BIOLOGICAL PEST CONTROL USING PATHOGENS
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Extensive discussions of the use of pathogens in biological control in tropical climates were 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 relative humidity also affect 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.
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 .
Many viruses affect insects that 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.
Collecting infected individuals in the field and storing them with care to suppress bacterial growth may produce viruses locally. 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 are typically <1% (Kelly & Robertson 1973).
Cytoplasmic polyhedrosis viruses are occluded RNA viruses that 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 that 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 to kill 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. 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 does this. 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.
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 that 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. E. V. Talahaev discovered the B. thuringiensis subsp. dendrolimus isolate used in the product in 1954 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 that 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. Drying and grinding larvae then make powdered formulations of the bacterial spores. 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.
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 is 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 has 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 suggest 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 are 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 that was 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 that 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.
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 that 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 that 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 (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 juvenile 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.
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.
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 also may be found at MELVYL Library ]