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Microbial Processes: Promising Technologies for Developing Countries (1979)

Chapter: MICROBIAL INSECT CONTROL AGENTS

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Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Page 84
Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Page 86
Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Page 88
Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Page 89
Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Page 90
Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Page 91
Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Page 93
Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Suggested Citation:"MICROBIAL INSECT CONTROL AGENTS." National Research Council. 1979. Microbial Processes: Promising Technologies for Developing Countries. Washington, DC: The National Academies Press. doi: 10.17226/9544.
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Chapter 5 Microbial Insect Control Agents There are more than 1,500 naturally occurring microorganisms or their products that hold promise for the control of major insect pests. Microorgan- isms that affect insects are termed entomopathogens. They may be used to induce diseases in target insects or to suppress populations of insects directly or in combination with chemical insecticides. Energy-efficient pest-control approaches must be developed to reduce to a minimum the use of factory-produced, toxic, broad-spectrum chemical insec- ticides used in industrial countries in ever-increasing amounts. The microbial approach can be applied to agricultural practices of both developing and developed countries, and many of its techniques are ready for implementa- tion. By adopting a systems approach to "integrated pest management," using entomopathogens and other nonchemical factors for specific pests, develop- ing countries have an important opportunity to bypass the traditional chem- ical approach to insect control. All types of microorganisms are represented among the potential microbial control agents. As an example, nearly 100 species of bacteria and over 700 viruses have been isolated from arthropods and more are being discovered each year. AD classes of fungi are represented among the more than 750 known entomopathogenic fungi. Protozoa are also likely candidates as mi- crobial control agents because many insects not attacked by other entomo- pathogens are susceptible to at least one of the 300 known species of ento- mophilic protozoa. Development of Bioinsecticides In planning an approach to the use of microbial control agents, the most significant factors to be considered include production technology, safety and specificity, and efficacy. 80

MICROBIAL INSECT CONTROL AGENTS Production Technology 81 Entomopathogens are produced by fermentation methods (Figure 5.1), In living insects (Figure 5.2), and in cell tissue cultures. Fermentation technol- ogy is used for some bacteria and fungi, whereas living insects can be used for the production of obligatory parasitic viruses and protozoa. Both processes have been successfully used to produce commercial entomopathogenic prod- ucts. For example, submerged fermentation is generally used for commercial production of the entomogenous bacteria Bacillus thuringiensis and Bacillus moritc~i and the fungi Beauveria bassiana and Entomophthora virulenta. Sur- face fermentation is employed to produce pathogenic fungi, for example, IVomuraea ri~eyi and Metarrhizium anisopliae, and a combination of both surface and submerged techniques for Beauveria bassiana and Hirsutella thompsonii. Living insects are exclusively used as substrates for the produc- tion of the respective nucleopolyhedrosis viruses of Helio this zea, Porthetria dispar, and Hemerocampa pseudosugata. Cell tissue culture methods currently produce only small quantities of viruses, but they are considered to be the mass production method of the future. Safety and Specificity Entomopathogens are infectious, replicating living organisms that are a natural part of our environment. Evidence that microbial insecticides pose little human or environmental hazard has been demonstrated by laboratory animal testing data developed to support federal pesticide registration. Never- theless, safety cannot be absolutely guaranteed for all entomopathogens in every living system, and it is important that potential hazards for new ento- mopathogens be known prior to use. Informal guidance for evaluating the specificity and risks ~ the use of microbial agents has been provided by reg- ulatory agencies. Formal guidelines are now being developed by the U.S. Environmental Protection Agency (EPA). Severe baculow~ses have been tested in living organisms with no evidence of toxic or pathogenic effects on vertebrates or nontarget invertebrates. Baculoviruses do not appear to replicate in vertebrate embryos or in cell lines derived from birds, fishes, amphibians, or mammals. No deleterious effects at normal field-use rates were reported in tests with such bacteria as Bacillus thuringiensis, Bacillus popilliae, and Bacillus moritai. While allergens are encountered among the fungi (<Beau veria, Entomophthora, Hirsutella, Metarrhizium, and Nomuraea species), results indicate that these fungi are not toxic or infectious to vertebrates. Three entomopathogenic protozoa, one from grasshoppers (Nosema locustue), one from mosquitoes (~Nosema al- gerae), and one from beetles (Mattesia trogodermae), have been evaluated against nontarget organisms. Initial in vivo tests indicate no apparent risk to vertebrates.

82 Minerals ~ Growth factors ~ | . . .e M ixer C} T 1- ~ ~ 1 ' ' ` Test 500 ml 6 liter tube flask flask Incubating c ulture MICROBIAL PROCESSES _ Ca rbon sou rce r Nitrogen source Steri liner - T seed L Production ~r- r - ~ Tooler ~- Concentration\ | Standar' ization | a_ 1 Formulation 1 1 1 . Packaging FIGURE 5.1 Submerged fermentation pathway used to produce Bacillus thunngien- sis. (Photograph courtesy of C. M. Ignoffo) FIGURE 5.2 Scanning electron micrograph of entomocidal parasp oral crystals and spores of Bacillus thuringiensis. (Photograph courtesy of C. M. Ignoffo)

MICROBIAL INSECT CONTROL AGENTS Efficacy 83 Entomopathogens have been used to control mites, beetles, and caterpillar pests of agricultural crops, forests, and stored products with varying degrees of success. Microbial insecticides, like chemical insecticides, are usually sprayed or dusted on crops. Entomopathogens may also be successfully introduced and established in an ecosystem by other application methods to provide long term control of pest populations. For example, insects themselves can be used to disseminate entomopathogens. Virus or fungus epizootics might be in- duced in an insect population before crop-damaging proliferation takes place. It may also be possible to manipulate the environment to create conditions in which naturally occurring pathogens exert their greatest effect. Some of these approaches may provide levels of control equal to or better than those cur- rently obtained with chemical insecticides, but further research is needed to exploit their potential. The potential for substituting microbial control agents for chemical pesti- cides can be deduced from the following examples in the United States shown in Table 5.1. Development of the use of entomopathogens or their by-products for microbial control agents is underexploited. Safe, effective entomopathogens formulated as microbial control agents are being developed by governmental agencies and industry, and the commercial products are being effectively used by growers. The newer agents have not been brought to their fullest potential. TABLE 5.1 Potential Substitution of Chemical Pesticides by Microbial Control Agents in the United States Potential Replacement of Chemica] Pesticide Disease Control Agent (t per year) Cotton bollworm and budworm (Hello this zea) Citrus rust mite (Florida) {Phyllocoptruta oleivoraJ Cabbage looper (Florida) {Trichoplusia ni) Western-range grasshoppers Green peach aphid on Maine potatoes (Myzas persicae) Baculovirus heliothis >7,700 Hirsutella thompsonii 1,800-7,200 Bacillus thuringiensis 450-1,400 Nosema locustae 450- 900 EntomopAthora ignobilis 90- 310

84 Bacteria MICROBIAL PROCESSES Mary bacteria are associated with insects, most of which belong to the families Pseudomonadaceae, Enterobacteriaceae, Lactobacillaceae, Micrococ- caceae, and Bacillaceae (Table 5.29. Members of these families may be obli- TABLE 5.2 Examples of Bacteria Pathogenic for Insects* Bacteria Insects Pseudomonadaceae *Pseudomonas aeruginosa Pseudomonas septica Vibrio leonardia Enterobacteriaceae Serratia marcescens and *Escherichia cold Enterobacter aerogenes Proteus vulgaris, P. mirabilis, and P. retigeri *Salmonella schottmuelleri var. alvei *Salmonella enteritidis, *Shigella dysenteriae Lactobacillaceae Diplococcus and Streptococcus spp. *Streptococcus faecalis Micrococcaceae *A~icrococcus spp. Bacillaceae Bacillus thuringiensis and B. cereus Bacillus popilliae and B. Ientimorbus Bac~lus sphaericus Bacillus larvae Bacillus moritai Clostridium novyi and C. perfringens Grasshoppers (Orthoptera) Scarab beetles (Scarabaeidae), striped ambrosia beetle (Tripodendron lineatum) Greater wax moth (Galleria melonella), European corn borer (Ostrinia nubilalis) Varieties of butterfly, moth and skipper (Lepidoptera) Grasshoppers (Orthoptera), varieties of butterfly, moth and skipper (Lepidoptera) Grasshoppers (Orthoptera) Honeybees (Apidae), greater wax moth (Galleria melonella) Greater wax moth (Galleria melonella) Cockchafer (Melolontha melolontha), silkworm (Bombyx mori), gypsy moth (Lyman tria dispar), processionary moths (Thaumetopoeia spp.) Greater wax moth (Galleria melorcella) Green June beetle (so tinis nitida), sawflies (Tenthredinidae), houseflies (Muscidae), various Lepidoptera including nun moth (Lymantria monacha), European corn borer (Ostrinia nubilalis), and cutworms (Noctuidae) Varieties of butterfly and moth (Lepidoptera) Scarab beetles (Scarabaeidae) Mosquitoes (Culicidae) Honeybees (Apidae) Flies (Diptera) Greater wax moth (Galleria melonella) *Those asterisked may also be pathogenic for man.

MICROBIAL INSECT CONTROL AGENTS 85 gate or opportunistic entomopathogens, depending on their host association in nature. Obligate entomopathogens are generally fastidious and are re- stricted to growth in a living host insect. The occasional or opportunistic pathogens are free living in nature, although they may commonly be found associated with one or more hosts. About 100 bacteria have been reported as entomopathogens, but only four (<Bacillus thuringiensis, B. popilliae, B. Ienti- morbus, and B. sphaericus) have been closely examined as insect-control agents. These species are sporeformers: the first two produce, in addition to the spore, discrete crystalline inclusions within the sporulating cell; the last two do not. Production Fermentation processes are employed in the production of B. thuringiensis and B. sphaericus spores, whereas B. popilliae orB. Ientimorbus spores are produced exclusively in living insects Two types of fermentation methods are used for the production of B. thuringiensis and B. sphaericus: 1) surface or semisolid, and 2) submerged. In surface fermentation, the B. thuringiensis spores are inoculated on a semisolid medium composed of wheat bran, ex- panded perlite, soybean meal, glucose, and inorganic salts. The bran is har- vested after 36-48 hours and formulated to a product active against insects. Submerged fermentation of B. thuringiensis (Figure 5.1) is carried out in a liquid medium using standard fermentation protein sources (fish meal, soy flour) and carbohydrate sources (molasses, sucrose). After sporulation of the bacteria, the spent liquor is passed through a fine screen, the active ingredi- ents are centrifuged from the medium, mixed with stabilizing agents, and then packaged as either a powder or liquid. Similar materials may be used for 3. sphaericus. For the in vivo production of B. popilliae and B. Ientim orb us, third instar Japanese beetle (Popillia japonica) larvae are infected with spores and in- cubated in soil seeded with rye to feed the larvae. The infected larvae are harvested 16-21 days after infection. The infected larvae are pulverized, stabilizing agents are added, and the slurry is dried and packaged as a dry powder. Living insects must be used to propagate these bacteria because artificial culture methods that will support large-scale sporulation of the E. popilliae and B. Ientimorbus are not as yet available. Safety and Specificity B. thuringiensis, B. popilliae, B. Ientim orb us, and B. sphaericus have been subjected to many safety tests, with no harmful effects for animals or human beings. What impact these bacteria have on the environment when used as insecti- cides is difficult to predict, because little data are available on persistence and

86 MICROBIAL PROCESSES concentration of bacterial agents in the food chain. True parasites are ob- viously dependent for existence on the population of their hosts. E',. Trlcacy B. thuringiensis (Figure 5.2) is orate of the best-known and most widely used microbial control agents. It is pathogenic for lepidopteran larvae (Figure 5.3) affecting more than 150 larval species that include some of the most important economic pests listed in Table 5.3. Preparations of B. thuringzer~s~s can be mixed with a number of commercial insecticides, fungicides, and various adhesives and wetting agents. Commercial products are applied to field crops and stored commodities, employing the same methods used for chemical pesticides. B. popilliae and B. Ientimorbus are pathogens of various beetles. When ingested by beetle larvae, the bacteria invade the blood system, where they proliferate and sporulate, causing death of the larvae. The mass of spores that accumulates prior to death of the insect is ultimately released and survives for extended periods in the soil. These spores may be eaten by newly hatched beetle larvae and, upon germination and growth in the insect gut, begin the infectious process again. The name given to this infection is "milky disease" because the infected larvae take on a whitish or milky appearance. One appli- FIGURE 5.3 Diseased larvae of the cabbage looper with symptoms of B. thunngiensis poisoning. (Photograph courtesy of C.M. Ignoffo)

MICROBIAL INSECT CONTROL AGENTS TABLE 5.3 Some Insect Pests Susceptible to Control with Preparations of Bacillus thuringiensis Insect Pest Plants Affected Cabbage looper (Trichoplusia ni) Imported cabbageworm (Pieris rapae) Tobacco hornworm (Man duca sexta) Tobacco budworm (Hello this virescens) Tomato hornworm (Manduca quinquewlaculata) Alfalfa caterpillar (Colias eurytheme) Gypsy moth (Lyw~ntria dispar) European corn borer (Ostrinia nubilalis) Grape leaffolder (Desmia funeralis) Codling moth (Laspeyresia pomonella) Green cloverworm (Plathypena seabra) Orangedog (Pailio cresphontes) Range caterpillar (Henuleuca oliviae) Sugarcane borer (Diatraea saccharalis) Cotton bollworm (Heliothis zea) Spruce budworm (Choristoneura fumiferana) Indian meal moth (Plodia interpunctella) Broccoli, cabbage, cauliflower, celery, lettuce, potato, melon Broccoli, cabbage, cauliflower Tobacco Tobacco Tomato Alfalfa Forest trees Corn Grape Apples, pears Soybeans Citrus Range grass Sugarcane Cotton Forest trees Stored grains 87 cation of these bacteria lasts many seasons, although the physical and chem- ical properties of the soil as well as climatic conditions, agricultural practices, and larval population density influence their effectiveness in nature over pro- longed periods. Several strains of B. sphaericus have been isolated that are highly toxic and specific to larvae of disease-carrying mosquitoes. The infectivity of B. sphaeri- cus strains varies with the mosquito species; in general, larvae of the genus Aedes are the least susceptible, while those of the genus Cule~c are the most susceptible, at least at the current stages of strain development. Experi- mentally, this bacterium has been produced commercially at prices competi- tive with those of chemical insecticides. All isolates that have insecticidal activity have not been fully characterized.

88 Limitations MICROBIAL PROCESSES The basic premise on which the insecticide industry operates and the conditions under which the EPA win issue registrations is that the pesticide must be useful against the target species without adversely affecting man, nontarget animals, or plants. Other parameters to be considered for extensive use of bacteria to control insects are: 1) convenience of application (appli- cable as a dust, liquid spray, or bait); 2) possibility of integration with con- ventional chemical insecticides; 3) storage characteristics; 4)economics; 5) ease of production; and 6) safety. The success of a bacterial insecticide, as for any pesticide, also depends on a variety of interacting factors such as environmental and climatic conditions, commodity to be protected (field crop or stored product), mode and timing of application, behavior and habits of the target hostess, and defense mechanisms of the insect. As noted above, field testing of the efficacy of B. thuringiensis, B. popil- liae, B. Ientim orb us, and B. sphaericus has been done and preparations of all these organisms are commercially produced, with the exception of B. sphaer~- cus. Because the spore stage is packaged for dissemination, most of the above criteria have been met for B. thuringiensis, B. popilliae, and B. Ientimorbus. Although B. thuringiensis and B. sphaericus meet most of the above criteria, B. popilliae and B. Ientimorbus have some serious drawbacks; they have limit- ed commercial use because of the difficulty of producing spores in quantity. A much clearer understanding of the metabolism of both the insect host and the bacterial pathogen is needed. For example, to facilitate successful industrial fermentation of spores of B. popilliae and B. Ientimorbus, insight into the mechanisms that control bacterial spore formation during the infec- tious process is necessary. Research Neecis The following are some of the most pressing needs for better understand- ing and use of bacterial insect pathogens: · To identify new bacterial pathogens; · To develop in vitro production of B. popilliae and B. Ientim orb us and determine nutrients required for sporulation; · To identify possible biohazards; · To increase understanding of the mechanisms of insect infection; and · To encourage industrial research and development through appropriate incentives.

MICROBIAL INSECT CONTROL AGENTS viruses 89 Virus diseases have been described for most major arthropod pests. About 700 viruses have been isolated from insects and mites. Most viruses (83 per- cent of those described in Table 5.4) have been isolated from caterpillars, since many moth and butterfly larvae are serious economic pests. Viruses of flies and sawflies account for about 14 percent of those listed in Table 5.4; the other 3 percent are equally divided among viruses of beetles, grasshoppers, and mites. Insect viruses fall into five major groups: · nucleopolyhedrosis viruses (NPV) · cytoplasmic polyhedrosis viruses (CPV) · granulosis viruses (G V ~ · entomopox viruses (EPV) · non-inclusion viruses (N IV). Figure 5.4 shows electron micrographs of these viruses, and Table 5.4 gives examples of some host species that are being considered for control. The NPV and GV, because of their specificity, safety, virulence, and stabil- ity, are probably the most promising candidates for development as viral insecticides. The CPV, EPV, and NIV are not currently considered likely candidates because less is known about their host range, production feasi- bility, stability, and efficacy. Production Insect viruses are strict parasites and must be mass-produced in living hosts or cell cultures. This means rearing the target pest insect and producing the virus by artificial infection, harvesting the virus, and formulating an effective insecticidal preparation. During early phases of the development of viral insecticides, insects were collected in the field and fed contaminated foliage. Dying insects were then processed into virus preparations for subsequent use. Recently developed techniques and semisynthetic diets permit year-round virus production. For example, bollworms are mass-reared and the bollworm NPV grown in the larvae (Figure 5.5~. Tissue culture may be a virus-production technology of the future. Insect tissue cell-culture lines have been established in which insect viruses grow and multiply. In spite of the highly specialized technology needed to mass-produce viruses, every major group of insect virus has been produced and used in the field as an insecticide. In addition, more than a dozen commercial or experi-

9o MICROBIAL PROCESSES TABLE 5.4 Examples of Viruses and Their Hosts Nucleopolyhydron Viruses (NPV) Alfalfa looper (At~tographa calzfornica) Almond moth (Epl~estia cautella) Army worm (Pseudaletia unipuncta) Asiatic rice borer (C17ilo supressalis) Beet armyworm (Spodoptera e-Yi`~la) Bollworm (fleliothis ea) Cotton cutworm (Noctuidae) Cotton leafworm (Alaba77?a argillacea) Cotton leaf-perforator (B~`c c'~'la tri-Y tI7urherie11a ) Cabbage looper (Tricoplusia ni) Corn earworm (HeliotI7is zea) Douglas fir tussock moth (On'£ria ~'scaclotsus~ata) Asiatic rice borer (Chilo supressalis) Cabbage worm (Pieris spp.) Codling moth (I aspe!'resia ~70n70nella) Potato tuberworm (Pl2tI70ri~7~a`'a operculella) Cabba~eworm (Pieris spp.) Mamestra cabba~cworm (Man7estra brassica<~) Pink bollworm (Pe~ ti/7op1~ora 5 oss ~ ''iella) Asiatic rice borer (CI~ilo sc~pressalis) Citrus red mite (Pal20~7l'C'hUS c~itri) Ermine moth ( Ypo,20~7~cuta padella) l:orest tent caterpillar (Malacoso'?~a disstria) Gypsy moth (I ~'na'7tria `1is~'ar) Imported cabba~eworm (Pieris rapac~) Mamestra cabbageworm (Ma~nestra hrassicac) Pine sawflies (Neod!'prion spp.) Spruce budworm (Choristoneura fu'77ifera~7a) Tobacco budworm (Heliothis ~ irt>scens ) Variegated cutworm (Periciro'77a saucia) Wattle bagworm (KotocI7alia jz~ndoi) Whitemarked tussock moth (Or~<ia lcacostig''7a) (, r an U losis Virusc~s ( G I V ) Redbanded leafroller (A rL(r] rotoenia ~'elutii7ana) Saltmarsh caterpillar (] sti~r'77e~7e acrea) Spruce budworm (C170ristoneura fi~n7iferana) Ci to plasmic Polyhedron Viruses (C PV) Non-inclusion Liruses (NIV) Pine caterpillar (TI7aun7ctopc)eia spp.) Pine processionary caterpillar ( Thau''7etopoeia spp. ) Spruce budworm (Choristoneura fu'~7iferana) C~oconut rhinoceros beetle (X~ lor-~ etes ja'77aice~7sis)

MICROBIAL INSECT CONTROL AGENTS ~~,~ ~ ~ ~i ~ - (a) Nucleopolyhedrosis virus. (Photograph cour- (b) Granulosis virus. (Photograph courtesy of tesy of C. M. Ignoffo) C. M. Ignoffo) at ma_ ~~..~.... . ~.~.~ _ ~~'~:~. ' ''am .~..~.~. .~ I. ~ ~~ I. . ~ ~ ~ I. I. ~ ... .; :-: A::::: ~ OF ..~., ::::::. , ...... ., , ~ . ~ .. ~_` . ~ ~ ........... _ ;. i - ~ -_ _ I, I, ~ ~ ~ ~ ~ _ _~ (c) Cytoplasmic polyhedlosis virus. (Photo- (d) Entomopox virus. (Photograph courtesy of graph courtesy of J. Adams) M. Bergoin) (e) Non-inclusion virus. (Photograph courtesy of C. Vago) FIGURE 5.4 Electron micrographs of the live types of viruses associated with insects.

92 PUPAE ADULTS \~As | Bollworm -.~ cycle 26-36 days MATU RE I LARVAE EGGS NEONA TAL LARVAE _ .~ _ LARVAE ~ ingestion multiplication ~ r _ _ _ ~ Virus _ cycle cell ~ _ reinfection _ _ Released 5-7 days _ _ virus Virus _ Cell replication_ penetration vi,,,< MICROBIAL PROCESSES Carbohydrates_ Fats'~Vitamins Proteins—:~' I | I '~ Gel C} ~ 1 ,~ Cooler e n 1- Di et / /Concentration\ | Standar dization | ..... n ~ ~ Form~latinn [ - ] Packaging FIGURE 5.5 In vivo process used to produce a nucleopolyhedrosis virus. (Photograph courtesy of C. M. Ignoffo) mental preparations of entomopathogenic viruses have been evaluated suf- ficiently to warrant designation of a trade name. Safety and Specificity Insect viruses Would be tested to determine the effects they may have on man, other animals, and plants. There is indirect evidence to suggest that viruses are safe, since man is continually exposed to them on marketed fruits and vegetables, with no apparent ill effects. Further, there is no evidence of toxicity or pathogenicity to those handling the virus in research and produc- tion. Such evidence, however, must be corroborated by direct experimenta- tion. Although absolute safety is not a prerequisite for use, testing sufficient to demonstrate that the virus can be used without adverse effect should be completed prior to large-scale field use. Its use can then be monitored for several years to ensure that unforeseen environmental effects do not develop. In the light of present knowledge baculoviruses appear to be safe and specific.

MICROBIAL INSECT CONTROL AGENTS Efficacy 93 The occurrence of natural epizootics that severely reduce populations of insect pests are striking demonstrations of the effectiveness of insect viruses. These natural epizootics can occur in many insect populations; they are com- monly observed in field populations of sawflies, gypsy moths, tent cater- pillars, cabbage loopers, and alfalfa caterpillars. Unfortunately, most of this natural control comes after the crop damage occurs. The same control, timed to prevent crop loss, has been achieved by introducing appropriate insect viruses to attack field populations of insects (Table 5.5~. TABLE 5.5 Examples of Viruses Tested for Control of Arthropod Pests. Crop Attacked and Target Pest Virus Type Vegetables Cabbage looper (Trichoplusia ni) Cabbageworm (Pieris spp.) Orchard Crops Citrus red mite (Panonychus citric Codling moth (Laspeyresia pomonella) Forage Crops Alfalfa caterpillar (Colias eurytheme) Fiber and Grain Crops Boll- and budworms (Heliothis zea) Cotton leafworm (Alabama argillacea) Forest and Shade Trees ~ . . European pme processlonary moth (Thaumetopoeia spp.) Gypsy moth (Lyw~antria dispar) Spruce budworm (Choristoneura fumiferarza) Nucleopolyhedrosis Granulosis Non-inclusion Granulosis Nucleopolyhedrosis Nucleopolyhedrosis Nucleopolyhedrosis Cytoplasmic polyhedrosis Nucleopolyhedrosis Entomopox Limitations Viruses must be grown in living systems, which means that a technology for mass production must be developed for each type of virus. Each virus must then be individually evaluated to determine its specificity and its poten- tial environmental impact before field use is possible.

94 MICROBIAL PROCESSES Although protocols and guidelines are available for evaluating the risks of using insect viruses, only a few viruses (Baculoviruses) have been evaluated in this way. Some viruses have a short residual field life, which may ultimately reduce efficacy against a target pest. Few can be shown to persist in nature as reservoirs of infection apart from their hosts. Long-term storage may be a problem, especially at higher temperatures. Viruses must infect and grow in insects to be effective. Because they are relatively host-specific, they infect only one insect or those several closely related. This is an advantage from the viewpoint of environmental effects. As with all insecticides, thorough coverage of an infested area is required for effective control. Research Needs The more important research and development needs related to the use of viruses as bioinsecticides include the following: · Compiling and maintaining a repository of potential viruses for crop and forest and of vectors of diseases of man and animals; · Developing better techniques for the identification and detection of viruses; · Increasing the understanding of how insect viruses infect and replicate in their hosts; · Developing more definitive criteria for evaluating the possible bio- hazards of entomopathogens; · Improving technology for production, formulation, and use of viral entomopathogens; and · Encouraging industrial participation in the development of entomo- pathogens with subsidies, grants, licensing agreements, patent protection, and similar incentives. Protozoa Protozoa are primarily free-living, but many types are intimately asso- ciated with insects in relationships ranging from compatible to harmful. Most of the approximately 300 described species of entomophilic protozoa are included in the orders Microsporidia (Subphylum Cnidospora) and Neo- gregarinida (Subphylum Sporozoa). Microsporidia, particularly common in insects, generally possess the great- est potential as entomopathogens (Table 5.6~. Among the microsporidia,

MICROBIAL INSECT CONTROL AGENTS TABLE 5.6 Protozoa as Promising Candidates for Insect Control Agents Protozoa Insect Host(s) Adelina tribolii, Farinocystis tribolii Malameba locustoe Mattesia dispora, M. trogodermae Nosema locustoe Nosema aIgerae Nosema pyrausta Nosema fumiferanae Nosema gasti Nosema melolonthae Vairimorpha necatrzx Octosporea muscaedomesticae Lambornella stegomyiae, L. clarki Stored product pests Grasshoppers Lepidopterous and coleopterous pests Grasshoppers Anopheline mosquitoes European corn borer (Ostrinia nubilalis) Spruce budworm (Choristoneura fumiferana) Boll weevil (Anthonomus grandis) European cockchafer (Melolontha melolontha) Lepidopterous pests Muscoid flies Mosquitoes 95 I\iosema locustoe (a pathogen of various grasshoppers and crickets), Nosema algerae (a pathogen of anopheline mosquitoes), Nosema pyrausta (a patho- gen of the European corn borer, Ostrinia nubilalis), and Vairimorpha (Nosema) necatrDc (a pathogen of many species of caterpillars; see Figure 5.6) are currently under consideration as insect control agents. Production Almost all protozoa of potential value as entomopathogens must be pro- duced in living hosts. At least 15 different protozoa have been propagated in various hosts for experimental use against pest insects. The primary hosts are generally used to produce spores, but alternate hosts can sometimes be used. For example, diff~culties of producing 1~. algerae in the primary-host mos- quito led to the development and use of the corn earworm, Helio this zea, as an alternate host. One earworm larva, when infected with spores, produces as many spores as can be obtained from 2,000 mosquito larvae. At a proper stage of disease development, infected earworms are processed to yield puri- fied suspensions of spores. Fresh spore suspensions have been used in most field studies, but spores usually can be stored for several months in an aque- ous medium or in insect cadavers at low temperatures (0°-5OC). Spores of some species can withstand freeze-drying for longer periods of time, but some, like N. algerae, are inactivated upon drying.

96 MICROBIAL PROCESSES (a) Stunted and dead larvae of the corn ea~worm, Heliothis zea, infected with the microsporidium Vairimorpha necat~ix. _ (b) Companson of nature of the fat body tissue of healthy larva (A), with that of an infected larva (B). (c) Spores of F. necatrix from fat body of infected larva. FIGURE 5.6 Effects of protozoa on insects. (Photographs courtesy of W. M. Brooks) Safety and Specificity Until recently, most protozoa were considered relatively host-specif~c. More extensive study of host range has revealed, however, that many are infective not only for host species in closely related genera but also for species in other families and even other orders of insects. Generally, however, although the host range for protozoa species may be quite extensive (more than 60 species of grasshoppers and crickets are susceptible to N. Iocustoe), it is confined to closely related species. Plants, human beings, and animals seem to be resistant to infection by insect protozoa. In an extensive study of N. algerae, a pathogen of many species of anopheline mosquitoes, no evidence of infection was found in such nontarget organisms as crayfish, freshwater shrimps, mosquito fish, several aquatic entomophagous insects, mice, or chickens. Efficacy Field Infestations caused by protozoa have seldom been documented, but the efficacy of protozoa as control agents is readily apparent in laboratory

MICROBIAL INSECT CONTROL AGENTS 97 colonies of insects. Protozoa decimate insect colonies through gradual debili- tation of the host. Even in natural populations, protozoa are probably more important than we have recognized. Nosema pyrausta is credited with being the most important factor in maintaining corn borer populations at levels that facilitate economic control by other means. Another example is the collapse of a field population of the spruce budworm due to the debilitating effects of Nosema fumiferanae. Only limited field studies of-protozoa as bioinsecticides have been con- ducted. Most protozoa are considered more suitable for long-term suppression programs. The slower, debilitative effects of chronic infections, such as re- duced fertility and shortened life span, generally preclude their widespread use as quick-acting agents. Limitations The availability of many kinds of protozoa enhances their potential use as microbial control agents. Although protozoa may be significant as natural regulatory agents, their use as microbial insecticides has been limited, espe- cially by the fact that protozoa act slowly on their hosts in contrast to the rapid action of some viruses or bacteria. This means that the control of insect damage to crops during the season of treatment with a particular protozoa is usually not possible. Usually the degree of success of the treatment is directly related to timing of application. The expense and difficulty of producing and storing sufficient quantities of protozoan spores for field use is another important limiting factor. Large-scale field evaluations of efficacy have been undertaken only with Nosema locustoe, a pathogen of grasshoppers. Thus, the use of protozoa as bioinsecticides is in its infancy, and much additional research will be neces- sary before the potential of this method can be realized. Research Neecis Some of the more important research and development needs for the development of protozoa as bioinsecticides include: · Establishing and maintaining a repository of viable protozoa; · Evaluating host range and safety of promising candidates; · Developing technology for mass production and storage; and · Improving technology for increased field persistence and dispersal, as well as critical assessment procedures for evaluation of field efficacy.

98 Fungi MICROBIAL PROCESSES More than 750 fungal species representing approximately 100 genera have been reported to infect insects. Nearly all major fungal groups are repre- sented, and virtually every type of insect is represented. Although the poten- tial of fungi for control of insect pests in underexploited, a few isolates are being developed (Table 5.7~. One of these (Beauveria bassiana) is already in extensive routine use in agriculture In We Soviet Union for control of the Colorado potato beetle (Leptinotarsa decemlineata) and the coaling moth (Laspeyresia pomonella). A number of Russian industrial plants are producing an estimated 20 t annually of B. bassiana formulation for commercial use. Since little survey work has been done and few of the many known fungi have been studied extensively, it is obvious that other promising candidates can be expected in the future. Figure 5.7 shows the steps involved in a research and development pro- gram for using a fungus to control aphids on potatoes. Production Means of mass production of fungi vary, but in general the available pro- duction technology is simple and straightforward. Coelomomyces and some Entomophthora species grow poorly in laboratory media, but most pathogenic TABLE 5.7 Experimental Fungi for Insect Control Fungus Infective Stage Insect Hosts Habitat Chytridiomycetes Coelomomyces Motile planonts Oomycetes Lagenidium Motile zoospores Zygomycetes Entomophthora Deuterom ycetes Aschersonia Beau reria Hirsutella Metarhizium Nomuraea Paecilomyces Verticillium Mosquitoes Aquatic Mosquitoes Aquatic Conidia or resting spores Conidia Conidia Conidia Conidia Conidia Conidia Conidia Caterpillars, aphids White flies Beetles, caterpillars Mites Froghoppers, leafhoppers, beetles, mosquitoes Caterpillars Beetles Aphids, white flies Foliage Foliage Foliage Foliage Foliage, soil, aquatic Foliage Foliage Foliage, greenhouses

MICROBIAL INSECT CONTROL AGENTS Exploratory Research Safety + Monitonng Identification Biological Specificity ll Environmental Monitoring Safety Guidelines Human Safety Efficacy Bioassay Method Small Plot Tests 11 Experimental Use Permit Farrn Tests Efficacy Experiment Station Tests Extension Service __ 99 Production Pilot Plant ForrnulatK>n Application for Registration 11 Registration Granted POTATO GROWER | FIGURE 5.7 R. S. Soper) Plant Design Construction Commercial Production _ Distribution Development of fungal control agent for aphids. (Flow chart courtesy of fungi, particularly species of the Fungi Imperfecti, can be mass produced on sterilized bran, grain, or beans. Semisolid fermentation or two-stage fermenta- tion (deep fermentation for mycelial growth followed by incubation in shal- low pans for spore production) have been successfully used for large-scale production of Beauveria and Metarhizium. Conidia (spores) normally are not produced in deep fermentation, but recently devised media enable pro- duction of Beauveria conidia. Thus, fungi can be produced in virtually any locale with technology geared to available facilities and staff.

100 MICROBIAL PROCESSES The conidia of Entomophthora, because they are fragile and short-lived, are difficult to store. Many species, however, produce resting spores that survive for years. Until recently, methods for germinating these spores were unavailable, but high levels of germination now have been obtained with several species, and this opens the possibility of commercialization of these r lungs. Safety and Specificity The host specificity of entomopathogenic fungi range from a single insect species to most members of a family or order, sometimes extending to several orders of insects and mites, and even to plants. Specificity varies not only with fungal species, but also with physiological races within species. Although B. bassiana occurs worldwide, isolates from one location have been shown t be more pathogenic for the same host elsewhere. The same effect has been shown for E. sphaerosperma. In general, fungi have been used to control a single pest, but recent work with lVomuraea rileyi has demonstrated control of several caterpillar pests of soybeans by conidia (Figure 5.8~. With most pathogenic fungi, beneficial insects are not harmed. With a single known exception, fungi with high virulence for insects do not cause disease in animals. Only Conidiobolus coronatus has shown possible infectivity to mammals. Its use as an insecticide is, therefore, not recom- mended. Some isolates of Aspergillus paves that infect insects may also pro- duce metabolites the+ are toxic to human beings, but extensive toxicological studies have not been conducted. Since pathogenic fungi usually do not grow in nature except in or on their insect hosts, toxin build up in the environment after fungi are introduced is not expected. Efficacy Some promising field results have been obtained with fungi. As noted, the types of insects involved in recent work are listed in Table 5.7. Fungi used in most microbial control studies were originally isolated from the pest insect or a closely related insect. In two recent studies (Hirsutella thompsonii vs. the citrus rust mite and Nomuraea rileyi vs. caterpillars), outbreaks of the fungi were artificially induced early enough to protect the crop. This procedure may be applicable to other fungus-host combinations. For example, the genus Entomophthora frequently causes dramatic reductions in older populations of grasshoppers, aphids, caterpillars, and flies (Figure 5.99. Control of the coaling moth (Laspeyresia pomonella) comparable to that obtained with chemical pesticides has been reported from Eastern Europe and Asia with Beauveria bassiana. Long-term effects (2-3 years) on the Colorado potato beetle (Leptinotarsa decemlineata) population through application of B. bassiana have been reported.

MICROBIAL INSECT CONTROL AGENTS 101 ,, . (a) Conidia of the entomopathoge~c fungus Nomuraea rileyi on the surface of m- fected cotton bollworm larva (below). (b) Cotton bollworm larva infected with Nomuraea rileyi. FIGURE 5.8 Effects of fungi on insects. (Photographs courtesy of C. M. Ignoffo) Small-scale field tests indicate that B. bassiana, B. brongniartii, and M. anisopliae have good potential for control of soil-inhabiting insects such as wireworms and cockchafers, and of others such as lepidopterous larvae. The soil environment, because the humidity is usually high, is considered favor- able for infection of insects by fungi. M. anisopliae is more frequently collecl;- ed in nature from beetle grubs in the soil than from any other source, and this fungus may yield specific strains that will prove effective for control of soil insects.

~ .,, it: ',:'"..:..: ' MICROBIAL PROCESSES FIGURE 5.9 Sarcophaga aldrischii infected with the fungus Entomophthora bullata. (Photograph courtesy of R. S. Soper) Little is known concerning interactions between soil microorganisms and pathogenic fungi, but mortality rates can be considerably lower in unsteril- ized than sterilized soil. More information is needed on soil inhibitors in order to select fungal pathogens that have increased ability to infect insects in the presence of soil microorganisms. The aquatic habitat is another promising place to use fungi. At least three fungi (Lagenidium, CuZicir~omyces, and Metarhizium species) have been devel- oped for small-scale held trials against mosquito larvae. All have broad host ranges within the mosquito family Culicidae. Limitations Current limitations to using fungi are related to lack of knowledge rather than to negative characteristics of the fungi. Although it was generally felt that fungal control would be effective only under conditions of high tempera- ture and humidity, it is now recognized that microclimate is more important than geo graphical climate, and that the microclimate can be manipulated (by irrigation, for instance, or by spacing plants to provide closed canopies).

MICROBIAL INSECT CONTROL AGENTS 103 The supposition that inoculum is already present in the environment, mak- ing introduction of fungi unnecessary, is also erroneous. Numerous field experiments have demonstrated that applications of fungal materials, particu- larly to foliage, greatly increase disease incidence. A final limitation is the incomplete safety information on entomopatho- genic fungi. Current data indicate that safe products can be produced, but most fungi require more extensive research before large-scale fieldwork can be conducted. Research bleeds Several areas of research need to be explored to develop the full potential of fungal insecticides. These include: · Conducting exploratory work to isolate new entomopathogenic fungi and improving methods for distinguishing fungal species and strains; · Developing bioassay techniques; · Selecting strains with increased virulence or other traits that will in- crease their effectiveness as fungal insecticides; · Documenting the modes of disease induction and development; · Developing predictive techniques so that insecticide applications can be eliminated where conditions indicate that fungi will soon significantly reduce the pest population; · Developing methods for encouraging natural epizootics; · Improving fungal insecticide production, formulation, and stabilization technology; · Devising field application and evaluation methods specifically for fungal insecticides; · Integrating fungus insecticides into pest-management systems by explor- ing compatibility and synergism with current chemical pesticides and cultural techniques; and · Initiating more extensive safety tests with fungal insecticides. References and Suggested Reading Bacteria Afrikian, E. G. 1973. Entomopathogenic bacteria and their significance. Yerevan: Armenian S.S.R. Academy of Sciences. Bulla, L. A., Jr.; Costilow, R. N.; and Sharpe, E. S. 1978. Biology of Bacillus popilliae. Advances in A pplied Microbiology 23 :1-18. ; Rhodes, R. A.; and St. Julian, G. 1978. Bacteria as insect pathogens. Annual Review of Microbiology 29:163-190.

104 MICROBIAL PROCESSES Falcon, L. A. 1971. Use of bacteria for microbial control of insects. In Microbial control of insects and mites, H. D. Burges and N. W. Hussey, eds., pp. 67-95. New York: Academic Press. St. Julian, G.; Bulla, L. A., Jr.; Sharpe, E. S.; and Adams, G. L. 1973. Bacteria, spiro- chetes, and rickettsia as insecticides. Annals of the New York Academy of Sciences 217:65-75. Somerville, H. J. 1973. Microbial toxins. Annals of the New York Academy of Sciences 217:93-108. Viruses Falcon, L. A. 1976. Problems associated with the use of arthropod viruses in pest control. Ann ual Review of En tomology 21: 305-324. Ignoffo, C. M. 1973. Development of a viral insecticide: concept to commercialization. Parasitology 33: 380-406. . 1973. Effects of entomopathogens on vertebrates. Annals of the New York Academy of Sciences 2 1 ~1 :14 1-1 64. McClelland, A. J., and Collins, P. 1978. UK investigates virus insecticides. I\lature 276:548-549. Stairs, G. R. 1971. Use of viruses for microbial control of insects. In Microbial control of insects and mites, H. D. Burges and N. W. Hussey, eds., pp. 97-124. New York: Academic Press. Summers, M. D.; Engler, R.; Falcon, L. A.; and Vail, P. V 1975. Baculoviruses for insect pest control: safety considerations. Washington, D.C.: American Society for Micro- biology. Summers, M. D., and Kawanishi, C. Y. 1978. Viral pesticides: present knowledge and potential effects on public and environmental health. Report EPA-600/9-78-026. Washington, D.C.: U.S. Environmental Protection Agency. World Health Organization. 1973. The use of viruses for the control of insect pests and disease vectors. Report of Joint FAD/WHO Meeting on Insect Viruses. WHO Techni- cal Report Series No. 531. Geneva: World Health Organization. Protozoa Brooks, W. M. 1974. Protozoan infections. In Insect diseases, G. Cantwell, ea., pp. 237-300. New York: Marcel Dekker. McLaughlin, R. E. 1971. Use of protozoans for microbial control of insects. In Microbial control of insects and mites, H. D. Burges and N. W. Hussey, eds., pp. 151-172. New York: Academic Press. Sprague, V. 1977. Systematics of the microsporidia. In comparative pathobiology, Vol. II: Systematics of the microspondia, L. A. Bulla, Jr., and T. C. Cheng, eds., pp. 1-510. New York: Plenum Publishing Corporation. Tanada, Y. 1976. Epizootiology and microbial control. In Comparative pathobiology, Vol. I: Biology of the microsporidia, L. A. BuLla, Jr., and T. C. Cheng, eds., pp. 247-279. New York: Plenum Publishing Corporation. Weiser, J. 1961. Die mikrosporid~en als parasiten der insekten. A monograph published by Zeitschrift fur Angewandte Entomologies, Supplement to No. 17. Hamburg: P. Parey. Fungi Ferron, P. 1975. Les champignons entomopathogens: evolution des recherches au cours des dix derrieres annees. SROP-Section Regionale Ouest Palearctique (Journal pub- lished by O.I.L.B.—Organisation Internationale de Lutte Biologique Contre les Ennemis des Cultures, Swiss Federal Institute of Technology, Zurich, Switzerland) No. 3. McCoy, C. W. 1974. Fungal programs and their use in the microbial control of insects and mites. In Proceedings of the summer institute on biological control of plants,

MICROBIAL INSECT CONTROL AGENTS 105 insects and diseases, F. G. Maxwell and F. A. fIarris, eds., pp. 564-575. Jackson Mississippi: University of Mississippi Press. Muller-Kogler, E. 1965. PilzkranEheiten bei insekten. Hamburg: P. Parey. Roberts, D. W., and Yendol, W. G. 1971. Use of fungi for microbial control of insects. In Microbial control of insects and mites, H. D. Burges and N. W. Hussey, eds. pp. 125-149. New York: Academic Press. Research Contacts Bacteria K. Aizawa, Institute of Biological Control, Kyushu University, Fukuoka, Japan. J. N. Aronson, Department of Chemistry, State University of New York, Albany, New York 12246, U.S.A. L. A. Bulla, Jr., U.S. Grain Marketing Research Laboratory, U.S. Department of Agricul- ture, Science and Education Administration, Manhattan, Kansas 66502, U.S.A. C. M. Ignoffo, Biological Control of Insects Research Unit, U.S. Department of Agricul- ture, Science and Education Administration, Federal Research, North Central Re- gion, P. O. Box A, Columbia, Missouri 65201, U.S.A. M. M. Lecadet, Institut de Recherches en Biologic Moleculaire, Centre National de la Recherche Scientifique, Universite de Paris VII, 2 place Jussieu, 75221, Paris, France. A. A. Yousten, Department of Biology, Virginia Polytechnic Institute and State Univer- sity, Blacksburg, Virginia 24061, U.S.A. viruses H. C. Chapman, Gulf Coast Mosquito Research Laboratory, U.S. Department of Agricul- ture, Science and Education Administration, Lake Charles, Louisiana 70601, U.S.A. L. A. Falcon, Department of Entomology and Parasitology, 333 Hilgard Hall, University of California, Berkeley, California 94720, U.S.A. C. M. I~offo, Biological Control of Insects Research Unit, U.S. Department of Agricul- ture, Science and Education Administration, Federal Research, North Central Re- gion, P. O. Box A, Columbia, Missouri 65201, U.S.A. G. R. Stairs, Department of Entomology, The Ohio State University, Columbus, Ohio 43210, U.S.A. C. Vago, Station de Recherches de Pathologie Comparee INRA-CNRS-EPHE,Universite des Sciences, Place Eugene Bataillon, 34060 Montpellier, France. Protozoa W. M. Brooks, Department of Entomology, North Carolina State University, Raleigh, North Carolina 27650, U.S.A. E~ I. Hazard, Insects Affecting Man Research Laboratory, U.S. Department of Agricul- ture, Science and Education Administration, Gainesville, Florida 32604, U.S.A. J. E. Henry, Rangeland Insect Laboratory, U.S. Department of Agriculture, Science and Education Administration, Montana State University, Bozeman, Montana 59715, U.S.A. J. V. Maddox, Section of Economic Entomology, Illinois Natural History Survey, Ur- bana, Illinois 61 801, U.S.A. J. Weiser, Laboratory of ~sect Pathology, Institute of Entomology, Academy of Sci- ences, Flemingovo nam 2, Praha 6, Czechoslovakia. Fungi J. P. Latge, Parasitologie Vegetale, Institut Pasteur, 25 Rue du Dr. Roux, Paris, France. C. W. McCoy, Department of Entomology, University of Florida, Lake Alfred, Florida 33850, U.S.A.

106 MICROBIAL PROCESSES D. W. Roberts, Boyce Thompson Institute for Plant Research, Tower Road, Ithaca, New York 14853, U.S.A. R. S. Soper, U.S. Department of Agriculture, Science and Education Administration Insect Pathology Research Unit, Boyce Thompson Institute, Cornell University, Tower Road, Ithaca, New York 14853, U.S.A. D. Tyrrell, Great Lakes Forest Research Centre, P. O. Box 490, Sault Ste. Marie, Ontario P6A 5M7, Canada. J. Weiser, Laboratory of Insect Pathology, Institute of Entomology, Academy of Sci- ences, Flemingovo nam 2, Praha 6, Czechoslovakia. N. Wilding, Rothamsted Experiment Station, Harpenden, Herts. AL5 2JQ, England.

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