present (Bernhard 1986).
Another unusual aspect is that NaBr- or alkali-dissolved crystals
recrystallize into flat squares when NaBr is removed or the pH is lowered. Beegle & Yamamoto (1992) noted that years ago host
spectral differences were known with B.
thuringiensis (Burgerjon
& Grison 1959). It was thought that such differences in activity spectra
of some isolates might have taxonomic value and possibly could be used in
classification (Burgerjon & Biache 1967). Some specialists believed that
the crystal toxin with differing host spectra were the same, thus the use of
the singular term "the delta-endotoxin," and that the spectral
differences were due to differences in crystal digestibilities. This belief
was based n the report of Lecadet & Martouret (1964) that there was a
direct relationship between the speed of enzymatic hydrolysis of B. thuringiensis crystals in gut juice of Pieris brassicae L. and their toxicity, and that when the
crystals of a less effective subspecies reached the same degree of
dissolution as those of a more effective subspecies, they were equally toxic.
The report of Aronson et al. (1991) confirmed that there can be a
relationship between toxicity of intact crystals and their digestibility.
They found that when the cryA(b)
gene was lost by a subsp. aizawai
isolate, its crystals were less soluble and less toxic to highly susceptible
insects such as Manduca sexta (L.) and T. ni, but solubilized protoxins from those crystals were
still fully active. But those effects were not observed with subsp. kurstaki crystals or with the
much less susceptible species Spodoptera
frugiperda J.E. Smith).
Also, Haider et al. (1986) found that the host spectrum of a subsp. aizawai isolate was determined
by differential proteolytic processing of the protoxin. When the subsp. aizawai solubilized protoxin
was activated by P. brassicae gut extracts, the
resulting active fragments were active against three dipterous cell lines and
only one lepidopterous (S. frugiperda) cell line. When the
protoxin was activated by A.
aegypti gut extracts, the
active fragments were active only against the four lepidopterous cell lines. The contention that host spectra were determined only by
crystal digestibility was probably the basis of Monsanto's expectations in
their early project involving B.
thuringiensis. In 1983
Monsanto announced that they had successfully incorporated the subsp. kurstaki crystal toxin gene
into corn root-colonizing Pseudomonas
fluorescens (Trevisan), which
then produced the soluble toxin (active fragment). Their expectation was that
Diabrotica spp. and Agrotis ipsilon (Hufnagel) larvae feeding on corn plants would be
killed because they would be ingesting the soluble toxin. This did not occur
at any level that was significantly effective against those insects. It
became widely accepted that there is a receptor binding domain in the
activated toxin that determines the specificity of the toxin, and that active
fragments with different activity spectra differ in their receptor domains
(Beegle & Yamamoto 1992). It was believed for quite sometime that the activity of B. thuringiensis spore crystal complexes toward insect larvae
was due entirely to the crystal toxins. Some insects as B. mori
(Angus 1954), Ephestia cautella (Walker) (McGaughey
1978), Simulium vittatum Zetterstedt (Lacey et
al. 1978), and L. decemlineata (Riethmuller &
Langenbruch 1989) seem susceptible only to the crystal toxin, the spore
having no effect. Other insects such as Colias
eurytheme Boisduval, T. ni, Pseudaletia
unipunctata (Haworth)
(Somerville et al. 1970), Pieris
rapae (L.) (Soliman et al.
1970), Plodia interpunctella Hübner)
(McGaughey 1978), Laspeyresia
pomonella (L.) (Roehrich
1962; Vervelle 1975), O. nubilalis (Mohd-Salleh & Lewis
1982, Sutter & Raun 1966), G.
mellonella (Burges) et al.
1976), Li et al. 1987), and S.
exigua (Moar et al. 1989)
are maximally sensitive to mixtures of spores and crystals. In O. nubilalis, the response to B. thuringiensis
spores and crystals is isolate dependent. Modh-Salleh & Lewis (1982)
found that subsp. galleriae
and kurstaki mixtures of
spores and crystals were significantly more toxic than crystals alone; but
with subsp. kenyae and tolworthi, mixtures of spores
and crystals were not significantly more toxic than crystals alone. In Choristoneura fumiferana (Clemens) some
controversy exists as to the relative importance of B. thuringiensis
spores and crystals. Yamvrias & Angus (1970) and Smirnoff & Valero
(1979) both found that mixtures of spores and crystals were the most toxic to
C. fumiferana larvae. Fast (1977) though concluded that
spores played little or no role in mortality of spruce budworm larvae by B. thuringiensis. Fast's data show that the LC-50s of
crystals alone and spore crystal mixtures were nearly identical, whereas the
LC-95 of the spore-crystal mixture was nearly half that of the LC-95 of
crystals alone. It is not known whether the increases in activity that result
when spores are present is due to the additional crystal protein present in
the spore coat (Delafield et al. 1968, Somerville et al. 1968, Somerville et
al. 1970, Somerville & Pockett 1974, 1975; Sutter & Raun 1967,
Lecadet et al. 1972, Scherre & Somerville 1977, Short et al. 1974, Tyrell
et al. 1981, Li et al. 1987), or to another factor such as infection by the
resulting vegetative cells. Two findings arguing against the former are
Burges et al. (1976) who found that a 1:1 mixture of spores and crystals of
subsp. galleriae was about
10,000-fold more toxic to G.
mellonella larvae than were
crystals alone; Li et al. (1987) reported that spores of an acrystalliferous
mutant of subsp. aizawai did
not contain any crystal protein and did not cause any significant mortality
when fed alone to G. mellonella larvae. However,
when the acrystalliferous spores were mixed 1:1 with subsp. aizawai crystals, the activity
was about 200-fold higher than for crystals alone. Reports that antibiotics
decrease B. thuringiensis activity when fed
with spore-crystal complexes to insect larvae (Affify & Merdan 1969,
Soliman et al. 1970, Somerville et al. 1970, Ignoffo et al. 1977a,b; Beegle
et al. 1981, Li et al. 1987) suggest that the enhancement of activity by
spore presence is due to a biological rather than a toxin factor. The degree
to which spores play a role in the pathology of B. thuringiensis
in any particular insect is probably determined by whether the insect in
question is a Type 1, 2, or 3 insect as per Heimpel & Angus (1959), which
may itself be influenced by larval age (Beegle et al. 1981). The need for
spore presence for maximum B.
thuringiensis activity with
some pest insects may be a limiting factor where plants have been engineered
to produce B. thuringiensis crystal toxin or
with products such as Mycogen's M-CAP which does not contain spores (Beegle
& Yamamoto 1992). A cherished attribute of B. thuringiensis
was the absence of resistance in target pests to bacteria/spore-crystal
complexes for >25 yrs of commercial use. However in the early 1980s
differences appeared in susceptibility to subsp. kurstaki spore-crystal complexes in populations of P. interpunctella in different storage bins (see Beegle &
Yamamoto 1992). Susceptibility decreased almost 30 times after two
generations of spore-crystal exposure and 100X after 15 generations of
exposure. McGaughey (1985) found that B.
thuringiensis resistance was
inherited as a recessive trait, and was stable after selection was
discontinued. Soon thereafter, resistance was found in populations of Cadra cautella (Walker) (McGaughey & Beeman 1988), H. virescens (Stone et al. 1989), and Plutella xylostella
(L.) (Tabashnik et al. 1990). In H.
virescens, resistance
developed to a genetically engineered P.
fluorescens containing a
130-kDa subsp. kurstaki
protoxin. Van Rie et al. (1990) and Ferre et al. (1991) showed that
resistance of P. interpunctella and P. xylostella, respectively, was correlated with a reduction
in affinity of the midgut epithelium membrane receptors for crystal toxin
proteins. This offers not only an explanation for the mechanism of
development of resistance, but also the mode of action of the crystal toxins
(Beegle & Yamamoto 1992). Heat-tolerant Exotoxins (Beta-Exotoxins).--These
exotoxins were discovered by McConnell & Richards (1959), being known as
Beta-exotoxin, fly factor, heat-stable toxin, thermostable toxin and
thuringiensin. The thuringiensin in the literature is produced by vegetative
cells during growth and is a water-soluble, dialyzable nucleotide composed of
adenine, ribose, glucose, and allaric acid with a phosphate group (Farkas et
al. 1969). The use of "stable" is somewhat misleading because the
heat-tolerant exotoxin activity of some subspecies (e.g., tolworthi) degrades slowly with
autoclaving, but others (e.g., thuringiensis)
contains isolates that produce exotoxin whose activity is not reduced by
autoclaving at 125°C for 30 min
(Mohd-Salleh et al. 1980). It was believed that the heat-tolerant exotoxins
produced by different subspecies of B.
thuringiensis were
identical. But studies by Mohd-Salleh et al. (1980) and Gingrich et al.
(1922a, 1922b) revealed the existence of more than one species of
heat-tolerant exotoxin. Levinson et al. (1990) chemically confirmed the
existence of a second heat-tolerant exotoxin, which they called II
Beta-exotoxin. They found the new exotoxin more specific than I
Beta-exotoxin, and very active against L.
decemlineata. The mode of action of type I Beta-exotoxin is inhibition
of DNA-dependent RNA polymerase (Sebesta & Horska 1970), thus it has a
very wide host range. Two Beta-exotoxin-containing products are produced and
used in the former USSR, Turingin 1 and 2 (2 and 10% B-exotoxin,
respectively) and Bitoxibacillin (0.6- 0.8% B-exotoxin) (Weiser 1986). The
products are used effectively against several species of red mites, as well
as the larvae of house flies and blow flies. Detectable levels of
Beta-exotoxin are not allowed in B.
thuringiensis products in
Western Europe and North America. Abbott Laboratories developed the product
D-Beta based on Beta-exotoxin, and has attempted to obtain registration.
Various methods have been used to detect and quantify heat-tolerant exotoxins
of B. thuringiensis, such as larval fly, bacterial, and biochemical
assays. The method of choice by 1992 was high-performance liquid
chromatography (HPLC) (Campbell et al. 1987) . Heat-labile Exotoxin (alpha-Exotoxin).--A heat-labile
insecticidal exotoxin produced by B.
thuringiensis was reported
by Toumanoff (1954). It was found that a precipitated protein from subsp. alesti culture filtrates was
toxic to G. mellonella larvae. Smirnoff
(1964) found a heat-labile substance in Thuricide product filtrate (0.3
millimicrons) that was toxic to Lepidoptera, Coleoptera, Diptera, Orthoptera
and Hymenoptera. Krieg (1971a) observed that sterile filtered supernatants
from subsp. alesti and galleriae broths were toxic per
os to Plutella xylostella (L.) larvae. He
found that the toxic substance was precipitated by two-thirds ammonium
sulfate saturation, and that activity was destroyed by autoclaving and
trypsin. Krieg (1971b) determined that the substance was not a lecithinase
(also called phospholipase or more accurately phosphatidylcholine choline
phosphohydrolase). Heimpel (1967) had coined the term alpha-exotoxin for the B. thuringiensis heat-labile exotoxin, and he assumed that it
was lecithinase C. Ivinskiene (1978) conformed that alpha-exotoxin and
lecithinase from B. thuringiensis were not the
same. Krieg & Lysenko (1979) believed that alpha-exotoxin was only
demonstrable by bioassay and that it could not be chemically isolated or
purified. But Krieg (1986) estimated that the size of alpha-exotoxin was
45-50 kDa, as estimated by gel filtration. One of the deficiencies of the
work with alpha-exotoxin is the failure to consider the extensive B. cereus toxin literature, as B. thuringiensis
is similar, if not identical, to B.
cereus except for an
insecticidal crystal. Among the B.
cereus toxins listed by
Turnbull (1981) are several that are lethal to mice, a trait also of the B. thuringiensis alpha-exotoxin. The one that appears to be
most like alpha-exotoxin is the "diarrheagenic" toxin, which as a
size of ca. 50 kDa and a pI of 4.85. Pathogenic Modes.--Although the spore may play a role in the pathogenicity
of B. thuringiensis, the parasporal body causes the rapid
paralysis and final death of insects (Aronson et al. 1986, Hofte &
Whiteley 1989, Huber & Luthy 1981, Moar et al. 1989). In the subspecies kurstaki, the parasporal body
dissolves after ingestion when coming into contact with the alkaline
environment of the midgut (pH 8-10). Many of the toxins have been found to be
actually protoxins of ca. 133 kDa (e.g., CryI, CryIVA, CryIVB) from which
active toxins of 60-70 kDa are cleaved by proteases. The activated toxin
molecules pass through the paratrophic membrane and bind to specific
receptors on the microvilli of the midgut epithelium. Binding is an essential
step in intoxication, and in susceptible insects the toxicity of a particular
B. thuringiensis protein is correlated with the number of
specific binding sites on microvilli and the affinity of the Bacillus molecules for these
sites (Hoffmann et al. 1988, Van Rie et al. 1989, 1990). Nevertheless,
binding alone does not always lead to toxicity, which suggests that insertion
and probably some kind of processing in the midgut membrane is necessary to
achieve toxicity (Wolfersberger 1990). The microvillii lose their typical
structure within minutes of binding, the cells becoming vacuolated and they
swell (Huber & Luthy 1981, Luthy & Eberrsold 1981). Swelling
continues until the cells lyse and detach from the basement membrane of the
midgut epithelium. Alkaline gut juices leak into the hemocoel as the cells
are detached, which causes the hemolymph pH to rise by ca. 0.5, ending in the
paralysis and death of the insect (Heimpel 1967, Heimpel & Angus 1963). The actual process of intoxication at the molecular level
is as yet unknown, especially the events occurring after the toxin binds to
the receptor. Evidence suggests an immediate influx of potassium and calcium,
after which the cell takes in water to balance these cations. As an
explanation of this cationic influx, it was proposed that B. thuringiensis molecules either act directly on a potassium
pump (Wolfersberger 1989), or insert into the microvillar membrane forming
transmembrane cation pores (Chilcott et al. 1990, Knowles & Ellar 1987).
Other evidence suggests that the Bacillus
molecules may act inside the cell (Schwartz et al. 1991), so that the mode of
action may be even more complex (Gill et al. 1991). Bacillus thuringiensis
for Insect Control.--Previously discussed attributes of fast action, low cost
and ease of mass production and deployment, and safety for nontarget organisms,
have caused the production of many commercially available products based on
different isolates and subspecies. The most successful isolate has been the
HD 1 isolate of B. thuringiensis kurstaki, which is the active
ingredient in many commercial types used to control larvae of Lepidoptera in
varied habitats (Morris 1982). For mosquito and simulid control, a range of
commercial products based on ONR 60A isolate of B. thuringiensis
israelensis are available
(Mulla 1990). The latter has been used by the World Health Organization for
onchocerciasis control in West Africa (Guillet et al. 1990, Lacey &
Undeen 1986). Also available are commercial products based on isolates of B. thuringiensis morrisoni
for Coleoptera, specifically larvae and adults of the Colorado potato beetle,
Leptinotarsa decemlineata (Say). Production typically involves submerging cultures on
protein rich corn steep media in large fermentation vats (Dulmage et al.
1990). A slurry is produced from fermentation, which contains sporulated,
lysed cells, which are frequently spray dried before formulation. A less
sophisticated method of production may simply involve bacteria grown on grain
and grain hulls in open shallow trays. Formulations include dusts, wettable powders or
emulsifiable concentrates that are applied as chemical insecticides. The
dosage varies within the range of 250-500 grams per ha., of which only 25-30%
consists of active proteins. Resistance to B.
thuringiensis is a reality
(McGaughey 1985, Tabashnik et al. 1990), and it is not known as of 1992
whether new strains might be able to keep ahead of the process as usage
expands. Standardization of B.
thuringiensis Products.--As previously discussed there were early problems with
standardization of the killing power of B.
thuringiensis-based products
because of the unfortunate use of spore counts. There is no relationship
between the number of spores in a preparation and its insect killing power,
which is true for both lepidopterous (Dulmage & Rhodes 1971) and
dipterous (Smith 1982) active isolates. Bonnefoi et al. (1958) published a
description of a standardization procedure using insect bioassay and
comparison of the resulting LC50s and LC50 of a concurrently bioassayed
standard to correct for daily LC50 fluctuations. They also mentioned
generating "biological units" as a measure of the potency of a
preparation based on comparison with a standard. Menn (1960) first published
a procedure in North America for bioassaying B. thuringiensis.
Mechalas & Anderson (1964) of Nutrilite Products first advanced in North
America the use of standard and potency ratios. Much of the basic bioassay
methodology advanced by Mechalas while at Nutrilite is still sued in
standardization. The first international effort came in 1964 at the
International Symposium on the Identification and Assay of Viruses and Bacillus thuringiensis Berliner Used for Insect Control, held in
London, England. Two notable resolutions adopted were (1) that spore count
was not sufficient and (2) the recommendation that LC50s and standard
preparations be used. The second B.
thuringiensis
standardization meeting was the 1966 Symposia on the Standardization of
Insect Pathogens held in Wageningen, Netherlands. It was recommended that a
preparation of B. thuringiensis subsp. thuringiensis produced by
Institut Pasteur in Paris, be adopted as the international primary standard
for the bioassay of B. thuringiensis preparations. The
standard was assigned a potency of 1000 international units per milligram,
and designated as E-61 (Burges et al. 1966). Many researchers contributed to
solving the standardization problem, including Bonnefoi & Burgerjon of
France, Krieg & Herfs of Germany, and Mechalas of Nutrilite Products and
Fisher of IMC in the United States. But the one probably most responsible in
Europe was Denis Burges of England and Art Heimpel in North America (Beegle
& Yamamoto 1992). There have been a number of B. thuringiensis
standards developed sine E-61. E-61 is still viable and available from the
Institut Pasteur, and was used in the standardization of both HD-1-S-1971
(Dulmage 1973) and HD-1-S-1980 (Beegle et al. 1986). The HD-1-S-1971
preparation was the U.S. kurstaki
reference standard in use from 1971 to 1980. By 1992 the U.S. kurstaki standard was
HD-1-S-1980. it is used to determine potencies of lepidopterous-active B. thuringiensis-based products. Unfortunately, the publicly
held supply of HD-1-S-1980, not at the USDA Northern Regional Res. Cent. in
Peoria, IL. has lost about 2000 IU per milligram and thus is assaying about
14,000 IU per milligram (P. Martinat, 1989, pers. commun. to C. Beegle &
T. Yamamoto). The potency loss is either the result of improper storage at
Weslaco, TX, or from being transported several times between 1986 and 1988.
But, there are a number of laboratories where HD-1-S-1980 was received before
1986, has been stored properly and is fully active. Salama et al. (1989)
developed a subsp. entomocidus-based
standard, HD-635-S-1987, for use in bioassays against Spodoptera spp., as HD-1-S-1980 has only small activity
against this genus. Another lepidopterous-active standard is CSBt5ab-87,
subsp. galleriae, developed
in the People's Republic of China (Beegle et al. 1991). There have been four
subsp. israelensis-based
standards developed for use in standardizing dipterous-active B. thuringiensis products. ÍPS-78 and IPS-80 lost potency in
storage and were replaced by IPS-82, which has been stable; and the
HD-968-S-1983 standard was lost, which leaves IPS-82 as the only subsp. israelensis-based standard available
in 1992 (Beegle & Yamamoto 1992). Two lepidopteran and two dipteran standard bioassay
techniques have been proposed. Additionally, two bioassay techniques have
been described and compared for use in bioassaying subsp. tenebrionis-based products against
coleopterous larvae (Riethmuller & Langenbruch 1989). The first
lepidopteran bioassay, developed by a joint industry and U.S. Dept.
Agriculture effort, was based on T.
ni larvae as the bioassay
insect and a diet-incorporation technique using semisynthetic diet (Dulmage
et al. 1971). But this assay had two drawbacks; antibiotic was specified to
be used in the semisynthetic diet, which can have variable effects depending
on the age of the bioassayed larvae (Beegle et al. 1981), and the ability of
a standard to correct for differences in assay methods was overestimated.
Later it was found that the use of a standard does not in all cases correct
for differences in assay techniques (Beetle 1990). Therefore, a new bioassay
for determining potencies of lepidopterous-active B. thuringiensis
preparations was developed jointly by the U.S. Dept. Agriculture and U.S. B. thuringiensis producers (Beegle et al. 1991). There are
two standardized bioassays for mosquito-active preparations. The first is the
World Health Organization 1981 bioassay with flexible protocols to allow for
differing conditions and materials available in different parts of the world,
and the second is the U.S. standard bioassay with specific protocols
(McLaughlin et al. 1984). Bioassay of B.
thuringiensis products is
costly, time consuming, prone to problems. Therefore, it has been suggested
that insect bioassays be replaced with chemical or in vitro assays. Winckler
et al. (1971) published a description of an immunochemical technique to
determine the amount of crystal protein and its relationship to insecticidal
activity as determined by insect bioassay. The technique takes 48 h and has a
98-107% agreement with insect bioassay, depending on the number of
determinations and samples. Andrews et al. (1980) modified this technique to
shorten the required time to 4 h. Smith & Ulrich (1983) developed a
non-competitive ELISA technique for quantitative detection of B. thuringiensis crystal protein, which also takes 4 h and
gives closer agreement with insect bioassay values than does rocket
immunoelectrophoresis. Brussock & Currier (1990) reported on using
SDS-PAGE to measure successfully the amount of crystal toxin protein in
production samples. There are some important cautions to be aware of when considering
using chemical assays to standardize B.
thuringiensis products. The
desired information is the killing power of the preparation toward target
pests. The host spectrum of a preparation is determined by the type of
crystal toxin present in the preparation, often differing by only a few amino
acids in the N-terminal region of the active fragment from other crystal
toxins with different host spectra. Isolates with multiple crystal toxin
genes, such as the HD-1, can yield products with somewhat differing host
spectra in different fermentation batches. The killing power of a preparation
is determined by both the quality and quantity of crystal toxin present.
Chemical methods only measure the quantity of toxin present and not its
quality. All published chemical methods use crystal toxin produced, harvested
and stored under certain often ideal conditions. In commercial production,
very large batches are produced under varying conditions, different media,
recovered by techniques such as spray drying and in the course of formulation
can be ground or milled, some of which may damage the crystal protein.
Possibly with the exception of work by Tyski (1989), chemical assays cannot
reliably distinguish between undamaged and damaged crystal protein (Beegle
& Yamamoto 1992). Also three are a number of pest insects that require
the presence of spores for maximum toxin activity, as previously discussed.
Chemical methods cannot measure the presence, number or viability of spores.
Significantly, Abbott Laboratories, pioneers in the development of chemical
assays of crystal toxin and the world's largest producer of B. thuringiensis products, still standardizes their
preparations by insect bioassay. Biotechnology or Genetic
Engineering.--Zakharyan et
al. (1976) first reported the presence of plasmids in B. thuringiensis.
Stahly et al. (1978) heat-shocked subsp. kurstaki
spores and isolated a small number (0.5-1.0%) of Spo+ Cry- colonies that had
lost all of their plasmids. This suggested involvement of the lsot plasmids in
crystal formation. Gonzales et al. (1981) isolated a series of mutants from
several different B. thuringiensis isolates, and
demonstrated that production of crystal protein ceased when a large plasmid
was lost. The presence or absence of crystal toxin activity in Cry+ and Cry-
variants was confirmed by insect bioassay. Gonzalez et al. (1982) further
reported that the plasmids bearing the crystal genes were transmissible to B. cereus, which then produced crystals. Schnepf & Whiteley (1981) cloned one of the subsp. kurstaki crystal toxin genes
into the pES1 plasmid in Echerichia
coli. The 130-kDa crystal
protein was expressed as shown by a positive antibody reaction to crystal
protein and toxicity to M. sexta larvae. Klier et al.
(1982) cloned a crystal gene from a strain of subsp. thuringiensis. A large number of reports of the cloning of
crystal toxin genes followed. Because crystal toxin proteins are highly
homologous, a cloned crystal toxin gene can be used to find or probe other
crystal toxin genes. Crystal toxin genes of the other two pathotypes also
were cloned. Ward et al. (1984) first cloned one of the crystal toxin genes
of subsp. israelensis
(pathotype B), and three different groups published simultaneously reports of
cloning the crystal toxin gene of subsp. tenebrionis
(pathotype C) (Sekar et al. 1987, Hofte et al. 1987, Jahn et al. 1987). Li et al. (1991) reported on the structure of subsp. tenebrionis toxin called
CryIIIA worked out by David Ellar at the University of Cambridge. They used
X-ray crystallography to resolve the structure of 2.5 Ang. The CryIIIA toxin
is made of 644 amino acid residues that are divided in three domains called
Domain I, II, III. Domain I, an assembly of several alpha-helices, is thought
to be involved in the membrane-spanning (poreforming) function. Domain II is
considered to be the receptor binding domain and has repeating beta-sheet
structures. Domain III is also rich in the beta-sheet structure, and is
thought to protect the other domains from proteinase digestion. It is
imperative to have the structural information when attempts are made to
engineer a new toxin protein. The CryIIIA structure determined by Ellar's
group clearly shows the orientation of each amino acid residue. This
information makes it possible to determine the involvement of each amino acid
residue in the insecticidal activity. Four research groups by 1987 reported success in obtaining
the expression of B. thuringiensis crystal toxin in
plants: Adang et al. 1987, Barton et al. 1987, Vaeck et al. 1987 in tobacco
and Fischhoff et al. 1987 in tomato. Initially the level of expressed crystal
toxin was very low, ca. 10,000-fold less than that produced in native B. thuringiensis cells. These low levels (<0.001% of total
soluble plant protein) were only effective against Manduca spp. larvae, which are extremely sensitive to the
crystal toxin. Monsanto scientists raised the expression level of crystal
toxin to 0.1% by modifying the nucleotide sequence of the structural subsp. kurstaki gene. The 0.1% level
of crystal toxin was reportedly sufficient to control such economically
important pests as Heliothis
spp. larvae (Fuchs et al. 1990, Perlak et al. 1990). Other plants such as
cotton, soybean and cabbage also have been transformed with B. thuringiensis crystal toxin genes. Several biotechnology companies attempting to create novel
B. thuringiensis products, have produced some interesting
genetic engineering results. Crop Genetics International placed the crylA(c)
gene from a kurstaki isolate
into Clavibacter xyli subsp. cynodontis Davis (Cxc), which
is a fastidious, Gram-positive, coryneform bacterium that exists only in the
xylem of Bermuda grass in nature (Dimock et al. 1988). Corn plants inoculated
with Cxc containing the B. thuringiensis crystal toxin
gene (Cxc/Bt) develop populations of up to 1 X 1010 Cxc/Bt cells
per ml. xylem sap in the vascular system of the stem at the soil line. In
vitro production of B. thuringiensis crystal toxin in
Cxc/Bt is ca. 0.1% of cell protein; less is produced in the xylem of corn
plants. Crop Genetics International estimated that ca. a 10-fold increase in
toxin expression is necessary for satisfactory control of O. nubilalis larvae. If that is achieved they may market seed
corn impregnated with Cxc/Bt (Beegle & Yamamoto 1992). Ecogen has constructed isolates containing new gene
combinations using a natural conjugal plasmid exchange system which exists in
B. thuringiensis (Carlton 1988). This has led to the products
Condor and Foil. With Condor they selected a plasmid-cured isolate having ca.
2X the activity of HD-1, and mated it to another isolated containing a
plasmid that coded for a crystal toxin having high activity toward gypsy moth
larvae. This resulted in a transconjugant having ca. 7.5-fold higher activity
than HD-1 against gypsy moth larvae. Because potato plants have both
lepidopteran and coleoptran pests, and because there are no native B. thuringiensis isolates that possess spore-crystal complex
activity against both groups, Ecogen developed a transconjugant isolate active
against both lepidopteran and coleopteran larvae. They mated a
coleopteran-active isolate found in Kansas soybean storage dust to a variant
of a wild type subsp. kurstaki
isolate having ca. 3X higher activity towards European corn borer larvae,
which isolate is the basis of their product Foil (Beegle & Yamamoto
1992). Mycogen produced two biologically encapsulated products
called MVP and M-Trak, active against lepidopteran and coleopteran pest
larvae, respectively (Gelernter 1990). They transferred the lepidopteran- and
coleopteran- active crystal toxin genes into a non-pathogenic strain of Pseudomonas fluorescens. The transformed P. fluorescens cells are grown in submerged culture, and after
they have formed toxic crystal inclusion bodies within the cells, they are
killed by heat and chemical treatments. The dead Pseudomonas cell walls are reported to be more rigid due
to cross linking of the cell walls components, resulting in a protective
microcapsule which encloses the respective crystals. The encapsulated
products reportedly have about 2X the activity persistence on field crops
compared with conventional products (Beegle & Yamamoto 1992). Relatively low levels of B. thuringiensis
crystal toxin is produced in transformed organisms, especially plants,
therefore quantification of the toxin is difficult. Monsanto found that the
low toxin levels in transformed plants precluded the incorporation of a
graded series of dilutions of homogenized plant parts into insect diet (Fuchs
et al. 1990). Depending on the sensitivity of the test insect, it was found
possible to obtain responses using detached leaf assays. Instead of using a
graded series of dilutions (ca. 7) to obtain a dosage mortality response,
they used larvae of seven different insect species which varied in their
sensitivity to the crystal toxin to obtain a graded response. This technique
enabled them to quantify toxin levels in transformed plant parts. Monsanto
compared their leaf assay with ELISA and Western blot techniques, and found
that the leaf assay was the most and Western blotting the least sensitive
(Beegle & Yamamoto 1992). Transconjugate and genetically engineered strains with
unique properties are approaching the market. He mentioned that Ecogen, Inc.
of Langhorne, Pennsylvania combined two different plasmids (one encoding
protein CryI toxic to Lepidoptera, another, CryIII, toxic to Coleoptera, into
a single bacterial strain. The transconjugate strain has an expanded host range
including both orders of insects. Also, Mycogen Corp. of San Diego, Calif.,
received registration for the first genetically engineered bacterial
insecticide, MVP, a killed strain of Pseudomonas
fluorescens that contains a B. thuringiensis toxin active against Lepidoptera (Feitelson
et al. 1990). This strain shows greater residual activity in the field,
obtained by fixing the Pseudomonas
cell wall around the Bacillus
protein. Because B.
thuringiensis genes may be
manipulated easily using recombinant DNA technology, probably a variety of
different approaches to controlling insects based on insecticidal proteins
will develop. An example is the fusion proteins with an expanded host range
that were made by fusing two B.
thuringiensis genes (Honee
et al. 1990). Also, some rather controversial developments include the
successful engineering of B.
thuringiensis genes into
plants such as cotton, tomato, tobacco, potato, walnut, etc. (Fischhoff et
al. 1987, Gould 1988a,b; Perlak et al. 1990, Vaeck et al. 1987). In these
only a single B. thuringiensis gene was
transformed into the plant species, and Stone et al. 1989) pointed out that
single genes are prone to develop resistance rapidly. This work was
controversial principally from the standpoint of the high risk that the
release of such plants would pose for resistance development in both
conventional and engineered products, which are applied as microbial
insecticides. Although there is no direct evidence that transgenic plants are
unsafe for human consumption, the question of their safety was raised
(Goldburg & Tjaden 1990). Beta-exotoxin.--Many isolates of B.
thuringiensis secrete a
thermostable exotoxin during vegetative growth, named variously as
Beta-exotoxin, Thuringiensin or "fly factor." This toxin is an unusual
nucleotide which acts as a competitive inhibitor of messenger RNA polymerase
(see Lecadet & DeBarjac 1981, Sebesta et al. 1981). The beta-exotoxin is
a teratogen, causing abnormal development and death in many different
insects. It is also harmful in high concentrations on mammals and other
vertebrates, and thus has not been registered in the United States. However,
in Finland and parts of Africa it is allowed in formulations for control of
filth breeding flies and in the former Soviet Union for the Colorado potato
beetle. Beegle & Yamamoto (1992) concluded that in 90 years B. thuringiensis has gone from a laboratory curiosity to by
far the most successful microbial pest control agent. It is the basis of the
vast majority of biotechnology efforts with insect pathogens. This progress
has resulted from numerous researchers efforts. Because of the attention that
geneticists, biochemists and protein chemists have given go B. thuringiensis, enormous progress has been made in the
toxin's genetics, identity and mode of action. They speculated that we may be
close to realizing the dream of designing B.
thuringiensis crystal toxins
that will be effective against pest insects which historically have been
unaffected by the spore-crystal complexes. Other Bacteria
Species Isolate 2362 of B.
sphaericus Neide was being
developed in 1992 for control of mosquito larvae and the path+ isolate of Serratia entomophila, a non-sporeformer for control of the grass
grub, Costelytra zealandica, a scarab pest in New
Zealand. Similar to B. thuringiensis, B. sphaericus produces a protein toxin that kills larvae by
cytolysis of the midgut epithelium (see Baumann et al. 1991, Davidson &
Younsten 1990, Singer 1990). The genes encoding the toxin have been cloned and
sequenced, and analyses suggests that the toxin is not related to any of
those known from B. thuringiensis (Baumann et al.
1988). The strain of S. entomophila under study causes
and infectious disease in scarabs, entering the larva through the gut and colonizing
the hemolymph, causing death. However, unlike B. popilliae,
S. entomophila is easily mass cultured on artificial media. Generalizations.--A large group of
organisms belonging to the Phycomytes, Oomycetes, Zygomycetes and Imperfect fungi
cause diseases of insects and can reduce their numbers. Many are easily
cultured and several are in use for biological control. Among the Phycomycetes the fungus Myiophagus ucrainicus
plays an important role in natural control of citrus mites and scales in
humid tropical regions and plantations with overhead sprinkling. The
yellowish infected scales are located on the sides of leaf veins. During
rain, the sporangia burst open and release flagellate zoospores which infect
new scales. This process was used for the artificial spread of the fungus
when branches with infected scales were brought into uninfected plantations
and attached to the tops of trees. During rainy periods and under sprinklers,
the zoospores invade the new habitat. The Entomophthoraceae belonging to the Zygomycetes often
cause large epizootics among insects of one species over a broad area of
several thousand hectares. This is the case with Noctuids or grasshoppers or
in many forest pests. The fungi can be isolated on coagulated egg yolk from
fresh infected insects where conidia are spread around the infected animal.
Old contaminated insects are not good for such isolation. The isolated fungi
can be maintained on artificial media and even mass produced in fermentors,
but the application of the fungus is difficult due to low infectivity of such
isolates. Entomophthora virulenta isolated in this way
was distributed in cultures in plastic cups inverted over alfalfa in
California thereby infecting aphids in this crop. Infected grasshoppers, caterpillars
and houseflies with the fungus were released and infections were initiated in
dense host populations. This is also the case in Strongwellsea castrans
infecting the beet fly. Adults with apparent infection have an opening in the
abdomen wherefrom conidia are dusted about during mating and flight. The most developed of microbial insecticides involve the
fungi imperfecti. These fungi grow well on artificial media and can be mass
produced for field application. In deep fermentation on liquid media short
hyphal cells, the blastospores, are formed. These are less resistant, and
survive only 3-4 months when properly stored. The airborne conidia are
produced only on stagnant media such as sterilized plant materials such as
cooked grain, rice and potatoes, or liquid artificial media. This production
is adaptable for local or cottage production. A transient method between
large scale and cottage type is the production in plastic bags on rice or on
liquid medium. With some modifications all deuteromycetes can be produced in
this way in rather large quantities (Weiser 1984). Beauveria bassiana, the white muscardine fungus, is an insect pathogen with
a broad host range. It kills adult houseflies, different beetle grubs and
adults. Examples are the Colorado potato beetle, snout beetles on roots of
strawberries or seedlings, different scale insects and white flies. The
fungus is not commercially produced , but cottage production is available. In
the greenhouse optimum temperatures of 20-25°C and a high RH for the period after application should be
guaranteed. Use of these fungi in the field is more complicated due to low
RH. Verticillium is useful
in dense cultures during a rainy period. Applications of insecticides with or
independently of the fungi is possible. Fungicides may reduce the speed of
action, but with the exception of Captan, Clorothalonin, imazalil, maneb and
thiram, they can be used independently or in treatments postponed for five
days. Other imperfect fungi such as Metarhizium anisopliae,
the green muscardine fungus or Paecilomyces
farinosus, and the rosy
muscardine fungus have an overlapping range of hosts with Beauveria. They are produced
under local conditions on plant materials, such as grains and rice, on traces
or in plastic bags inoculated with sterile conidia. Harvesting after 14 days
yields conidia together with remains of the nutrient and conidia that are
washed off with water or collected with an exhaust apparatus. The temperature
range of Metarhizium is
lower than Beauveria (15-20°C) and the range of Paecilomyces
drops to 12-18°C. Both are
typical soil inhabiting fungi. The low temperature activity range of Paecilomyces is useful in its
main habitat, the soil of coniferous forests where it infects hibernating
insect larvae (Weiser 1984). Nemourea rileyi is a common fungus infecting noctuids in alfalfa fields
and other crops in America. It is developed there as a microbial insecticide.
Another fungus, Hirsutella thompsoni is used for control of
mites under the trade mark Mycar. It has a short shelf life but is a rather
efficient acaricide. The produce is on the market only as an experimental
formulation pending improvement of storage. Fungi comprise a large group of eucaryotes that are
distinguished by the presence of a cell wall, as in plants, but
lacking in chloroplasts. They live either as saprophytes or parasites
of plants and animals, requiring organic food for growth which obtained by
absorption from the substrate. The vegetative phase is known as a thallus
and can be either unicellular, as in yeasts, or multicellular and
filamentous, forming a mycelium, the latter being characteristic for most of
the fungi attacking insects. During vegetative growth the mycelium consists
primarily of hyphae which may be either septate or non-septate,
which grow throughout the substrate in a quest for nutrients. Reproduction
may be either sexual or asexual, during which phase the
mycelium produces specialized structures such as motile spores, sporangia and
conidia which are usually the agents by which infection occurs. Fungi are
best adapted to wet or moist habitats, and are usually easily cultured on
artificial media. There are five major subdivisions of fungi, reflecting the
evolution of the biology of fungi from aquatic to terrestrial habitats. Salient Biological
Properties.--Fungi usually
infect insects by active penetration through the cuticle, which makes them
attractive for control of insects with sucking mouthparts. Typically the life
cycle begins when a spore, either motile or a conidium, alights on the
cuticle. Spores usually germinate quickly, producing a germ tube that grows
and penetrates the cuticle, entering the hemocoel (St. Leger 1992). Hyphal
bodies bud from the penetrant hyphae and either continue to grow and divide
in a yeast-like way or elongate, forming hyphae that grow throughout the
insect. Complete colonization of the body usually requires 7-10 days, after
which the insect dies. Some fungi produce peptide toxins during vegetative
growth, and in these strains death may occur within 48 hrs. If conditions are
favorable, the mycelium forms reproductive structures and spores, thereby
completing the life cycle. These may be produced either internally or
externally, and can be motile spores, resistant spores, sporangia or conidia,
according to the species. For microbial control, the ability of fungi to
infect insects through the cuticle gives them advantage over viruses,
bacterial and protozoa. Effective development would make them useful against
the wide range of important insect pests with sucking mouthparts such as
whiteflies, leafhoppers, scale insects and aphids. Detailed
Characteristics.--Fungi are
common pathogens which cause insect diseases in the field, and outbreaks of
fungal diseases under favorable conditions often lead to spectacular
epizootics that reduce populations of specific insects over large areas
(Andreadis & Weseloh 1990, Carruthers & Soper 1987). Therefore, there
has been great interest in using fungi to control insects for over a century,
with the first efforts deploying Metarhizium
anisopliae (Metchnikoff) for
wheat cockchafer, Anisoplia austriaca Hubst. control. in
Russia (Krassilstschik 1888, Steinhaus 1949). Numerous attempts since then to
develop fungi as microbial insecticides have, however, not been very
successful. There are presently (1992) no commercially available fungal
insecticides in industrialized countries. But, fungi imperfecti such as M. anisopliae and Beauveria
bassiana (Balsamo) are
produced and used in several developing areas of the world. A "cottage
industry" in Brazil and China and a semi commercial product
"Boverin" in the former Soviet Union are present. Efforts to find
alternatives to chemical insecticides have intensified research on fungi,
which could lead to the successful use of fungi as both classical biological
control agents and microbial insecticides. (Please refer to the following for
additional information on fungi: Ferron 1978, Hall & Papierok 1982, McCoy
1990, McCoy et al. 1988 Several examples to illustrate the advantages and
disadvantages of this group of organisms as biological control agents as
follows: Aquatic Fungi.--Coelomomyces
& Lagenidium.--The genus Coelomomyces
comprises >70 species of obligately parasitic fungi with complex life
cycles involving alternation of sexual (gametophytic) and asexual
(sporophytic) generations (Couch & Bland 2985, Whisler 1985). The sexual
phase parasitizes a microcrustacean host, typically a copepod, while the
asexual generation develops, with rare exception, in mosquito larvae. A
biflagellate zygospore invades the hemocoel of a mosquito larva where it
produces a sporophyte that colonizes the body, forming resistant sporangia.
The larva dies and subsequently the sporangia undergo meiosis, producing
uniflagellate meiospores that invade the hemocoel of a copepod host, where a
gametophyte develops. At maturation the gametophyte cleaves, forming
thousands of uniflagellate gametes. Cleavage results in death of the copepod
and escape of the gametes which fuse, forming biflagellate zygospores that
seek out another mosquito host, completing the life cycle. The life cycles of
these fungi are highly adapted to those of their hosts, and as obligate
parasites they are very fastidious in their nutritional requirements, so that
no species of Coelomomyces
has been cultured in vitro. Coelomomyces is the largest genus of insect parasitic fungi, being
reported from numerous mosquito species many of which are vectors of malaria
and filariasis. In some species epizootics caused by Coelomomyces may kill >95% of the mosquito larval
population (Chapman 1985, Couch & Umphlett 1963). Considerable interest developed in Lagenidium giganteum,
an oomycete fungus with two important advantages over Coelomomyces: ease of culture on artificial media and no
need for an alternate host (Federici 1981). In the life cycle, a motile
zoospore invades a mosquito larva through the cuticle; and once within the
hemocoel, the fungus colonizes the body ind 2-3 days, producing an extensive
mycelium consisting mainly of non-septate hyphae. Near the end of growth, the
hyphae do become septate, and out of each segment an exit tube forms which
grows back out through the cuticle, forming zoosporangia at the tip. Zoospores
quickly differentiate in these, exiting through an apical pore to seek out a
new substrate. Also, thick walled resistant sexual oospores may be formed
within the mosquito cadaver. Both zoosporangia and oospores may be produced in vitro. But field trials in California and North Carolina
have revealed that the zoosporangia are too fragile for routine use in
practical control. However, the oospore is quite stable even though
germination is unpredictable. Field tests show that germination of even a small
percentage of oospores can result in epizootics, leading to season-wide
mosquito control (Kerwin & Washino 1987). Lagenidium remains a promising candidate for successful
commercial development. Because oospores can overwinter, less frequent
applications my be necessary in subsequent years. The aquatic hyphomycete fungi, Culicinomyces clavosporus
Couch and Tolypocladium cylindrosporum have also been
considered for use in mosquito control (Federici 1981, Soares & Pinnock
1984). At present high production costs and unclear control results in the
field have curtailed their use. Terrestrial
Fungi.--Terrestrial
fungi have received the most attention in biological control, with most
emphasis being placed on species of hyphomycetes such as M. anisopliae
and B. bassiana. Also, the more specific and nutritionally
fastidious entomophthoraceous fungi continue to receive attention, but rather
for their potential use as classical biological controls than as microbial
insecticides. Entomophthorales.--This is a large order of zygomycete fungi containing
numerous genera, many species of which are found parasitizing insects and
other arthropods. They routinely cause localized and in some cases widespread
epizootics in populations of Hemiptera and Homoptera, particularly aphids and
leafhoppers, but also in grasshoppers and caterpillars. A few species of the
genus Conidiobolus cause
mycoses in some mammals, including humans (Humber 1989, Humber et al. 1989).
However, most of the entomophthoraceous fungi are highly specific, obligate
parasites of insects and therefore pose no threat for non-target organisms.
Complex nutritional requirements have made culture in vitro
impossible, and these fungi are highly host specific. The conidia are
fragile, and the resistant spores, such as the oospores of Lagenidium are not easily
germinated. By providing an optimum habitat for the natural occurrence of
these fungi, they may be used in integrated control. There is also promise
from exotic strains and species, for classical biological control. Infection and host colonization by the Entomophthorales is
similar to that for other fungi, even though the types of reproductive
structures formed and the specific details of life cycles differ (MacLeod
1963). If environmental conditions are favorable after infection (e.g., high
RH), the mycelia generate conidiospores, typically growing ut of the insect
cadavers and form fragile primary conidia at their tips. Such conidia
are discharged in order to settle on another insect, and form a distinctive halo
of conidia on the substrate around a dead insect. This is an effective method
of dissemination where insects live in groups or develop high population
densities. A secondary conidium may be generated if the primary
conidium fails to locate a suitable substrate, repeating the process. This
can continue for several times or until the conidial nutrient reserves are
depleted. There can also be formed within the insect, thick-walled resistant
sexual zygospores and asexual azygospores, both capable of
surviving in the dead insect or in soils, germinating years later. Common genera found attacking insects are Entomophthora on flies and
aphids, Conidiobolus on
aphids, Erynia on aphids, Zoophthora on beetles, aphids
and caterpillars and Entomophaga
on caterpillars and grasshoppers. However, Wilding (1981) considered that
none of these fungi offered much promise for commercial development.
Classical biological control with these fungi may offer more consideration,
as for example the introduction of Erynia
radicans from Israel to
Australia to control the spotted alfalfa aphid, Therioaphis maculata
(Buckton), has resulted in a permanent population drop (Milner et al. 1982).
A cultural modification enhanced control of alfalfa weevil, Hypera postica (Gyllenhal), where the first cutting of alfalfa
was moved forward in the growing season in order to concentrate hosts under
windrows when the air was warm and humid (Brown & Nordin 1982). Voronina
(1971) distinguished zones based on moisture and temperature, which led to
the development of a model that predicted epizootics by Entomophthora spp. in populations of the pea aphid, Acyrthosiphon pisum (Harris). Like most
integrated control, success depends on a thorough knowledge of ecology and
epizootiology. Hyphomycete Fungi.--These fungi belong to the subdivision Deuteromycotina
(Fungi Imperfecti), which was designated to include fungi for which the
sexual phase, or perfect state, is unknown. The group contains the species
that are considered to have the highest potential for development as
microbial insecticides, such as Beauveria
bassiana and Metarhizium anisopliae, causing,
respectively, the white and green muscardine diseases of insects. These
species have a broader host range and are thought capable of infecting most
orders of insects. Therefore, there is concern about the safety to non-target
organisms, such as parasitoids and predators, although natural occurrence in
these groups is rare. Workers working in mass culture of these fungi often
develop allergic reactions to the conidia. Life cycles are similar to that described previously for
other fungi (Roberts & Humber 1981). During invasion and colonization,
some species also produce peptide toxins that hasten the death of the host.
The conidium is the infectious stage, and the taxonomy for Hyphomycetes is
based mainly on the morphology of the reproductive structures, in particular
conidiophores and conidia (Samson 1981). Most species under commercial
development are easily grown on a variety of artificial media. Generally, B. bassiana and M.
anisopliae are being used
against insects in cooler and humid climates, such as beetle larvae in soil
and planthoppers in rice. Several other species with narrower host ranges are
considered potential useful, including Paecilomyces
fumoso-rosea for whiteflies, Verticillium
lecanii for aphids and
whiteflies indoors, Nomurea rileyi for noctuid larvae, and Hirsutella thompsonii for Acarina. There are several reasons for the
current lack of availability of these fungi in industrialized countries. Steinernematids &
Heterorhabitids Although
different groups of
nematodes are involved in the biological control of plant pests, in only two
instances has a commercial system of production been developed. Nematodes of
the group of Steinernematidae including the genera Steinernema (= Neoaplectana)
and Heterorhabditis. The
Steinernematidae are typical bacteria bearing nematodes which bring a
specific bacterium within the host along with an invasive larva. When inside
the host's body cavity, the larva releases the bacteria and grows to the
adult using the bacteria as food. The first parasites are a giant female and
male generation, while the next generation consists of normal males and females
which produce larvae which in turn leave the host and crawl around seeking
another host. The invaded host dies in 24-48 hrs. It is critical that the
larvae do not dry out. This group having a wide host range, includes Steinernema kraussei, the most cold
tolerant, attacking sawflies in forest soils and a group of former Neoaplectana: S. glaseri, S.
carpocapsae, S. bibionis, etc. adapted to higher temperatures. They all
attack Lepidoptera, Coleoptera, sawflies and Hemiptera. However, they do not
enter fly maggots, the latter feeding rather on the nematodes. They are very
efficient control factors of soil inhabiting stages of pests, especially of
the Colorado potato beetle, different grubs and hibernating noctuids. Once
found in a host, they can be cultivated on different caterpillars, usually on
Galleria larvae. The
invasive larvae are collected by washing the culture and are stored in bits
of foam plastic in a water suspension. They are introduced with these
substrates or washed out from the foam and sprayed in water over infected
plants with pests during the early morning or late afternoon. Invasive larvae
in foam plastic can be stored in closed containers with adequate moisture at
4°C for >1-yr.
Transportation in moist foam at temperatures over 25°C is not recommended. In the tropics, Heterorhabditis
is the most adapted genus. It has a higher working temperature of 20-30°C, but does not survive long at 4°C. It has to be maintained at room temperatures. Insects infected
by this nematode turn a brown color due to the activity of the bacterium
symbiont, which participates also in some insecticidal activity. The nematode
can be reared and produced in large quantities in the same way on
caterpillars, and does not survive desiccation. Nematodes are able to act independently of former
insecticide applications, and are able to find their hosts by the sense of
smell. They play important long lasting, self regulating roles in plantations
where they control the hosts in soil. Nematodes are safe for humans and vertebrates.
Nematodes are diverse but simple multicellular eucaryotes
in the Phylum Nematoda, which are the roundworms. There is a lot of variation
in biologies within the phylum. Many species are free-living, while others
are either facultative or obligate parasites. Generally, nematodes are
bilaterally symmetrical, elongate and vermiform, tapering at both ends, and
covered by a cuticle which is molted during development. Most species have a
stylet plus specialized feeding glands and an alimentary tract. Life stages
include an egg, several juvenile stages (larvae) and adults. Adults may be
sexually dimorphic as well as hermaphroditic. Species attacking insects vary
in size from less than a millimeter in length to 30 centimeters!. Biological Characteristics.--The groups of nematodes which have been most thoroughly
studied are the obligately parasitic mermithids (Family Mermithidae)
attacking mosquitoes, the facultatively parasitic steinernematids (Family
Steinernematidae) and heterorhabditids (Family Heterorhabditidae), which have
been considered for control of insects in soil or within trees, and the
facultatively parasitic neotylenchid nematodes (Family Neotylenchidae) of the
genus Deladenus that have
been developed for control of Sirex
wood wasps. The facultative parasites appear to hold the most potential for
biological control, which is due to the development of techniques for mass
culture on artificial (please see Gaugler & Kaya 1990, Petersen 1982
& Poinar 1979). Registration of nematodes as insecticides is not required
by the Environmental Protection Agency. Mermithids.--These nematodes are among the largest attacking
insects, adult females measuring from 5-20 cm. or more in length. The
advanced stages of developing nematodes can often be seen within the host
hemocoel where they appear as long, thin, white worms. Mermithids are
obligately parasitic, having been reported from many orders of insects and
from other arthropods such as crustaceans and spiders. Romanomermis culicivorax
and R. iyengari are the only species that were seriously
considered for biological control. These are capable of parasitizing many
species of mosquito larvae (Petersen 1982). The life cycle of R.
culicivorax illustrates the
biological control potential of these nematodes: Females are found in wet
soil at the bottom of aquatic habitats in which mosquitoes breed. After
mating, females lay thousands of eggs. The embryo develops into a first
instar juvenile over ca. one week, and then molts to a second stage juvenile
while still within the egg. The second, or preparasitic, stage hatches and
swims to the surface of the water where it seeks out, and with the aid of the
stylet, invades early instar mosquito larvae through the cuticle. The
immature larva grows over a period of 7-10 days within the hemocoel by
absorbing nutrients through its cuticle, and molts once during this time.
Upon completion of the parasitic phase, the third stage juvenile punctures
its way out through the cuticle of the host, thereby killing the mosquito. It
then descends to the bottom of the aquatic habitat where it matures without
feeding over 7-10 days, and molts to the adult stage. Adults mate and the
females lay eggs, completing the life cycle. Petersen (1982) and associates were able to mass culture R. culicivorax. In field studies, parasitism, and mortality,
of anopheline larvae ranged as high as 85% when pre-parasites were applied at
a rate of 2,400/m2. Levy & Miller (1977) obtained higher
levels of 96% parasitism when preparasites were applied at a rate of 3,600/m2
for control of floodwater mosquitoes in Florida. Romanomermis culicivorax
is able to recycle, providing some level of control in subsequent generations
of mosquitoes, but the level is usually not high enough to reduce the vector
potential of mosquitoes or alleviate the annoyance problem. This nematode is
very sensitive to chloride ions, and the methods developed for mass
production, storage, shipment and use were not cost-effective, especially
when compared to commercial formulations of B. thuringiensis
israelensis. As was true
with the furtherance of other organisms, such as planaria and hydra for
mosquito control, B. thuringiensis apparently has
halted efforts towards developing nematodes for biological control. It
remains to be seen whether the development of resistance to Bacillus will follow the same
route as with the chemical pesticides. Steinernematids
& Heterorhabitids.--Both are
small (<1-3 mm) terrestrial nematodes occurring most often in nature as
parasites of soil-inhabiting insects. They have simple and typical nematode
life cycles that include an egg, four larval stages and the adult. This group
of nematodes is distinguished by the fact that they have established a
mutualistic relationship with bacteria that are harbored within their alimentary
tracts. It is actually the bacteria that kill insects! The bacteria have
evolved specific relationships with individual species of nematodes, as in
the bacterial species Xenorhabdis
nematophilis (Thomas &
Poinar). This bacterium is associated with the steinermatid, Steinernema carpocapsae whereas X. luminescens is associated with the heterorabditid, Heterorhabditis bacteriophora. These nematodes
also produce an unusual quasi-resistant larval stage, or dauer larva,
the insect-infective stage, which is actually the 3rd instar juvenile
surrounded by the molted cuticle of the 2nd stage. The dauer larva finds and
infects the insect through the mouth, anus or the spiracles. Within the
hemocoel the nematode feeds on hemolymph and defecates, releasing symbiotic
bacteria. The latter colonize the insect, with death occurring 1-3 days
later. The nematodes feed on the bacteria and tissues of the dead larva,
mature and undergo 2-3 generations within the dead insect over a period of
1-2 weeks. Thousands of dauer larvae are produced, which leave the dead
insect in search of new hosts. The experimental insect host range of these nematodes is
broad, including >200 species of Coleoptera, Lepidoptera and Orthoptera. However,
they do not survive well in dry or unprotected environments. Best results are
obtained against beetle grubs or caterpillars in cryptic habitats, with
applications of 1-7 x 109 dauer larvae/ha. Control levels range from 70-99%
(Georgis & Hague 1991). Mass culture has been facilitated because the symbiotic
bacteria and nematodes can be grown on a variety of artificial media in vitro (Friedman 1990). Neotylenchid
Nematodes (Genus Deladenus).--The facultative parasitic nematode Deladenus siricidicola
was used to control Sirex
wood wasps in exotic pine forests in Tasmania and Victoria, Australia. Two
phases occur in the life cycle, a free-living phase that feeds on fungi in
trees, which are transmitted by the Sirex
wasps, and a parasitic phase that invades wasp larvae, develops in larvae and
adults, and destroys the eggs of adult females. The latter phase consists of
females that first develop as free-living nematodes, and then invade the wasp
larvae to complete their parasitic development. The free-living phase can be
mass cultured by feeding the nematodes the fungi grown in vitro.
They can then be inoculated into holes bored in trees in infested forest
areas. Bedding (1974) was able to attain parasitism rates of >90% of Sirex wasps over four years,
which reduced the number of trees killed from 200 to zero over that time
period and in a 1,000 acre forest in Tasmania. ------------------------------------- Van Driesche & Bellows Account Nematode Pathogens of
Arthropods Nematodes That Attack
Arthropods Steinernematidae
and Heterorhabditidae. Van Driesche & Bellows (1996) noted that nematodes
represent a single phylum, the Nematoda, within which about 9 families occur
that are parasitic on insects and have potential for use as biological
control agents. Nematodes are translucent, usually elongate, and cylindrical
in form. The body is covered with an elastic, noncellular cuticle, but is not
segmented. Unlike bacteria, viruses, and protozoa, nematodes are
multicellular animals that possess well-developed excretory , nervous,
digestive, muscular, and reproductive systems. However, they do not have
circulatory or respiratory systems. The digestive system consists of a mouth,
buccal cavity, intestine, rectum, and anus. Nematode taxonomy is based
largely on sexual characters of adults; so that, immature stages are
difficult or impossible to identify Nematodes are diverse, being found in nearly all habitats.
Nematodes may be free- living or parasitic on either plants or animals.
Nematode associations with insects range from phoresy to parasitism. Some
nematodes, such as Beddingia siricidicola, have complex life histories
with both parasitic and free living cycles that may continue indefinitely. Many nematodes have relatively simple life cycles with
three life stages: eggs, juveniles, and adults. Mated female nematodes
deposit eggs in the environment: the first juvenile stage usually molts
inside the egg and emerges as a second stage juvenile. Most nematodes molt
four times. In many groups, the third stage juvenile remains ensheathed in
the cuticle of the second stage, which provides it with increased resistance
to adverse conditions. This third stage form is called a dauer juvenile,
dauer being the German word for durability. Molting to the adult stage may
occur inside the host or free in the environment. All nematode stages, except
the egg, are mobile. Most nematodes have separate, single-sexed individuals
and mating is required. Nematode infections usually occur in the hemocoel, but in
some groups such as the Phaenopsitylenchidae (e.g., Beddingia) and
Iotonchiidae (e.g., Paraiotonchium ) nematodes may invade the sexual
organs. Nematode infections may severely affect the host, causing
debilitation, castration, or death. Most of the obligatory parasitic
nematodes are relatively host specific and are associated with one or a small
group of hosts. However, some groups such as the steinernematids and
heterorhabditids, often have broad host ranges under laboratory conditions.
But, such laboratory host ranges are typically broader, as is true in many
kinds of natural enemies, than actual host ranges in nature because of the
absence in such tests of ecological factors that restrict host contacts to
species found only in certain habitats. In nematodes, unlike other pathogens, host finding may be
an active process in which nematodes move towards and recognize hosts using
cues such as bacterial gradients, host fecal components, or carbon dioxide
(Grewal et al. 1993a). Nematode species vary in their host searching
strategies, with some being ambush predators and others actively moving in
search of hosts (Kaya et al. 1993). Host entrance may be a passive process,
as when nematode eggs or juveniles of Tetradonematidae are ingested by larvae
of sciarid flies. However, most of the time host penetration is an active
process in which juveniles nematodes penetrate hosts through the integument
or natural openings (mouth, anus, spiracles). In the cases of natural
openings, nematodes seeking entrance have only to move through the opening,
avoiding efforts of the host to brush them aside (in the case of the mouth).
Once inside the gut, nematodes use mechanical devices such as stylets, and
spears, to puncture the gut wall and enter the hemocoel, where infection occurs.
Stylets and spears may also be used externally to perforate the cuticle to
penetrate directly to the hemocoel in some groups. Other kinds of nematodes,
such as the Sphaerulariidae, may use adhesive materials that attach the
nematode to the host=s cuticle
assisting in cuticle perforation with stylets. Nematode infections produce relatively few external signs
other than, sometimes, distended abdomens or changes in color. An exception
to this is the formation of intercaste or intersex individuals infected by
mermithids. Internal effects of infection my be profound. Sterility is
induced by several groups of nematodes, including Mermithidae.
Phaenopsitylenchidae, and Iotonchiidae. Molting may be inhibited in some
cases. Behaviors of nematode-infected hosts may be abnormal. Infected
individuals may have difficulty walking or flying normally, or may show
unnatural phototropisms. Mermithids differ from other nematodes because they leave
their hosts before reaching the adult stage. Postparasitic juveniles exit
from hosts and then molt to adults that mate and produce progeny as
free-living stages. Steinernematids and heterorhabditids, the groups of
nematodes used most extensively in augmentative biological control, kill
their hosts in 2-3 days, a shorter time than for other groups of nematodes.
This happens because these families have mutualistic bacteria in their
intestines (Xenorhabdus spp. & Photorhabdis spp.) That kill
hosts by septicemia. Juvenile nematodes reach the hemocoel by penetrating the
midgut wall after being ingested by the host, or by penetrating the host
integument. Xenorhabdus spp. Or Photorhabdis spp. bacteria are
then released into the host hemocoel by defecation of the juvenile nematodes.
Juveniles feed saprophytically on the dead host=s tissues and then mature to adults which reproduce. When
a new generation of the dauer stage is attained, they leave the host cadaver. Nematodes in the families Phaenopsitylenchidae and
Iotonchiidae include both facultative and obligate parasites. The
phaenopsitylenchid Beddingia siricidicola has two life cycles. One is
free living and feeds on a fungus that is mutualistic with the insect host.
This fungus is spread by the host (a siricid wood wasp) and grows in the
cambium of the host tree attacked by the wasp. In the free-living cycle,
juvenile nematodes feed on fungus, become adults, and lay eggs. In the
parasitic life cycle, adult female nematodes penetrate the cuticle of wood
wasp larvae which themselves feed on the fungus. After the host insect has
pupated, nematodes develop in the hemocoel and produce offspring that invade
the developing eggs of the wood wasp. When the wood wasp oviposits in new
fungal patches, it deposits nematode-infected eggs rather than healthy ones.
The eggs are killed by the nematodes, which emerge and continue their
development, either through the fungus-based life cycle or the insect-based
life cycle depending on the presence or absence of insect hosts on the patch.
Similarly, the iotonchiid Paraiotonchium autumnale, a parasite of Musca
autumnalis, invades ovaries of its host and is dispersed in the habitat
by the fly=s oviposition
attempts, as does Paraiotonchium muscaedomesticae Color & Nguyen,
a parasitoid of Musca domestica (Coler & Nguyen 1994). Please
refer to Gaugler & Kaya 1990, Kaya 1993 and Tanada & Kaya (1993) for
further details. Nematodes That Attack Arthropods Van Driesch & Bellows (1998) reported that the
classification of entomopathogenicc nematodes at the family level has changed
significantly since the mid 1900's. Recent discussions are given by Maggenti
(1991) and Remillet and Laumond (1991). Reviews of nematodes associated with
anhropods were provided by Poinar (1986), Kaya (1993), Kaya ind Gaugler
(1993), and Tanada and Kaya (1993). Of the 30 or more families of nematodes
associated with insects, only nine have members with any potential as
biological control agents: Tetradonematidae, Mermithidae, Steinernematidae,
Heterorhabditidae, Phaenopsitylenchidae, Iotonchiliidae, Allantonematidae,
Parasitylenchidae and, Sphaerulariidae. Most attention has beem focused on
two families, the Steinermeatidae and Heterorhabditidae. These are associated
with pathogenic symbiotic bacteria that enable them to rapidly kill a wide
range of hosts. Members of five other families (Tetradonematidae,
Mermithidae, Phaenopsitylenchidae, Iotonchiidae and also merit attention. Steinernematidae and Heterorhabditidae Several species in the genera Steinernematidae and
Heterorhabditidae have been the focus of most efforts to develop commercial
uses of nematodes (Gaugler & Kaya 1990; Kaya 1993; Kaya and Gaugler 1993;
Tanada and Kaya 1993). Synonomies of' species names in these genera
complicate the interpretation of literature citations. Ten species are
recognized in Steinernema (Doucet and Doucet 1990) and three in Heterorhabditis
(Poinar 1990). Kaya and Gaugler 1993) listed these and discussed their
nomenclature, and Smith et al. (1992) gave a bibliography of research on
these These families have been used as commercial pest control agents
because they have the following atttributes (Poinar 1986): a wide host ringe;
an ability to kill the host within 48 hours; a capacity for growth on
artificial media; a durable infective stage capable of being stored; a lack
of host resistance; and the apparent safety to the environment. These nematodes invade hosts through natural openings
(mouth, spiracles, anus) or woundsand penetrate into the haemocoel. Bacteria
in the genera Xenorbabdus or Pbotorbabdus, syimbiotic to the nematode
but pathogenic to the host, are released into the hemocoel and quickly kill
the host. The nematodes then develop saprophytically on the decomposing host
tissues. Gaugler and Kaya (1990) and Kaya and Gaugler (1993) provide
information on rearingand using these groups of nematodes for pest control.
These nematodes work best in moist environments such as soil. Commercial
markets for some strains have been established and large scale introduction
systems developed (Kaya 1985: Gaugler and Kaya 1990). In addition to the augmentative use of nematodes in these
two families, some species with more narrow host ranges have been imported
for the control of immigrant pests; for examplee, Steinernema scapterisci was
introduced into Florida in a program to provide biological control of immigrant
species of Scapteriscus mole crickets (Parkman et al. 1993). Tetradonema plicans is a tetradonematid that infects larvae of Sciara
(Diptera: Sciaridae), which are pests in glasshouse and mushroom crops.
Effective control of these root gnats was obtained by Peloquin and Platzer
(1993) with applications of T. plicans eggs at a ratio of 10 eggs per
host larva. Cultures of the nematode can he maintained on hosts reared on a
composted media of sphagnum moss, shredded paper, and commercial rabbit food.
Host larvae ingest eggs or, perhaps, young larvae. Nematode larvae then
penetrate the gut and enter the hemocoel, where they mature and cause the
death of the host. Adult nematodes mate in the host and females either exit
and lay eggs outside the host or remain in the host, in which case eggs are
released into the soil as the host and female nematode decay. The nematode Romanomermis culicivorax Ross &
Smith attacks larvae of various mosquitoes. The life cycle is divided between
stages that occur within the host and others that occur outside of the host.
Infective nematode larvae find hosts, penetrate the integument, and enter the
host's hemocoel. Larvae partially mature inside hosts, penetrate through the
host integument, and emerge into the environment as fourth stage larvae. They
later transform into adults, mate, and lay eggs, which hatch and produce
infective juveniles to resume the cycle. This nematode was developed as a
commercial product under the name Skeeter DOOM, but the product failed
commercially because of storage and transportation problems (Poinar 1979). The nematode Beddingia siticidicola was introduced
from New Zealand into Australia where it contributed substantially to the
suppression of a major pest of conifer plantations, the European wood wasp Sirex
noctilio (Bedding 1984). The nematode invades the ovaries of the adult
wood wasp, killing the eggs. The wasp, however, continues to oviposit, with
the result that nematodes rather than eggs are deposited in new trees,
spreading the nematode. The iotonchiid Paraiotoncbium aut mnaic, (Nickle),
a native of Europe, was discovered in New York in 1964 attacking the face
fly, Musca a t mnalis I)e Geer. The nematodes m@tte in dling and enter
fly larvae. Nematodes develop through two generations but do not kill the
host, which develops to the adult stage. At this point nematodes are present
in the fly's ovaries, reducing egg production. The adult fly, instead of
laying eggs, deposits nematodes, A laboratory production method for this
species was developed by Stoffolano (1973). A species of iotonchiid attacking
Musca domestica has recently been recorded from Brazil (Coler and
Nguyen 1994). The nematode Tripius sciarae (Boiven) attacks
sciarid and mycetophilid flies in Europe and North America. The nematode
infects its host in the larval stage but does not immediately kill it. Some
iiifected hosts develop to the adult stage, but their reproductive systems
are destroyed by nematodes that are spread by oviposition attempts of
infected flies. The nematode is easily reared and capable of controlling
fungus gnat infestations in glasshouses (Poinar 1965). A number of plant-parasitic nematodes in the Tylenchina,
especially the family Anguinidae, induce galls on plant foliage. The taxonomy
of this group is reviewed by Siddiqi (1986). Some nematodes in this group are
resistant to dehydration, a feature that enhances their survival. A number of
species in the group have been considered for use in biological control
through augmentation (Parker 1991). One species (Subanguina piciidis Kidanova
& Ivanova) has been introduced against an adventive plant, Centaurea
diffusa Lamarck (Julien 1992). Included are a diverse group of eucaryotes in the Kingdom Protista,
which are unicellular and motile (Levine et al. 1980). Included are species
that are free-living and saprophytic, commensal, symbiotic or parasitic. The
cell contains a variety of organelles, but no cell wall, and they vary
in size and shape. They feed by ingestion or adsorption, and vegetative
reproduction is by binary or multiple fission. Both asexual and sexual
reproduction occur, with the latter often very complex and used for
identification. Many protozoa possess a resistant spore stage that is also
useful in taxonomy. The kingdom is divided into a series of phyla based
primarily on the mode of locomotion and structure of locomotory organelles,
which includes the Sarcomastigophora (flagellates & amoebae), Apicomplexa
(sporozoa), Microspora (microsporidia), Acetospora (haplosporidia) and
Ciliophora (ciliates). Some protozoa, such as amoebae and ciliates, are
easily cultured in vitro, while many of the
obligate intracellular parasites have not been grown outside of cells. Biological Characteristics.--Many species of protozoans are associated with insects and the
biology of these associations includes a range from symbionts to parasites.
Parasitic species typically cause chronic diseases. Many parasites,
especially microsporidia, increase slowly in insect populations, eventually
causing epizootics that are followed by a rapid decline in the insect
population. Nevertheless, research has shown that protozoa have only a small
potential for use as fast-acting microbial insecticides, because of the
chronic nature of the diseases they cause and the lack of a mass culture
procedure. Some protozoans, microsporidia in particular, may be used as
classical biological control agents or in intermediate and long range pest
population management schemes. There is considerable diversity in life cycles and
biologies (see Brooks 1988, Canning 1982, Henry 1990, & Maddox 1987).
However, the group with the most potential, the microsporidia, deserve a more
detailed treatment here. Microsporidia.--These protozoans, in the Phylum Microspora, are the most
common and widely studied of protozoans that cause important insect diseases.
Over 800 species are known, most having been described from insects (Brooks
1988). They are found principally in Coleoptera, Diptera, Orthoptera and
Lepidoptera, but are probably found in all orders. Protozoan epizootics in
insects populations are usually caused by microsporidia. All microsporidia are obligate intracellular parasites,
which lack mitochondria. They produce spores that are distinguished by
the presence of a polar filament, or long coiled tube within the spore that
is used to infect hosts with the sporoplasm (Vavra 1976). A typical life
cycle begins with the ingestion of the spore by a susceptible insect. Once in
the midgut, the polar filament extrudes, rapidly injecting the sporoplasm
into host tissue. The sporoplasm is unicellular but may be uni- or
binucleate. Upon entry into the cytoplasm of a host cell, such as the fat
body in many insects, the sporoplasm forms a plasmodium, or meront,
which undergoes many cycles of vegetative growth, or merogony. During
such cycles, the cells multiply repeatedly, dividing by binary or multiple
fission, and spreading to other cells, and in many species to other tissues
of the host. After several merogonic cycles, the microsporidian undergoes
sporulation, consisting of two principal phases, sporogony (a terminal
reproductive division committed to sporulation) and spore morphogenesis.
In sexual phases, meiosis occurs early during sporogony. The spores,
generally measuring several microns in diam. and length, have thick walls and
are highly refractile when viewed by phase microscopy. The disease often
lasts for several weeks, during which spores accumulate in the tissues of
infected hosts yielding billions per individual. Systematics of microsopridia
is based on the size and structure of the spores, life cycles and host
associations. Many microsporidia are transmitted vertically from adult
females to larvae via the egg (transovarially) in addition to
ingestion. Some species are host specific whereas other occur in many species
of the same insect family or order. Biological Control Attributes.--Insect populations are often effectively reduced by naturally
occurring epizootics of microsporidia. However, accurate prediction is not
possible. For example, the epizootics caused by Nosema pyrausta
(Paillot) in the European corn borer, Ostrinia
nubilalis (Maddox 1987) are
helpful in pest control, but cannot be depended upon. Therefore, research
emphasis has been on inundative releases. Due to the absence of mitochondria, microsporidia cannot
be cultured on artificial media, and spores must be grown on living hosts.
Yields can be high (10 9-10 spores/host). The number of larvae that have to
be grown to treat a hectare and infect most of the target host population is
similar to the requirements for nuclear polyhidrosis viruses. However,
because of the chronic nature of the infections, practical control is usually
not possible (Maddox et al. 1981). Vairimorpha necatrix, a dimorphic species having a broad host range among
noctuid larvae, is an example of problems one encounters when microsporidia
are used as microbial insecticides. Fuxa & Brooks (1979) found that
application of spores at a rate of 1012 per acre reduced feeding
damage as effectively as Dipel (B.
thuringiensis, while rates
as high as 1013 spores/acre often do not give adequate control.
When the same rate was used against Heliothis
zea on soybeans, even though
99% of larvae became infected, thee was only a small reduction in feeding
damage. Nosema locustae
Canning, when used
against grasshoppers in a population management sense, reduced grasshopper densities
by 50% 28-30 days after application of 2.5 x 109 spores per
hectare. Production costs for this application were very low, as the yield of
spores from Melanoplus differentialis averaged 6.4 x
109 spores per grasshopper, or about 1/6th a grasshopper per acre!
Nosema locustae is the only protozoan registered by the
Environmental Protection Agency for use in insect control. REFERENCES: Please refer to: <bc-50.ref.htm> and <vandries.ref.htm>
[References
also may be found at MELVYL Library ] |