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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 |