The potential of pathogens for pest control

Agriculture, Ecosystems and Environment, 10 (1983) 101--126

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Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

THE POTENTIAL OF PATHOGENS FOR PEST CONTROL

ROBERT P. JAQUES

Research Station, Agriculture Canada, Harrow, Ontario NDR 1GD (Canada) (Accepted 2 June 1983)

ABSTRACT

Jaques, R.P., 1983. The potential of pathogens for pest control. Agric. Ecosystems Environ., 10: 101--126. A large number of viruses, bacteria, fungi and protozoa can kill or incapacitate insects. Some of these have the potential for a significant role in the management and regulation of pest species o f insects, either as naturally occurring entomopathogens or as applied or introduced insecticidal agents. The usefulness of entomopathogens, especially the feasibility of development as microbial insecticides, is examined b y consideration of their effectiveness, safety and specificity, production and propagation, and marketability and profitability. Effectiveness, a major consideration in assessing potential, is influenced b y several factors including efficacy, dissemination, persistence, compatibility with other regulatory agents, and establishment in the host population, and by factors such as humidity, temperature, chemicals, and formulation, that directly influence the activity of the pathogen.

INTRODUCTION

The increasing dissatisfaction with the dependence on chemical insecticides for protection of crops against pest insects has led to more serious consideration of the potential role of biological agents for the regulation of insects. Whereas a substantial proportion of research has emphasized exploitation of parasitic and predaceous species of arthropods, it is becoming increasingly apparent that entomopathogens have real potential as components of integrated pest management systems for certain species of pest insects or groups of pest insects. A large number of microorganisms, including bacteria, fungi, viruses and protozoa, have been found to cause demonstrable harm or death to insects, that is, to cause disease, by infection of toxemia or other means. Many of these are accidental or potential pathogens, that is, microorganisms that cause a pathological condition only under exceptional circumstances, and would n o t be expected to have potential as naturally occurring or applied mortality agents. A substantial proportion of the fungi and bacteria that are pathogenic to insects are facultative pathogens; these 0167-8809/83/$03.00

© 1983 Elsevier Science Publishers B.V.

102 are m i c r o o r g a n i s m s t h a t i n f e c t t h e insect causing a pathological c o n d i t i o n in t h e insect a n d / o r p r o d u c e t o x i n s or a n t i m e t a b o l i t e s t h a t can have a d e t r i m e n t a l e f f e c t o n t h e h o s t ; these m i c r o o r g a n i s m s can also m u l t i p l y and grow o u t s i d e t h e insect t o live s a p r o p h y t i c a l l y . T h e viruses, p r o t o z o a and s o m e e n t o m o p a t h o g e n i c fungi and b a c t e r i a are obligate parasites, n o t multip l y i n g o u t s i d e the living tissue o f t h e h o s t insect or closely related insects. O n l y a few o f t h e k n o w n facultative and obligate p a t h o g e n s o f insects are c o n s i d e r e d t o have a significant role, or the p o t e n t i a l for a significant role, in t h e r e g u l a t i o n o f i n s e c t pests e i t h e r b y natural o c c u r r e n c e or as i n t r o d u c e d p a t h o g e n s . S o m e are listed in T a b l e I. TABLE I Some entomopathogens of interest in management of pest insects VIRUSES Nuclear polyhedrosis viruses Agrotis segetum NPV Autographa californica NPV Choristoneura fumiferana NPV Gilpinia (= Diprion ) hercyniae NPV Hadena sordida NPV Heliothis NPV L yrnantria dispar NPV Mamestra brassicae NPV Neodiprion lecontei NPV Neodiprion sertifer NPV Orgyia pseudotsugata NPV Oryctes rhinoceros NPV Pseudaletia unipuncta NPV Spodoptera frugiperda NPV Spodoptera exiqua NPV Trichoplusia ni NPV

Granulosis viruses Cydia (= Laspeyresia ) pomonella GV Pieris brassicae GV Pieris rapae GV Cytoplasmic polyhedrosis viruses Choristoneura fumiferana CPV Dendrolimus spectabilis CPV Thaumetopoea pityocampa CPV Entomopox viruses Choristoneura fumiferana EPV Melolontha melolontha EPV

BACTERIA Bacillus Bacillus Bacillus Bacillus Bacillus Bacillus

lentirnorbus popilliae sphaericus thuringiensis var. israelensis thuringiensis var. kurstaki thuringiensis var. thuringiensis

FUNGI Aschersonia spp. Beauveria bassiana Coelomomyces spp. Conidiobolus osrnodes Entomophlhora spp. Hirsutella thompsonii Metarrhizium anisopliae Nomuraea rileyi Verticillium lecanii Zoophthora (= Entomophthora) aphidis Zoophthora (= Entomophthora) phytonorni

PROTOZOA Nosema fumiferanae Nosema locustae Nosema pyraustae Vairimorpha necatrix

P a t h o g e n s are a t t r a c t i v e f o r m a n a g e m e n t o f pest insects because: t h e y are effective, n a t u r a l l y o c c u r r i n g or i n t r o d u c e d p a t h o g e n s , and are c o m p e t i t i v e with o t h e r m e t h o d s o f c o n t r o l ; t h e y are specific f o r t a r g e t pest insects or groups o f species, have little or n o adverse i m p a c t on t h e beneficial and nont a r g e t a r t h r o p o d f a u n a and are n o t h a z a r d o u s t o h u m a n s , m a m m a l s or o t h e r i m p o r t a n t animals; t h e y are n a t u r a l l y o c c u r r i n g and d o n o t p o l l u t e t h e field h a b i t a t ; t h e y m a i n t a i n effectiveness because pest insects d o n o t develop

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resistance or tolerance; and they are compatible with many chemical pesticides and with other means of pest control. ASSESSMENT OF ENTOMOPATHOGENS

The role that an e n t o m o p a t h o g e n has or could have in the regulation of populations of an insect pest is influenced by several factors {Table II). Some of these factors are especially applicable to the evaluation of a naturally occurring pathogen, whereas others have application only to assessment of the benefit and feasibility of mass-producing an entomopathogen for introduction into the habitat of the pest insect in order to regulate it or its damage to an economically acceptable level. Ranking of the factors b y importance as criteria in assessing the potential role of an e n t o m o p a t h o g e n or a group of pathogens is n o t a t t e m p t e d because the relative importance of such factors or criteria tends to vary with the particular circumstance. TABLE II Criteria applicable in assessing the potential role of an entomopathogen in management of pest insects 1. Effectiveness Regulation and/or suppression compared to that required or desired. Efficacy compared to other control agents available. Dissemination in population of target insects and carry-over in population. Persistence in habitat o f target insects, especially on substrate contacted by target insect. Effect o f limiting environmental factors, e.g., humidity and temperature. Compatibility with pesticides and probability of enhanced effectiveness b y integration. Compatibility with parasitic and predaceous arthropods and probability of enhanced effectiveness by integration. Techniques, including microenvironmental manipulations, to increase effectiveness o f natural or introduced entomopathogen. Enhancement of effectiveness by formulation. 2. Safety and specificity Host range and specificity. Probability of impact on nontarget fauna and flora in the habitat. Safety for humans, mammals, other vertebrates and invertebrates. Historical evidence of safety. Cost of safety testing. 3. Production and propagation Feasibility of:mass-propagation; availability of improved techniques. Shelf-life and formulation. 4. Marketability and profitability Niche to be filled; competing insecticides or techniques. Size o f market; number o f target insects; economic importance. Cost--benefit relationship for user. Public reaction.

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I. Effectiveness Effectiveness in suppressing the population of the insect pest and/or protecting the crop or animal or other substrate against the pest is the major consideration in the assessment of the potential role of most naturally occurring or applied entomopathogens in pest management. Effectiveness of an insecticide is based on a composite of several factors, but primarily it is a function of ability to kill, cause demonstrable harm to or otherwise impair a pest insect, and of the probability of the insect contacting the insecticide. In addition, effectiveness is an expression of suppression of the population by a mortality agent considering the degree of suppression required or desired.

(i) Regulation and suppression required The degree and extent of suppression of the population required to give acceptable regulation of the pest is a significant consideration in assessing the effectiveness of an entomopathogen. Generally, populations of pests t h a t feed directly on the portion Of the plant or animal that is to be used or t h a t is critical to the development or survival of the plant or animal must be regulated at lower densities than populations of pest insects t h a t do not feed on the portion to be utilized or that can be tolerated in substantial numbers w i t h o u t causing harm to the plant or animal. For example, populations of the European spruce sawfly, Diprion (=Gilpinia)hercyniae (Hartig) (Bird and Elgee, 1957), are maintained at low densities in New Brunswick, Canada by a nuclear polyhedrosis virus t h a t was introduced and subsequently became established. The degree of suppression of the population of the sawfly by this virus is acceptable for this pest whereas a similar degree of suppression of other pests, such as the codling moth, Cydia (=Laspeyresia) pomonella (L.), a major pest of apple, would n o t be considered to be satisfactory. Similarly, infection of up to 90% of adults of the European corn borer, Ostinia nubilalis (Hiibner), by microsporidia may sufficiently reduce damage of maize grown for livestock feed, but n o t o f peppers in which no damage by the corn borer can be tolerated. Nevertheless, the natural infection of O. nubilalis adults by microsporidia is helpful in the protection of the peppers because suppression of the adult population reduces the potential damage to peppers, thus reducing the need for chemical insecticides.

(ii) Efficacy of entomopathogens Efficacy, a measure of the ability of a pathogen to kill, inactivate or disrupt the individual host insect, is a valuable tool in assessing entomopathogens, but, because efficacy is determined under laboratory conditions or under controlled or unnatural field conditions, data may not be closely related to actual effectiveness in the natural field habitat. Efficacy data expressed as LDso, the dose t h a t is lethal to 50% of exposed individuals, or as LCs0, the concentration on or in a substrate t h a t is lethal to 50% of

105 exposed individuals, are, nevertheless, very valuable in expressing or comparing activities of pathogens. For example, the similarity of the LDs0 values for the single-embedded nuclear polyhedrosis virus of Trichoplusia ni (TNNPV-SEV) and of the nuclear polyhedrosis virus of Autographa californica (ACNPV) against Trichoplusia ni (Hiibner) in laboratory tests suggested similar activity in the field against T. ni larvae, but field plot tests were required to determine the protection of cabbage that would result from application of a dosage in the field (Jaques, 1977a). Likewise larvae of Pieris rapae (L.) were more susceptible than larvae of T. ni to the crystal endotoxin of Bacillus thuringiensis (B. t. } in the laboratory and were controlled in the field by lower dosages of formulations of B.t. than were T. ni larvae (Jaques, 1973a). On the other hand, reproducible LCs0 and LDs0 data for fungal pathogens have been obtained for only a few host--pathogen systems (Champlin et al., 1981; Tedders et al., 1982) and appear to have a limited relation to effectiveness of the fungi in the field.

( iii) Dissemination Dissemination and/or establishment in the population of the pest substantially affect the ability of many pathogens, particularly naturally occurring pathogens, to regulate a pest species. The effectiveness of TNNPV or ACNPV against the cabbage looper (Jaques, 1972b, 1974a, 1974b), or of Hirsutella thompsonii against the citrus rust mite, Phyllocoptruta oleivora (Ashmead) (McCoy, 1981), results partially from the establishment of the pathogen in the field habitat following application. Similarly, the effectiveness of Zoophthora (= Entomophthora) p h y t o n o m i and Conidiobolus osmodes, naturally occurring pathogens of the alfalfa weevil, Hypera postica (GyUenhal), is totally dependent on their transmission within populations of the weevil (Puttler et al., 1978; Ben-Ze'ev and Kenneth, 1980). On the other hand, dissemination within the population has little or no influence on the effectiveness of B.t. against target insects, and its use is similar to that of a chemical insecticide. It would appear that facultative pathogens which can exist saprophytically between and within generations of a host, and which may, therefore, have a high capacity for dissemination, should be more effective as naturally occurring pathogens than should obligate pathogens, such as viruses, especially if the obligate pathogenicity is highly specific. This reasoning would suggest, for example, that naturally occurring Beauveria bassiana should have a substantial impact on populations of several species, but, on the contrary, diseases caused by this fungus are usually enzootic (Ferron, 1981), partially due to low efficacy. Similarly, diseases caused by Bacillus cereus, a saprophyte with a wide insect host range, are usually enzootic. Conversely, infection of adults of the corn borer, O. nubilalis, by microsporidia is widespread, reaching an incidence of 90%, largely because of the capacity of the microsporidium for dissemination and because of its high infectivity. Other arthropods and animals may contribute to the dissemination of

106 pathogens thus influencing effectiveness, especially of naturally occurring pathogens. The dissemination of viruses by parasitic spe_cies (e.g. Pieris ra_pae granulosis virus (P. rapae GV) by Apanteles glomeratus (Levin et al., 1979)) enhances the effectiveness of the virus. In addition, the carrying of viruses by animals such as birds (e.g. Choristoneura fumiferana NPV (Bird, 1955) and TNNPV (Hostetter and Biever, 1970)) is a means of introducing the virus into populations of the host.

(iv) Persistence o f the pathogen Persistence of the entomopathogen in the habitat of the host insect, particularly the ability to remain active on a substrate from which infection of the host may occur readily, is a very important factor affecting the effectiveness of naturally occurring and most introduced pathogens. The majority of the pathogens of current interest in pest management are applied to foliage of plants, or contact the target insect on plants, where the pathogen is exposed to sunlight. Most pathogens are adversely affected by exposure to sunlight, presumably by the ultraviolet portion of sunlight (Ignoffo et ai., 1977; Jaques, 1977b; Maddox, 1977; Pinnock et al., 1977; Roberts and Campbell, 1977; Leong et al., 1980; Kreig et al., 1981). Data on the relative susceptibility of B. t., viruses, fungi, and microsporidia to inactivation by sunlight vary, but most pathogens appear to have a half-life of 1--2 days when exposed to sunlight. For example, Beegle et al. (1981) found that the halflife of insecticidal activity of B.t. on soybean foliage was 1.5--2 days. Ignoffo and Batzer (1971) found that Heliothis NPV (HNPV) retained little activity 2 days after application to cotton foliage. TNNPV or P. rapae GV retained about 50% of original activity in a similar period after application to cabbage (Jaques, 1975), whereas Lymantria dispar NPV (Podgwaite et al., 1979) and Neodiprion sertifer NPV (Mohamed et al., 1982) applied to forest trees were inactivated more slowly. Spores of Vairimorpha necatrix were 50% inactivated within 0.6, 1.9 and 2.2 days on cotton, tobacco, and soybean foliage, respectively (Fuxa and Brooks, 1978). Inactivation of deposits of entomopathogens may be more detrimental to effectiveness against pest species that feed on an exposed substrate for a comparatively small portion of their life cycle. Larvae of P. rapae and T. ni that escape or otherwise survive an application of TNNPV or P. rapae GV, perhaps because they did not emerge from the egg until the residues of the spray were practically inactivated, could be killed by a subsequent application. On the other hand, larvae of the codling moth, Cyclia pomonella, that escaped an application of C. pomonella GV would have burrowed into the apple or nut and would not be exposed to a subsequent application. Additives to formulations of entomopathogens to protect deposits against inactivation by sunlight has extended the period of activity of the pathogens, enhancing activity. Early tests indicated that some dark-colored proteinaceous materials (Jaques, 1971, 1972a) or carbonaceous materials (Ignoffo and Batzer, 1971) extended the activity of viruses. Additives developed to

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protect entomopathogens are now included in some formulations to enhance effectiveness. It is significant that selection of C. pomonella GV increased tolerance to ultraviolet radiation to some extent (Brassel and Benz, 1979), but selection of ACNPV (Witt and Hink, 1979) increased tolerance only slightly suggesting that this technique may not be as feasible as physical or chemical protection procedures, especially incorporation of ultraviolet-screening materials into formulations and/or spray mixtures (Couch and Ignoffo, 1981). Viral deposits protected by the cadaver of the host or by decomposed body fluids appear to retain activity for extended periods (e.g.L. dispar NPV (Podgwaite et al., 1979)), increasing intergeneration transmission of naturally occurring viruses. Similarly, viruses and other pathogens deposited in soft or in the debris on the soil surface may retain activity for long periods (Jaques, 1964. 1967b, 1974a, 1974b; Young and Yearian, 1979; Thompson et al., 1981; McLeod et al., 1982) and serve as a reservoir from which pathogens are transferred by wind and rain to foliage of plants or trees to initiate epizootics. Podgwaite et al. {1979) found that L. dispar NPV persisted between generations in cadavers and on the bark of trees. Although spores of B.t. remain active for long periods in soil (Saleh et al., 1970), the concentration of the bacterium accumulating from killed host insects is not sufficient to initiate disease or toxemia. Treatment of soil at transplanting (Jaques, 1973a, 1973b) or treating plants before transplanting (Ignoffo et al., 1980) with NPV were found to be effective techniques for introducing the virus into the field to control T. ni, taking advantage of the persistence of the virus in the soil. The prolonged persistence of spores of Bacillus popilliae in soil following inoculation (Hutton and Burbutis, 1974) substantially enhances the effectiveness of this pathogen in the control of larvae of the Japanese beetle, Popilliae japonica Newman (Klein, 1981). The importance of the pathogen remaining active in a substrate accessible to the target insect is also applicable to the need for persistence of spores of Bacillus thuringiensis vat. israelensis (B. t.i. ) in water, preferably near the surface, to be effective against mosquito larvae. (v) Effect o f microenvironment -- humidity Humidity and surface moisture affect the effectiveness of some entomopathogens, and in some cases are limiting factors. For example, desiccation is a major factor limiting the usefulness of nonsporeforming bacteria as microbial insecticides, whereas desiccation has little effect on the activity of spores of B. popilliae and B.t. (Pinnock et al., 1977). Desiccation has little effect on NPV and GV viruses (Jaques, 1977b), but reduces the activity of microsporidia such as V. necatrix (Chu and Jaques, 1981). On the other hand, high humidity, particularly with alternate wetting and drying of surface moisture (Maddox, 1977), may reduce the period of activity of spores of some microsporidia by promoting extrusion of the polar filament, stimulating germination of the spore.

108 Although humidity has little effect on the persistence of viruses on foliage it has been found that the alkaline reaction of dew on cotton foliage contributed to inactivation of HNPV (Andrews and Sikorowski, 1973). Furthermore, wet smears of TNNPV were inactivated more readily than dry smears by exposure to ultraviolet light (Jaques, 1967a). The effectiveness of fungi is affected directly by humidity and surface moisture. Infection by most fungi is favored by higher humidity (Roberts and Campbell, 1977; Dedryver, 1979; Ignoffo, 1981; McCoy, 1981), but surface moisture may be slightly detrimental except that rain contributes to dissemination of spores. Allen and Kish (1978), for example, considered that a 70% relative humidity is minimum for germination of spores of Nomuraea rileyi and that alternating wet and dry conditions were necessary for infection and epizootic development with dry conditions and light wind favoring conidial dispersion. Likewise, higher humidity enhanced the effectiveness of Verticillium lecanii in populations of whiteflies and other insects in glasshouses (Hall, 1981). Inhibition of entomofungi by low humidity may be offset by manipulation of the microenvironment of the pest and pathogen. For example, Burleigh {1975) showed that infection of Heliothis species by N. rileyi was greater on cotton that grew with a closed foliage canopy that promoted a higher humidity than on varieties Of cotton that grew with an open canopy. Similarly, mortalities of H. zea and Plathypena scabra (Fabricius) larvae by N. rileyi on soybean were increased by reducing the space between rows thereby increasing the relative humidity of the microhabitat by retarding loss of moisture from dew and protecting the fungus from inactivation by solar radiation (Jaques, 1978). (vi) Compatibility with chemical pesticides Compatibility with other agents applied for control of a pest or with naturally occurring mortality agents acting on the pest species is a major consideration in assessing efficacy of an entomopathogen. The numerous studies on compatibility of entomopathogens with chemical pesticides have been reviewed elsewhere (Benz, 1971; Burges, 1981a; Jaques and Morris, 1981) and only a few examples are discussed here. The activity of bacteria, notably B.t., was not adversely affected by most pesticides at the rates used in agriculture or forestry. Some insecticides (e.g. naled and parathion (Dougherty et al., 1971)) slightly reduced the activity of B.t., but most other insecticides had a potentiating or additive effect on B.t. (Morris, 1977a). Similarly, entomoviruses, especially the Baculoviruses, are generally compatible with most common chemical pesticides. On the other hand, entomofungi are adversely affected by the majority of fungistatic and fungicidal chemicals, but not by most insecticides. For example, the fungicides benomyl, maneb, captan, ferbam and chlorothalonil inhibited many fungi as did the insecticides methyl parathion and phenoate (Ignoffo, 1981; McCoy, 1981). Other laboratory tests showed that benomyl was among the most

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inhibitory chemicals against Beauveria bassiana (Olmert and Kenneth, 1974) and N. rileyi (Ignoffo et al., 1975), but dinocap had little effect on B. bassiana. Tedders (1981) found that dinocap and sulphur were least toxic to B. bassiana and Metarrhizium anisopliae, whereas triphenyltin hydroxide was the most toxic. The value of selecting fungicides compatible with entomofungi is well illustrated by the inhibition of mortality of Psylla mali Schmidb. by Entomophthora sphaerosperma in orchards sprayed with certain fungicides (Jaques and Patterson, 1962). Likewise, mortality of aphids on potatoes caused by Entornophthora spp. was inhibited by use of certain fungicides, and application of methidathion to citrus trees increased populations of mites that were normally controlled by naturally occurring Hirsutella thompsonii (McCoy, 1981). Some mixtures of entomopathogens and chemical insecticides are not only compatible but additive in effect against the pest species, enhancing the potential role of these entomopathogens in integrated pest management. For example, combinations of B.t. and carbaryl or carbofuran appeared to have a synergistic or additive effect against larvae of Heliothis virescens (F.) (Chen et ah, 1974}. Likewise, some low
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The combination of chemical and microbial insecticides sequentially in treatment regimes has potential as an effective use of entomopathogens in integrated pest management. Treatment regimes including applications of viruses, bacteria and a chemical were very effective in protecting cabbage plants in studies by Jaques (1972b, 1973a, 1977a).

(vii) Compatibility with parasitic and predaceous arthropods The compatibility of most entomopathogens with parasitic and predaceous arthropods enhances their effectiveness and contributes significantly to their potential usefulness in integrated pest management (Jaques and Morris, 1981). Forsberg (1976), in a review of the environmental impact of B.t., did not find evidence that B.t. or the crystal endotoxin was directly antagonistic or toxic to parasitic species of insects. Although several nonlepidopterous insects have been reported to have been infected by B.t. (Kreig and Langenbruch, 1981), the effects on field populations have been negligible. For example, the population of only one non-lepidopterous insect was suppressed by applications of B.t. to a Canadian apple orchard in a 4-year period (Jaques, 1965). It is suggested that reduced incidence of parasitism of species of lepidopterous pests observed following treatment with B.t. (Vail et al., 1972; Reardon et al., 1979) was due to indirect effects of B.t., such as reduced density of the populations of the insect hosts of the parasite. Weseloh and Andreadis (1982) suggested that application of B.t. enhanced parasitism of L ymantria dispar larvae by Apanteles melanoscelus because the bacterium delayed deveiopment of host larvae. Similarly, the incidence of NPV was positively correlated with the incidence of parasitoids in populations of L. dispar in eastern U.S.A., indicating mutually advantageous joint action (Reardon and Podgwaite, 1976). Not all entomopathogens are compatible with parasitic or predaceous arthropods. For example, Levin et al. (1981) found that time of infection of P. rapae larvae by GV relative to time of oviposition by A. glomeratus influenced the competitive position of the parasite. Beegle and Oatman (1975) made similar observations on the relationship of the parasite Hyposoter exiguae and TNNPV in T. ni larvae. An additional type of competition was demonstrated by the finding that Baculoviruses of Mythimna (= Pseudaletia ) unipuncta (Haworth), Spodoptera exigua (Hiibner) and T. ni impaired parasitism by Apanteles spp., apparently by producing a toxin that inhibited growth and development of the parasite (Kaya and Tanada, 1973). Parasitic and predaceous species may increase the effectiveness of entomopathogens by transmitting pathogens within populations of pest species. For example, P. rapae GV was transmitted within populations of P. rapae larvae by ovipositing A. glomeratus adults (Levin et al., 1979), and L. dispar NPV was transmitted-by A. melanocelus (Raimo et al., 1977). Biever et al. (1982) exploited the ability of predators to transmit TNNPV to T. ni by releasing virus-contaminated predators into the field habitat. The transmission of

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pathogens by parasites could play a substantial role in the dissemination of pathogens of insects that feed alone on a substrate that is not exposed (e.g. leafrollers and boring insects). (viii) Techniques to increase effectiveness The probability of increasing the effectiveness of a pathogen is a significant consideration in assessing potential for use. As indicated previously, effectiveness of naturally occurring pathogens may be enhanced by procedures or manipulations that protect the pathogen from inactivation, that provide a more favorable microenvironment or microhabitat for infection and disease development, or that increase dissemination of the pathogen (Allen et al., 1978). In addition, effectiveness of introduced pathogens may be enhanced by selection and genetic manipulations, formulation, and method of introduction. Selection of TNNPV by Potter et al. (1978) did not yield strains of TNNPV that were more active or that killed more quickly. However, Wood et al. (1981) selected a mutant of ACNPV that killed T. ni larvae more quickly than did the naturally occurring ACNPV. Serial passage of C. fumiferana NPV in alternate hosts altered the pathology of disease caused by the virus in C. fumiferana larvae (Stairs et al., 1981), but did not appreciably alter its efficacy. Likewise, serial passage of ACNPV in O. nubilalis did not enhance infectivity for this secondary host (Lewis et al., 1977), but passaging ACNPV and TNNPV in S. exigua did increase virulence for T. ni (Tompkins et al., 1981). Nutrition of T. ni larvae in which ACNPV was propagated appeared to influence the virulence of the virus produced (Baugher and Yendol, 1981). Although the results of selection for viruses of enhanced activity have been variable, this technique remains a feasible possibility as a means of increasing effectiveness. Screening and selection yielded high potency strains of B.t., notably the H.D.-1 strain of B.t. vat. kurstaki, and has the potential of identifying isolates that have a higher activity against certain species (Dulmage, 1981). In addition, Martin and Dean (1981} were very optimistic about the probability of producing strains of B.t. with higher efficacy by genetic manipulation. Similarly, there is believed to be potential for increasing infectivity of fungi by genetic manipulation although this is not well supported by current studies (A1-Aidroos and Roberts, 1978; A1-Aidroos and Seifert, 1980; Hall, 1980}. (ix) Formulation The formulation of entomopathogens has a substantial influence on their effectiveness in management of pest species. Requirements to maintain efficacy of the product and to enhance distribution, persistence, activity and host attractiveness of the pathogen have been reviewed by Couch and Ignoffo (1981) and other authors. Equally important, the method of application must be suited to the specific requirements of the entomopathogen (Smith et al., 1978; Smith and Bouse, 1981). In addition, the additives included in formulations must increase the suitability of the pathogen to

112 the substrate to be treated and should protect the pathogen against inactivation b y environmental factors in order to prolong activity. For example, c o m p o n e n t s of formulations of B.t.i. should aid in maintaining the pathogen in pools of water to facilitate ingestion by mosquito larvae (Ramoska et al., (1982), whereas formulations of Cydia pomonella GV, applied for control of the codling moth, should enhance uniform distribution on fruit and leaves of apple (Jaques et al., 1981) and should contain additives that protect the virus from inactivation by sunlight. Cornmeal bait formulations were an effective m e t h o d of distributing spores of Vairimorpha necatrix against Heliothis larvae on t o b a c c o (Fuxa and Brooks, 1979), and bran-based baits were used for spreading spores of Nosema locustae for control of grasshoppers on rangelands (Henry and Oma, 1981). Feeding attractants and other adjuvants in sprays enhanced effectiveness of some entomopathogens (Ignoffo et al., 1976; Smith and Hostetter, 1982). The benefit of incorporating attractants and other adjuvants in formulations should be explored further. Formulation and application equipment are particularly important in the use of pathogens against insects in forests (Morris, 1977b; Cunningham, 1978; Cunningham and Entwistle, 1981) in which distribution and activity of small volumes of spray mixture are required for aerial application. 2. SAFETY AND SPECIFICITY Effects on the user, on the consumer of the crop or product treated and on the nontarget fauna and flora in the field habitat, and other side-effects of the entomopathogens are major criteria in assessing the potential role of an entomopathogen in integrated management. Safety is not absolute; a pathogen is considered safe if there is no evidence and/or no reason to believe that the pathogen can infect or be otherwise hazardous to humans, other nontarget animals and plants and if environmental impact is zero or negligible. The development of series of tests to assess the risk in the use of entomopathogens has been an arduous task, largely because of the requirem e n t to develop basic knowledge by which to assess the risk. The assessment and development of procedures for testing, and the development of protocols of test procedures, have been the subject of numerous articles over the past 15 years, some of the more recent of which are by Burges (1980, 1981a, 1981b), Burges e t al. (1980a, 1980b), Ignoffo et al. (1979), Lundholm and Stackerud (1980), Miltenburger (1980), Papworth (1980), and Rogoff (1980). The protocol developed by the Environmental Protection Agency of the United States in 1981 and the subsequent adaptation of the tier system of testing were considered as major advances in the development of entomopathogens. The tier approach reviewed by Rogoff (1980) is based on the fact that the entomopathogens have been in the environment for a long time, nullifying the need to carry out long-term exposure tests if acute toxicity is

113 n o t demonstrable. Therefore, the absence of unfavorable reactions in nontarget species in the first tier of tests, the maximum hazard tests of toxicity, is considered satisfactory p r o o f of safety to humans, other nontarget fauna and flora of the habitat, thus negating the need to carry o u t tests in subsequent tiers. The tests applied in granting exemption from tolerance and/or use permits in the U.S., Canada, U.K., France, and the Federal Republic of Germany for the early formulations of B.t. var. thuringiensis in 1965--67, and for formulations of B.t. vat. kurstaki and var. alesti in 1970--73, were modified to assess HNPV for use against Heliothis species on cotton. Further modifications of this protocol using a tiered system of testing were applied in the registration in the United States of L. dispar NPV, Orgyia pseudotsugata NPV, B.t. var. israelensis, N. locustae and Hirsutella thompsonii. Similar protocols are being developed and/or applied in assessing the safety of microbial agents in other countries. Safety of entomopathogens is a function of specificity. Pathogens such as Baculoviruses which infect and multiply only in living cells of insects, usually cells of only one species, should be less likely to be hazardous to nontarget organisms than should a pathogen such as Pseudomonas aeruginosa which can multiply in a variety of live and dead tissues and substrates. Nevertheless, B.t., for example, is a facultative pathogen that multiplies in a variety of insect hosts as well as in several saprophytic substrates, b u t B.t. has no pathogenicity for mammals and most nontarget nonpest species b y normal routes of entry, and thus it is not considered to be hazardous. Similarly, the fungi B. bassiana and Metarrhizium anisopliae which, like B.t., multiply in a variety of insect hosts, appear to pose little or no threat to mammals (Shadduck et al., 1982). The toxins produced by some bacteria and fungi must be considered in evaluating safety to nontarget organisms. The toxins produced by entomofungi have been found to have little or no effect on mammals and other nontarget species (Roberts, 1981). The Beta toxin of B.t., thuringiensin, may be somewhat toxic to humans, other vertebrates and nontarget invertebrates (Sebesta et al., 1981), b u t the hazard to higher animals may, nevertheless, be low, especially compared to that of some chemical insecticides, if normal precautions utiliz_ed in handling insecticides are exercised. A substantial consideration in the risk in the use of an entomopathogen is the probability that the pathogen introduced may adapt to n e w hosts b y mutation or selection. This concern is becoming less of a consideration in the development of entomopathogens as techniques to assess the risk are improved and as knowledge increases, especially as more is learned on muta, tion rates, identification, and specificity. 3. P R O D U C T I O N

AND PROPAGATION

OF PATHOGENS

The technical and economic feasibility of propagation and/or production of an entomopathogen at the scale required for commercial distribution as a

114 microbial insecticide is a major concern in assessing the potential of a pathogen. It is apparent that pathogens, such as B.t., B.t.i., B. bassiana, Verticilliurn lecanii and H. thompsonii, that can be propagated on artificial media and other readily available substrates using techniques adaptable to mass-production should be more attractive candidates for development than are obligate pathogens, such as viruses and microsporidia, or other pathogens which are aifficult to propagate in large quantities in a form suitable for formulation, distribution and use. The difficulties associated with obligate pathogenicity appear to limit development of some entomoviruses. For example, relatively low-cost mass-rearing of host insects permits masspropagation of HNPV, ACNPV and TNNPV, b u t the higher cost of production of C. pomonella GV and Choristoneura fumiferana NPV relative to the benefit and market m a y limit their development. On the other hand, respondents to the Stanford study (Ayers et al., 1977) suggest that the difficulties in mass-production may n o t be a limiting factor for entomoviruses provided that sufficient economic return is available to justify the development of the required production technology. Thus, it could be argued that development of Cydia pomonella GV is feasible and justified because of the need for the virus in integrated management of this world-wide pest of apples, pears and some n u t crops. Similarly, the economic benefits of control of the Japanese beetle using spores of Bacillus popilliae warrant the cumbersome labor-intensive techniques required in the production of spores (Klein, 1981), whereas such expensive techniques could n o t be justified for B.t., B.t.i. or HNPV. In addition, it is doubtful if the required scale of production of the latter entomopathogens could be met b y these techniques. The use of cell cultures for propagation of obligate entomopathogens, such as viruses that must be propagated in or on living tissue, is a promising alternative to propagation in live insects (Stockdale and Priston, 1981). Although tremendous advances have been made in increasing the yields of active viruses from cell cultures and in decreasing costs of cell propagation, the use of live insects remains the most economical procedure for massproducing viruses. In addition, certain viruses with potential for development (e.g. codling moth GV) have n o t y e t been propagated in cell culture. It is n o t e w o r t h y that the culture of entomoviruses in cells other than those of an insect, notably cells of yeast, has n o t been successful. Propagation of viruses in an alternate host that is more readily mass-cultured or that produces larger yields of virus appears to be a fruitful area of research. Recent improvements in propagation techniques have greatly enhanced the potential of some entomogenous fungi as candidates for development as microbial insecticides. For example, improved formulations of H. thompsonii for control of the citrus rust mite are conidia, n o t fragments of mycelia used in early formations (McCoy, 1978, 1981). Similarly, development o f procedures for propagating large numbers of spores of Zoophthora aphidis, Z. phytonomi and other fastidious fungi increases their potential for development.

115 4. MARKETABILITYAND PROFITABILITY The marketability and the profit expected from the marketing of a microbial insecticide are major factors influencing development by private enterprise. To be a marketable product the microbial insecticide should fill a niche not already adequately filled. For example, B.t. filled a niche in the management of pests of horticultural crops and cotton because an alternative insecticide that was effective and not detrimental to naturally occurring biological mortality agents was not available. For similar reasons B.t.i. has tremendous potential for extensive use in the control of mosquitoes, a universally important group of pests. HNPV was suggested by 79% of respondents in the Stanford study (Ayers et al., 1977) to have the best potential among the entomoviruses for commercialization, largely because of the large potential market in the management of H. zea and H. virescens on cotton. Because of the comparatively good effectiveness of other biological agents, notably B.t. and ACNPV, against these pests on cotton the full potential of HNPV may not be realized. Conversely, P. rapae GV has little potential for development because it is active against only one host, P. rapae, and because P. rapae on most crops is readily controlled by B.t. and wellestablished chemical insecticides. The NPV of the rhinoceros beetle, Oryctes rhinoceros, kills only one pest but is an attractive virus because, although the cost of release is high, the dissemination through populations of the host results in good crop protection at a relatively low cost per unit area compared to the benefit (Bedford, 1981). ASSESSMENT OF POTENTIAL OF ENTOMOPATHOGENS

Assessment of the potential role of entomopathogens in pest management is somewhat subjective even though objective criteria are applied. In addition, insufficient knowledge of the actual or potential impact of pathogens, especially naturally occurring pathogens, in integrated pest management hampers the assessment procedure. The following is an indication of some features that affect the potential role of some representative entomopathogens as regulatory agents in management of pest insects. The reader is referred to appropriate recent articles for a more complete assessment (Bulla, 1973; Ayers et al., 1977; Allen et al., 1978; Cunningham, 1978; Roberts, 1980; Singer, 1980; Burges, 1981a, 1981c; Dulmage et al., 1981). The bacteria

Relatively few of the bacteria that kill insects have potential for a significant role in pest management. The wide range of activity of some bacteria increases the hazard to mammals and nontarget arthropods reducing their suitability as introduced pathogens. The bacteria that are currently con-

116 sidered to have potential are sporeforming facultative pathogens that can be readily mass-produced and formulated in stable preparations. The usage of the three species of bacteria (B.t., B. popilliae and B. lentimorbus) currently registered for use indicates the substantial role that pathogens can have in integrated management of pest insects. Bacillus thuringiensis (i) Activity against several species of insects largely attributable to activity of toxins (Liithy, 1980; Dulmage, 1981; Fast, 1981); Krieg and Langenbruch (1981) listed susceptible host species; (ii) Formulations of B.t. var. thuringiensis, var. alesti, and/or var. kurstaki registered in several countries (e.g. Thuricide HPC, Dipel HD-1, Bactospeine, and Entobacterin) for use against several agricultural and forest pests; good use pattern and competitive effectiveness against several important pests, especially lepidopterous larvae ingesting exposed foliage; little or no carryover impact on pest populations except in forests; good competitive status in market; (iii) Further development of thuringiensin, the beta-exotoxin c o m p o n e n t of B.t. (now used in the U.S.S.R. (Sebesta et al., 1981)), would broaden the spectrum of activity,especially to coleopterous insects (Cantwell and Cantelo, 1981); and (iv) Bacillus thuringiensis var. israelensis (Serotype 14) registered for use (e.g. Teknar and Bactimos); highly effective against mosquitoes and blackflies (Davidson et al., 1981;Mulla et al., 1982); high potential for a significant role in control of these important pests indicating a high-volume market. Bacillus popilliae Registered in the U.S.A. for control of larvae of P. japonica in turf (Klein, 1981); good effectiveness resulted in highly successful use; long-term carryover impact on pest populations; labor-intensive production techniques warranted by marketability of product and excellent cost--benefit status. The viruses

The entomoviruses have a high potential as natural or introduced agents for the regulation of populations of pest insects (Ayers et al., 1977; Franz and Huber, 1979; Granados, 1980; Morris, 1980; Shieh and Bohmfalk, 1980; Katagiri, 1981). Some viruses are particularly effective as introduced or naturally occurring pathogens because they become well established in the host populations. Many are lethal to the host by small inocula, whereas others are less active. Except for susceptibility to inactivation by sunlight, viruses are little affected by environmental factors; potential would be increased by development of improved protectant materials and mechanisms. Because of their specificity, the viruses, especially the Baculoviruses (NPV and GV), are considered to present no hazard to nontarget species. Because viruses are obligate pathogens propagation is in living tissue, presently in

117 live host insects; the development of improved techniques for mass-propagation will greatly enhance the competitive market status of entomoviruses n o t only by enabling mass-production but also by improving the purity of formulations. Viruses enter the host by ingestion. Infection is in the larval stage but pupae and adults may be killed by carry-over infection. Only representative Baculoviruses are included in this appraisal; cytoplasmic polyhedrosis viruses (Katagiri, 1981) and nonoccluded viruses (Reed, 1981) are of less current interest for development. Species of insects susceptible to viruses are listed by Martignoni and Iwai (1981). Helio this NP V (Baculovirus heliothis) Registered for use against H. zea and H. virescens on cotton and other crops; potential for widespread use in pest management but variable effectiveness may reduce competitive status (Ignoffo, 1973; Ignoffo and Couch,

1981). Autographa californica N P V Moderate to excellent effectiveness as introduced pathogen against several pest species, especially T. ni, Heliothis sp. and Spodoptera sp., O. pseudotsugata; good potential and favorable economic competitiveness; good carryover; mass-propagation economically feasible; recent experimental use permit for use against O. pseudotsugata in the U.S.A. Trichplusia ni N P V Highly effective against T. ni, the cabbage looper, by natural occurrence or introduction; ACNPV may be preferred over TNNPV for development because of wider spectrum of activity. Cydia pomonella GV Good potential for use in management of C. pomonella, the codling moth; competitive status reduced by variable effectiveness and requirement for frequent application in some locations; little carry-over; improved masspropagation required to increase attractiveness. Choristoneura fumiferana N P V Development favored by high demand to improve control of C. fumiferana, the spruce budworm, a universal pest of forests; moderate effectiveness with good carry-over; high cost of production of effective dosages reduces competitive status (Cunningham, 1978). L y man tria dispar N P V Registered in the U.S.A. for use against L. dispar, the gypsy moth; good potential for use; good effectiveness compared to alternatives; carry-over impact; moderate market volume (Lewis, 1981).

118

Orgyia pseudotsugata N P V Registered in the U.S.A. and Canada (temporary) for use against O. pseudotsugata, the Douglas Fir tussock moth; effectiveness enhanced by carry-over; competitive status hampered by specificity for one pest species of limited distribution; ACNPV being developed in competition. Oryctes rhinoceros NP V Good effectiveness against O. rhinoceros, the rhinoceros beetle, establishment in population gives long-term effect; good potential for use in integrated management (Bedford, 1981). Neodiprion sertifer NPV Like Baculoviruses of other sawflies, good effectiveness but limited market (Cunningham and Entwistle, 1981). The fungi A relatively small proportion of fungi that kill insect pests are considered to have a potential for use in pest management. Many fungi are non-specific facultative pathogens that have wide host range but variable efficacy. All stages of the insect may be infected; entry of the fungus is through the body wall or, more rarely, by ingestion. Entomofungi are important naturally occurring mortality agents for many species of pest insects. Unfortunately, some fungi that are highly effective as natural pathogens are not as effective as introduced pathogens and may be difficult to mass-produce. This may suggest a fruitful area of research. Applied entomofungi usually become established in the environment to some extent, causing carry-over impact. Fungi are sensitive to fungicidal chemicals and to environmental conditions, especially humidity.

HirsuteUa thompsonii Exemption from tolerance in the U.S.A. for control of Phyllocoptruta oleivora, the citrus rust mite, and related pests; highly effective component of pest management program on citrus (McCoy, 1981); carry-over impact; potential enhanced by improved mass-propagation and formulation techniques. Nomuraea rileyi Infects many important pest species, mainly Lepidoptera (Ignoffo, 1981) such as T. ni, Spodoptera spp. and P. scabra; effective as applied and naturally occurring pathogen; excellent potential for development, especially with improvement in propagation techniques. Verticillium lecanii Registered in the United Kingdom; widespread natural occurrence, especially against aphids, scale insects and other small hemipterons, particularly in tropical and glasshouse environments (Hall, 1981); reliable effectiveness as applied agent in glasshouse environment.

119

Beauveria bassiana Registered in the U.S.S.R. (Boverin) and China; acceptable efficacy against some pest species (e.g. Melolontha melolontha, L. decemlineata and C. pomonella) (Ferron, 1981); mass-produced at competitive cost. The pro tozoa

The entomogenous protozoa mainly belong to the microsporidia and gregarinia with the microsporidia being of particular current interest (Brooks, 1980}. The microsporidia are obligate pathogens with varying specificity and are, therefore, propagated in living insect tissue. Although microsporidia may kill slowly, with some infections not causing death, they readily become established in host populations because of their facility for transmission. Infection is by ingestion or by transfer in the egg and is most c o m m o n in immature stages but pupae and adults are also killed. Nosema locustae Registered for use against grasshoppers on rangeland in the U.S.A. (Henry and Oma, 1981; Henry and Onsager, 1982); good effectiveness applied as a bait, especially combined with a low dosage of an insecticide; good potential for expansion of use. Vairimorpha necatrix Wide host range (Maddox et al., 1981) enhances attractiveness for development; good effectiveness; often established in population affording carryover. Nosema pyraustae Good potential against O. nubilalis, the European corn borer, a universal pest of maize (Lublinkhof and Lewis, 1980); high incidence of natural infection in adults may suppress populations; increased effectiveness of introduction and improved techniques for propagation would enhance feasibility of development. REFERENCES AI-Aidroos, K. and Roberts, D.W., 1978. Mutants of Metarhizium anisopliae with increased virulence toward mosquito larvae. Can. J. Genet. Cytol., 20: 211--219. AI-Aidroos, K. and Seifert, A~VI., 1980. Polysaccharide and protein degradation, germination, and virulence against mosquitoes in the entomopathogenic fungus Metarhizium anisopliae. J. Invertebr. Pathol., 36: 29--34. Allen, G.E. and Kish, L.P., 1978. The role of entomopathogens in an integrated pest management system in soybeans. In: G.E. Allen, C.M. Ignoffo and R_P. Jaques (Editors), Microbial Control of Insect Pests: Future Strategies in Pest Management Systerns. Proc. USDA--NSF--Univ. of Florida Workshop, Jan. 1978, pp. 164--185. Allen, G.E., Ignoffo, C.M. and Jaques, R.P. (Editors), 1978. Microbial Control of Insect Pests: Future Strategies in Pest Management Systems. Proc. NSF--USDA--Univ. of Florida Workshop, Jan. 1978, 290 pp.

120 Andrews, G.L. and Sikorowski, P.P., 1973. Effects of cotton leaf surface on the nuclearpolyhedrosis virus of Heliothis zea and Heliothis virescens (Lepidoptera: Noctuidae). J. Invertebr. Pathol., 22: 290--291. Ayers, J.H., Blue, T.A., Bramhall, R.R., Braunstein, T.J., Davis, E.E., De Graw, J.I., Elward, T.E., Inmaw, R.E., Johnson, O.H., Leaf, E.B., Offensend, F.L. and Stent, P.D., 1977. New Innovative Pesticides: An Evaluation of Incentives and Disincentives for Commercial Development by Industry. Stanford Research Institute, Menlo Park, California, U.S.A., 318 pp. Baugher, D.G. and Yendol, W.G., 1981. Virulence of Autographa californica Baculovirus preparations fed with different food sources to cabbage loopers. J. Econ. Entomol., 74: 309--313. Bedford, G.O., 1981. Control of the rhinoceros beetle b y Baculovirus. In: H.D. Burges (Editor), Microbial Control of Pests and Plant Diseases 1970--1980. Academic Press, L o n d o n and New York, pp. 409--426. Beegle, C.C. and Oatman, E.R., 1975. Effect of nuclear polyhedrosis virus on the relationship between Trichoplusia ni (Lepidoptera: Noctuidae) and the parasite, Hyposoter exiguae (Hymenoptera: Ichneumonidae). J. Invertebr. Pathol., 2 5 : 5 9 - 7 1 . Beegle, C.C., Dulmage, H.T., Wolfenbarger, D.A. and Martinez, E., 1981. Persistence of Bacillus thuringiensis Berliner insecticidal activity on cotton foliage. Environ. Entomol., 10: 400--401. Benz, G., 1971. Synergism of microorganisms and chemical insecticides. In: H.D. Burges and N.W. Hussey (Editors), Microbial Control of Insects and Mites. Academic Press, L o n d o n and New York, pp. 327--355. Ben-Ze'ev, I. and Kenneth, R.G., 1980. Zoophthora phytonomi and Conidiobolus osmodes [Zygomycetes: Entomophthoraceae], two pathogens of Hypera species [Col.: Curculionidae] coincidental in time and place. Entomophaga, 25: 171--186. Biever, K.D., Andrews, P.L. and Andrews, P.A., 1982. Use of a predator, Podisus maculiventris, to distribute virus and initiate epizootics. J. Econ. Entomol., 75: 150--152. Bird, F.T., 1955. Virus diseases of sawflies. Can. Entomol., 87 : 124--128. Bird, F.T. and Elgee, D.E., 1957. Virus disease and introduced parasites as factors controlling the European spruce sawfly, Diprion hercyniae (Htg.), in central New Brunswick. Can. Entomol., 89: 371--378. Brassel, J. and Benz, G., 1979. Selection of a strain of the granulosis virus of the codling m o t h with improved resistance against artificial ultraviolet radiation and sunlight. J. Invertebr. Pathol., 33: 358--363. Brooks, W.M., 1980. Production and efficacy of protozoa. Biotechnol. Bioeng., 22: 1415---1440. Bulla, L. (Editor), 1973. Regulation of insect populations by microorganisms. Ann. N.Y. Acad. Sci., 217: 1--243. Burges, H.D., 1980. Risk analysis in the registration of pesticidal bacteria: Pathogenicity and toxicological aspects. In: B. Lundholm and M. Stackerud (Editors), Environmental Protection and Biological F o r m s of Control of Pest Organisms. Ecol. Bull. (Stockholm), 3 1 : 8 1 - - 9 0 . Burges, H.D., 1981a. Strategy for microbial control of pests in 1980 and beyond. In: H.D. Burges (Editor), Microbial Control of Pests and Plant Diseases 1970--1980. Academic Press, L o n d o n and New York, pp. 797 -836. Burges, H.D., 1981b. Safety, safety testing and quality control of microbial pesticides. In: H.D. Burges (Editor), Microbial Control of Pests and Plant Diseases 1970--1980. Academic Press, L o n d o n and New York, pp. 737--767. Burges, H.D. (Editor), 1981c. Microbial Control of Pests and Plant Diseases 1 9 7 0 - 1 9 8 0 . Academic Press, L o n d o n and New York, 949 pp. Burges, H.D., Croizier, G. and Huber, J., 1980a. A review of safety tests on baculoviruses. Entomophaga, 25 : 329--340. Burges, H.D., Huber, J. and Croizier, G., 1980b. Guidelines for safety tests on insect viruses. Entomophaga, 25: 341--348.

121

Burleigh, J.G., 1975. Comparison of Heliothis spp. larvalparasitism and Spicaria infection in closed and open canopy cotton varieties.Environ. Entomol., 4: 574--576. Cantwell, G.E. and Cantelo, W.W., 1981. Bacillus thuringiensis, a potential control agent for the Colorado potato beetle. A m . Potato J., 58: 457--468. Champlin, F.R., Cheung, P.Y.K., Pekrul, S., Smith, R.J., Burton, R.L. and Grula, E.A., 1981. Virulence of Beauveria bassiana mutants for the pecan weevil. J. Econ. Entomol., 74: 617--621. Chen, K., Funke, B.C., Schulz, J.T., Carlson, R.B. and Proshold, F.I., 1974. Effects of certain organophosphate and carbamate insecticideson Bacillus thuringiensis.J. Econ. Entomol., 67 : 471--473. Chu, W.H. and Jaques, R.P., 1981. Factors affecting infectivity of Vairimorpha necatrix (Microsporidia: Nosematidae) in Trichoplusia ni (Lepidoptera: Noctuidae). Can. Entomol., 113: 93--102. Couch, T.L. and Ignoffo, C.M., 1981. Formulation of insect pathogens. In: H.D. Burges (Editor), Microbial Control of Pests and Plant Diseases 1970--1980. Academic Press, London and N e w York, pp. 621--634. Cunningham, J.C., 1978. The use of pathogens for spruce budworm control. In: G.E. Allen, C.M. Ignoffo and R ~u. Jaques (Editors), Microbial Control of Insect Pests: Future Strategies in Pest Management Systems. Proc. USDA--NSF--Univ. of Florida Workshop, Jan. 1978, pp. 249--260. Cunningham, J.C. and Entwistle, P.F., 1981. Control of sawflies by Baculovirus. In: H.D. Burges (Editor), Microbial Control of Pests and Plant Diseases 1970-1980. Academic Press, London and N e w York, pp. 379--407. Davidson, E.W., Sweeney, A.W. and Cooper, R., 1981. Comparative field trialsof Bacillus sphaericus strain 1593 and B. thuringiensis vat. israelensis commercial powder formulations. J. Econ. Entomol., 74: 350--354. Dedryver, C.A., 1979. D~clenchement en serre d'une ~pizootie a Entomophthora fresenii sur Aphis fabae par introduction d'une inoculum et r~gulation de l'humidit~ relative. Entomophaga, 24: 443--453. Dougherty, E.M., Reichelderfer, C.F. and Faust, R.M., 1971. Sensitivity of Bacillus thuringiensis vat. thuringiensis to various insecticides and herbicides. J. Invertebr. P a t h o l . , 17 : 292--293. Dulmage, H.T., 1981. Insecticidal activity of isolates of Bacillus thuringiensis and their potential for pest control. In: H.D. Burges (Editor), Microbial Control of Pests and Plant Diseases 1970--1980. Academic Press, London and New York, pp. 1 9 1 - 2 2 2 . Dulmage, H.T., Soper, R.S. and Smith, D.B., 1981. The use of bacteria and fungi in insect control. In: Application of Biorational Substances and Natural Enemies. Proc. First Japan/U.S.A. Symposium on Integrated Pest Management, Sept. 1981, pp. 112--127. Fargues, J., 1975. Etude exp~rimentale dans la nature de l'utilisation combin~e de Beauveria bassiana et d'insecticides a dose rdduite contre Leptinotarsa decemlineata. Ann. Zool.-l~,col. Anim., 7: 247--264. Fast, P.G., 1981. The crystal toxin of Bacillus thuringiensis. In: H.D. Burges (Editor), Microbial Control of Pests and Plant Diseases 1 9 7 0 - 1 9 8 0 . Academic Press, London and New York, pp. 223--248. Ferron, P., 1981. Pest control by the fungi Beauveria and Metarhizium. In: H.D. Burges (Editor), Microbial Control of Pests and Plant Diseases 1 9 7 0 - 1 9 8 0 . Academic Press, L o n d o n and New York, pp. 465--482. Forsberg, C.W., 1976. Bacillus thuringiensis: Its Effects on Environmental Quality. Natl. Res. Counc. Can. Publ., NRCC 15385, 134 pp. Franz, J.M. and Huber, J., 1979. Feldversuche mit Insekten pathogenen Viren in Europa. Entomophaga, 24: 333--343. Fuxa, J.R. and Brooks, W.M., 1978. Persistence of spores of Vairimorpha necatrix on tobacco, cotton, and soybean foliage. J. Econ. Entomol., 71: 169--172. Fuxa, J.R. and Brooks, W.M., 1979. Effects of Vairimorpha necatrix in sprays and corn meal on Heliothis species in tobacco, soybeans and sorghum. J. Econ. Entomol., 72: 462--467.

122 Granados, R.R., 1980. Infectivity and mode of action of Baculoviruses. Biotechnol. Bioeng., 22: 1377--1405. Hall, R.A., 1980. Effect of repeated subculturing on agar and passaging through an insect host on pathogenicity, morphology, and growth rate of Verticillium lecanii. J. Invertebr. Pathol., 36: 216--222. Hall, R.A., 1981. The fungus Verticillium lecanii as a microbial insecticide against aphids and scales. In: H.D. Burges (Editor), Microbial Control o f Pests and Plant Diseases 1970--1980. Academic Press, London and New York, pp. 481--498. Hamilton, J.T. and Attia, F.I., 1977. Effects of mixtures of Bacillus thuringiensis and pesticides on PluteUa xylostella and the parasite Thyraeella eollaris. J. Econ. Entomol., 70: 146--148. Henry, J.E. and Oma, E.A., 1981. Pest control b y Nosema locustae, a pathogen of grasshoppers and crickets. In: H.D. Burges (Editor), Microbial Control of Pests and Plant Diseases 1970--1980. Academic Press, London and New York, pp. 571--586. Henry, J.E. and Onsager, J.A., 1982. Large-scale test of control of grasshoppers on rangeland with Nosema locustae. J. Econ. Entomol., 75: 31--35. Hostetter, D.L. and Biever, K.D., 1970. The recovery of virulent nuclear-polyhedrosis virus of cabbage looper, Trichoplusia ni, from feces of birds. J. Invertebr. Pathol., 15: 173--176. Hutton, P.O. and Burbutis, P.P., 1974. Milky disease and Japanese beetle in Delaware. J. Econ. Entomol., 67 : 247--248. Ignoffo, C.M., 1973. Development o f a viral insecticide: Concept to commercialization. Exp. Parasitol., 33: 380--406. Ignoffo, C.M., 1981. The fungus Nomuraea rileyi as a microbial insecticide. In: H.D. Burges (Editor), Microbial Control of Pests and Plant Diseases 1970--1980. Academic Press, L o n d o n and New York, pp. 513--538. Ignoffo, C.M. and Batzer, O.F., 1971. Microencapsulation and ultraviolet protectants to increase sunlight stability of an insect virus. J. Econ. Entomol., 64: 850--853. Ignoffo C.M. and Couch, T.L., 1981. The nucleopolyhedrosis virus of HeUothis species as a microbial insecticide. In: H.D. Burges (Editor), Microbial Control of Pests and Plant Diseases 1970--1980. Academic Press, London and New York, pp. 3 2 9 - 3 6 2 . Ignoffo C.M., Hostetter, D.L. and Smith, D.B., 1976. Gustatory stimulant, sunlight protectant, evaporation retardant: three characteristics of a microbial insecticidal adjuvant. J. Econ. Entomol., 69: 207--210. Ignoffo C.M., Hostetter, D.L., Garcia, C. and Pinnell, R.E., 1975. Sensitivity of the entomopathogenic fungus Nomuraea rileyi to chemical pesticides used on soybeans. Environ. Entomol., 4: 765--768. Ignoffo C.M., Garcia, C., Kapp, R.W. and Coate, W.B., 1979. An evaluation o f the risks to mammals of the use of an entomopathogenic fungus, Nomuraea rileyi, as a microbial insecticide. Environ. Entomol., 8: 354--359. Ignoffo C.M., Garcia, C., Hostetter, D.L. and Pinnell, R.E., 1980. Transplanting: A method of introducing an insect virus into an ecosystem. Environ. Entomol., 9: 153--154. Ignoffo C.M., Hostetter, D.L., Sikorowski, P.P., Sutter, G. and Brooks, W~M., 1977. Inactivation of representative species of entomopathogenic viruses, a bacterium, fungus, and protozoan by an ultraviolet light source. Environ. Entomol., 6: 411--415. Jaques, R.P., 1964. The persistence of a nuclear-polyhedrosis virus in soil. J. Insect Pathol., 6: 251--254. Jaques, R.P., 1965. The effect of Bacillus thuringiensis Berliner on the fauna of an apple orchard. Can. Entomol., 97: 795--802. Jaques, R.P., 1967a. The persistence of a nuclear polyhedrosis virus in the habitat of the host insect, Trichoplusia ni. I. Polyhedra deposited on foliage. Can. Entomol., 99: 785--794. Jaques, R.P., 1967b. The persistence of a nuclear polyhedrosis virus in the habitat of the host insect, Trichoplusia ni. II. Polyhedra in soil. Can. Entomol., 99: 820--829.

123 Jaques, R.P., 1971. Tests on protectants for foliar deposits of a polyhedrosis virus. J. Invertebr. Pathol., 17: 9--16. Jaques, R.P., 1972a. The inactivation of foliar deposits of viruses of Trichoplusia ni and Pieris rapac and tests on protectant additives. Can. Entomol., 104: 1985--1994. Jaques, R.P., 1972b. Control of the cabbage looper and imported cabbageworm by viruses and bacteria. J. Econ. Entomol., 65: 757--760. Jaques, R.P., 1973a. Tests on microbial and chemical insecticides for control of Trichoplusia ni and Pieris rapac on cabbage. Can. Entomol., 105: 21--27. Jaques, R.P., 1973b. Methods and effectiveness of distribution of microbial insecticides. In: L.A. Bulla (Editor), Regulation of Insect Populations by Microorganisms. Ann. N.Y. Acad. Sci., 217: 109--119. Jaques, R.P., 1974a. Occurrence and accumulation of viruses of Trichoplusia ni in treated field plots. J. Invertebr. Pathol., 23: 140--152. Jaques, R.P., 1974b. Occurrence and accumulation of the granulosis virus of Pieris rapae in treated field plots. J. Invertebr. Pathol., 23: 351--359. Jaques, R.P., 1975. Persistence, accumulation, and denaturation of nuclear polyhedrosis and granulosis virus. In: M.D. Summers, R. Engler, L.A. Falcon and P.V. Vail (Editors), Baculoviruses for Insect Pest Control: Safety Considerations. Monogr. Am. Soc. Microbiol., pp. 90--99. Jaques, R.P., 1977a. Field efficacy of viruses infectious to the cabbage looper and imported cabbageworm on late cabbage. J. Econ. Entomol., 70: 111--118. Jaques, R.P,, 1977b. Stability of entomopathogenic viruses. In: D.L. Hostetter and C.M. Ignoffo (Editors), Environmental Stability of Microbial Insecticides. Misc. Publ. Entomol. Soc. Am., 10: 99--117. Jaques, R.P:, 1978. Manipulation of the environment to increase effectiveness of microbial agents. In: G.E. Allen, C.M. Ignoffo and R.P. Jaques (Editors), Microbial Control of Insect Pests: Future Strategies in Pest Management Systems. Proc. USDA--NSF-Univ. of Florida Workshop, Jan. 1978, pp. 72--85. Jaques, R.P. and Patterson, N.A., 1962. Control of the apple sucker, PsyUa mall Schmidb., by the fungus Entomophthora sphacrosperma (Fresenius). Can. Entomol., 94: 818--825. Jaques, R_P. and Laing, D.R., 1978. Efficacy of mixtures of Bacillus thuringiensis, viruses, and chlordimeform against insects on cabbage. Can. Entomol., 110: 443--448. Jaques, R.P. and Morris, O.N., 1981. Compatibility with other methods of pest control and with different crops. In: H.D. Bulges (Editor), Microbial Control of Pests and Plant Diseases 1970--1980. Academic Press, London and New York, pp. 695--715. Jaques, R.P., Laing, J.E., McLellan, C.R., Proverbs, M.D., Sanford, K.H. and Trottier, R., 1981. Apple orchard tests on the efficacy of the granulosis virus of the codling moth, Laspeyresia pomonella (Lepidoptera: Olethreutidae). Entomophaga, 26: 111--118. Katagiri, K., 1981. Pest control by cytoplasmic polyhedrosis virus. In: H.D. Burges (Editor), Microbial Control of Pests and Plant Diseases 1970--1980. Academic Press, London and New York, pp. 433--440. Kaya, H.K. and Tanada, Y., 1973. Hemolymph factor in armyworm larvae infected with a nuclear polyhedrosis virus toxic to Apanteles militaris. J. Invertebr. Pathol., 21: 211--214. Klein, M.G., 1981. Advances in the use of Bacillus popiUiac for pest control. In: H.D. Burges (Editor), Microbial Control of Pests and Plant Diseases 1970--1980. Academic Press, London and New York, pp. 183--192. Kreig, A. and Langenbruch, G.A., 1981. Susceptibility of arthropod species to Bacillus thuringiensis. In: H.D. Burges (Editor), Microbial Control of Pests and Plant Diseases 1970--1980. Academic Press, London and New York, pp. 835--896. Kreig, A., Groner, A., Huber, J. and Zimmermann, G., 1981. Inaktivierung von verschiedenen Insektenpathogenen durch ultraviolette Strahlen. Z . Pflanzenkr. Pflanzenschutz, 88: 38--48. Leong, K.L.H., Cano, R.J. and Kubinski, AM., 1980. Factors affecting Bacillus thuringiensis total field persistence. Environ. Entomol., 9: 593--599.

124 Levin, D.B., Laing, J.E. and Jaques, R.P., 1979. Transmission of granulosis virus by Apanteles glomeratus to its host Pieris rapae. J. Invertebr. Pathol., 34: 317--318. Levin, D.B., Laing, J.E. and Jaques, R~P., 1981. Interactions between Apanteles glomeratus (L.) (Hymenoptera: Braconidae) and granulosis virus in Pieris rapae (L.) (Lepidoptera: Pieridae). Environ. Entomol., 10: 65--68. Lewis, F.B., 1981. Control of the gypsy moth by a Baculovirus. In: H.D. Burges (Editor), Microbial Control of Pests and Plant Diseases 1970--1980. Academic Press, London and New York, pp. 363--377. Lewis, L.C., Lynch, R.E. and Jackson, J.J., 1977. Pathology of a Baculovirus of the alfalfa looper, Autographa californica, in the European corn borer, Ostrinia nubilalis. Environ. Entomol., 6: 535--538. Lublinkhof, J. and Lewis, L.C., 1980. Virulence of Nosema pyrausta to the European corn borer when used in combination with insecticides. Environ. Entomol., 9 : 6 7 - - 7 1 . Lublinkhof, J., Lewis, L.C. and Berry, E.C., 1979. Effectiveness of integrating insecticides with Nosema pyrausta for suppressing populations of the European corn borer. J. Econ. Entomol., 72: 880--883. Lundholm, B. and Stackerud, M. (Editors), 1980. Environmental protection and biological forms of control of pest organisms. Ecol. Bull. (Stockholm), 31 : 1--171. L~ithy, P., 1980. Insecticidal toxins of Bacillus thuringiensis. FEMS Microbiol. Lett., 8: 1--7. Maddox, J.V., 1977. Stability of entomopathogenic protozoa. In: D.L. Hostetter and C.M. Ignoffo (Editors), Environmental Stability of Microbial Insecticides. Misc. Publ. Entomol. Soc. Am., 10: 8--18. Maddox, J.V., Brooks, W.M. and Fuxa, J.R., 1981. Vairimorpha necatrix, a pathogen of agricultural pests: Potential for pest control. In: H.D. Burges (Editor), Microbial Control o f Pests and Plant Diseases 1970--1980. Academic Press, London and New York, pp. 587--594. Martignoni, M.E. and Iwai, P.J., 1981. A catalogue of viral diseases of insects, mites and ticks. In: H.D. Burges (Editor), Microbial Control of Pests and Plant Diseases 1970-1980. Academic Press, L o n d o n and New York, pp. 895--911. Martin, P.A.W. and Dean, D.H., 1981. Genetics and genetic manipulation of Bacillus thuringiensis. In: H.D. Burges (Editor), Microbial Control of Pests and Plant Diseases 1970--1980. Academic Press, L o n d o n and New York, pp. 299--311. McCoy, C.W., 1978. Entomopathogens in arthropod pest control programs for citrus. In: G.E. Allen, C.M. Ignoffo and R.P. Jaques (Editors), Microbial Control of Insect Pests: Future Strategies in Pest Management Systems. Proc. U S D A - - N S F - U n i v . of Florida Workshop, Jan. 1978, pp. 211--219. McCoy, C.W., 1981. Pest control by the fungus Hirsutella thompsonii. In: H.D. Burges (Editor), Microbial Control of Pests and Plant Diseases 1970--1980. Academic Press, L o n d o n and New York, pp. 497--512. McLeod, P.J., Young, S.Y. and Yearian, W.C., 1982. Application of a Baculovirus of Pseudoplusia includens to soybean. Efficacy and seasonal persistence. Environ. Entomol., 11: 412--416. Miltenburger, H.G., 1980. Viral pesticides: Hazard evaluation for non-target organisms and safety testing. In: B. Lundholm and M. Stackerud (Editors), Environmental Protection and Biological F o r m s of Control of Pest Organisms. Ecol. Bull. (Stockholm), 31: 57--74. Mohamed, M.A., Coppel, H.C. and Podgwaite, J.C., 1982. Persistence in soil and on foliage of nucleopolyhedrosis virus of the European pine sawfly, Neodiprion sertifer (Hymenoptera: Diprionidae). Environ. Entomol., 11: 1116--1118. Morris, O.N., 1977a. Compatibility of 27 chemical insecticides with Bacillus thuringiensis var. kurstaki. Can. Entomol., 109: 855--864. Morris, O.N., 1977b. Long-term study of the effectiveness of aerial application of Bacillus thuringiensis--acephate combinations against the spruce budworm, Choristoneura fumiferana (Lepidoptera: Tortricidae). Can. Entomol., 109: 1239--1248.

125 Morris, O.N., 1980. Entomopathogenic viruses: Strategies for use in forest insect pest management. Can. Entomol., 112: 573--584. Mulla, M.S., Federici, B.A. and Darwazeh, H.A., 1982. Larvicidal efficacy of Bacillus thuringiensis serotype H-14 against stagnant-water mosquitoes and its effects on nontarget organisms. Environ. Entomol., 11: 788--795. Olmert, I. and Kenneth, R.G., 1974. Sensitivity of the entomopathogenic fungi, Beauveria bussiana, Verticillium lecanii, and Verticillium sp. to fungicides and insecticides. Environ. Entomol., 3: 33--38. Papworth, D.S., 1980. Registration requirements in the U.S. for bacteria, fungi and viruses used as pesticides. In: B. Lundholm and M. Stackerud (Editors), Environmental Protection and Biological Forms of Control of Pest Organisms. Ecol. Bull. (Stockholm), 31: 135--143. Pinnock, D.E., Miistead, J.E., Kirby, M.E. and Nelson, B.J., 1977. Stability of entomopathogenic bacteria. In: D.L. Hostetter and C.M. Ignoffo (Editors), Environmental Stability of Microbial Insecticides. Misc. Publ. Entomol. Soc. Am., 10: 77--97. Podgwaite, J.C., Shields, K.S., Zerillo, R.T. and Bruen, R.B., 1979. Environmental persistence of the nucleopolyhedrosis virus of the gypsy moth, Lymantria dispar. Environ. Entomol., 8: 528--538. Potter, J.N., Jaques, R.P. and Faulkner, P., 1978. Modification of Trichoplusia ni nuclear polyhedrosis virus passaged in vivo. Intervirology, 9: 76--85. Puttler, B., Hostetter, D.L., Long, S.H. and Pinnell, R.E., 1978. Entomophthora phytonomi, a fungal pathogen of the alfalfa weevil in the mid-great plains. Environ. Entomol., 7: 670--671. Raimo, B., Reardon, R.C. and Podgwaite, J.D., 1977. Vectoring gypsy moth nuclear polyhedrosis virus by Apanteles melanoscelus. Entomophaga, 22: 207--215. Ramoska, W.A., Watts, S. and Rodriguez, R.E., 1982. Influence of suspended particulates on the activity of Bacillus thuringiensis serotype H-14 against mosquito larvae. J. Econ. Entomol., 75: 1--4. Reardon, R. and Podgwaite, J.D., 1976. Disease--parasitoid relationships in natural populations of Lymantria dispar [Lep.: Lymantriidae] in the northern United States. Entomophaga, 21: 333--341. Reardon, R., Metterhause, W. and Balaam, R., 1979. Impact of aerially applied Bacillus thuringiensis and carbaryl on gypsy moth and adult parasites. Entomophaga, 24: 305--310. Reed, D.K., 1981. Control of mites by non-occluded viruses. In: H.D. Burges (Editor), Microbial Control of Pests and Plant Diseases 1970--1980. Academic Press, London and New York, pp. 427--432. Roberts, D.W. (Editor), 1980. Pest Management with Microbial Agents: Recent Achievements, Deficiencies, and Innovations. Boyce Thompson Institute, Ithaca, New York, 71 pp. Roberts, D.W., 1981. Toxins of entomogenous fungi. In: H.D. Burges (Editor), Microbial Control of Pests and Plant Diseases 1970--1980. Academic Press, London and New York, pp. 441--464. Roberts, D.W. and Campbell, A.S., 1977. Stability of entomopathogenic fungi. In: D.L. Hostetter and C.M. Ignoffo (Editors), Environmental Stability of Microbial Insecticides. Misc. Publ. Entomol. Soc. Am., 10: 19--76. Rogoff, M.H., 1980. Testing requirements for registered biological pesticides in the United States -- Current status. In: B. Lundholm and M. Stackerud (Editors), Environmental Protection and Biological Forms of Control of Pest Organisms. Ecol. Bull. (Stockholm), 31: 111--134. Saleh, S~VI., Harris, R.F. and Allen, O.N., 1970. Recovery of Bacillus thuringiensis vat. thuringiensis from field soils. J. Invertebr. Pathol., 15: 55--59. Sebesta, K., Farkas, J., Horska, K. and Vankova, J., 1981. Thuringiensin, the Beta-exotoxin of Bacillus thuringiensis. In: H.D. Burges (Editor), Microbial Control of Pests and Plant Diseases 1970--1980. Academic Press, London and New York, pp. 249--279.

126 Shadduck, J.A., Roberts, D.W. and Lause, S., 1982. Mammalian safety tests of Metarhizium anosipliae: preliminary results. Environ. Entomol., 11: 189--192. Shieh, T.R. and Bohmfalk, G.T., 1980. Production and efficacy of Baculoviruses. Biotechnol. Bioeng., 22: 1357--1375. Singer, S., 1980. Bacillus sphaericus for the control of mosquitoes. Biotechnol. Bioeng., 22: 1335--1355. Smith, D.B. and Bonse, L.F., 1981. Machinery and factors that affect the application of pathogens. In: H.D. Burges (Editor), Microbial Control of Pests and Plant Diseases 1970--1980. Academic Press, London and New York, pp. 635--653. Smith, D.B. and I-Iostetter, D.L., 1982. Laboratory and field evaluations of pathogenadjuvant treatments. J. Econ. Entomol., 75: 472--476. Smith, D.B., Hostetter, D.L. and Ignoffo, C.M., 1978. Formulation and equipment effects on application of a viral (Baculovirus heliothis) insecticide. J. Econ. Entomol., 71: 814--817. Stairs, G.R., Fraser, T. and Fraser, M., 1981. Changes in growth and virulence of a nuclear polyhedrosis virus from Choristoneura fumiferana after passage in Trichoplusia ni and GaUeria mellonella. J. Invertebr. Pathol., 38: 230--235. Stockdale, H. and Priston, R.A.J., 1981. Production of insect viruses in cell culture. In: H.D. Burges (Editor), Microbial Control of Pests and Plant Diseases 1970--1980. Academic Press, London and New York, pp. 313--328. Tedders, W.L., 1981. In vitro inhibition of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae by six fungicides used in pecan culture. Environ. Entomol., 10: 346--349. Tedders, W.L., Weaver, D.J., Wehunt, E.J. and Gentry, C.R., 1982. Bioassay of Metarhizium anisopliae, Beauveria bassiana and Neoplectana carpocapsae against the larvae of the plum curculio, Conotrachelus nenuphar (Herbst.) (Coleoptera: Curculionidae). Environ. Entomol., 11: 901--904. Thompson, C.G., Scott, D.W. and Wickman, B.E., 1981. Long-term persistence of the nuclear polyhedrosis virus of the Douglas-fir tussock moth, Orgyia pseudotsugata (Lepidoptera: Lymantriidae), in forest soil. Environ. Entomol., 10: 254--255. Tompkins, G.J., Vaughn, J.L., Adams, J.R. and Reiehelderfer, C.F., 1981. Effects of propagating Autographa californica nuclear polyhedrosis virus and its Trichoplusia ni variant in different hosts. Environ. Entomol., 10: 801--806. Vail, P.V., Soo Hoo, C.F., Seay, R.S., Killinen, R.G. and Wolf, W.W., 1972. Microbial control of lepidopterous pests of fall lettuce in Arizona and effects of chemical and microbial pesticides on parasitoids. Environ. Entomol., 1: 780--785. Weseloh, R_M. and Andreadis, T.G., 1982. Possible mechanism for synergism between Bacillus thuringiensis and the gypsy moth (Lepidoptera: Lymantriidae) parasitoid, Apanteles melanoscelus (Hymenoptera: Braconidae) Ann. Entomol. Soc. Am., 75: 435--438. Witt, D.J. and Hink, W.F., 1979. Selection of Autographa californica nuclear polyhedrosis virus for resistance to inactivation by near ultraviolet, far ultraviolet, and thermal radiation. J. Invertebr. Pathol., 33: 222--232. Wood, H.A., Hughes, P.R., Johnston, L.B. and Langridge, W.H.R., 1981. Increased virulence of Autographa californica nuclear polyhedrosis virus by mutagenesis. J. Invertebr. Pathol., 38: 236--241. Young, S.Y. and Yearian, W.C., 1979. Soil application of Pseudoplusia NPV: Persistence and incidence of infection in soybean looper caged on soybean. Environ. Entomol., 8 : 860--864.