Ecologically safe control of insect pest: The past, the ...

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Emerging Concepts in Plant Health Management, 2004: ISBN: 81-7736-227-5 Editors: Robert T. Lartey and Anthony Caesar

Ecologically safe control of insect pest: The past, the present and the future Margarita V. Shternshis Department for Biological Control, Novosibirsk State Agrarian University 630039, Novosibirsk, Russia

Abstract This chapter focuses on the most widespread natural agents used for ecologically safe control of insect pests, such as Bacillus thuringiensis, baculoviruses, entomopathogenic fungi, and some microbial metabolites. Special attention is given to the enhancement of control agent insecticidal activity and relevant formulations. Possible ways to improve insect control using mixtures of particular entomopathogens with other synergistic entomopathogens or with nontoxic enhancers are discussed. In most cases, data obtained by Russian authors have been presented. The significance of the dual properties of some natural control agents for Correspondence/Reprint request: Dr. Margarita V. Shternshis, Department for Biological Control, Novosibirsk State Agrarian University, 630039, Novosibirsk, Russia. E-mail: [email protected]

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Margarita V. Shternshis

plant protection, and the role of host plant compounds for plant health management is emphasized, especially for future research. Retrospective analysis suggests ways to further increase ecologically safe insect control. Challenges for future research would include the development of a better understanding of the mechanism of interaction between insect control agents and their hosts at both molecular and physiological levels, as well as further consideration of environmental factors.

Introduction Phytophagous insects are responsible for significant damage to crops and timber, sometimes even to the extent of complete yield loss. Therefore, plant protection is an indispensable part of agriculture and forestry. In recent decades, plant protection has been dominated by chemical insect control. In spite of the benefits offered by chemical insecticides, they nevertheless have some serious disadvantages from an ecological perspective. These include adverse impact on non-target organisms (beneficial insects, birds, fish), environmental contamination, and the accumulation of poisonous compounds in fruits. Such negative consequences have spurred the development of alternatives for chemical pest control in plant protection. These alternatives include the use of biotic agents of compatible both with the plant species being attacked by insects and the environment. Natural insect enemies, such as beneficial insects and entomopathogenic microorganisms, are alternative biological control agents. It should be noted that the term “biological control” is generally considered in its broad sense. For example, it has been suggested that use of this term should be restricted to the use of living organisms [1]. In Russia and some other countries (members of Eastern Palearctic Regional Section of International Organization for Biological Control), however, the term “biological control” is applied to the use of living organisms as well as their products [2]. This latter connotation of the term is broader and seems to be warranted, especially in the light of the wide use of biological preparations based on Bacillus thuringiensis (Bt) spore-crystal complex. Although spores are viable living organisms and thus are considered biological control according to the more restricted definition, it has been found that the crystals of Bt containing the Cry and Cyt toxins are responsible for the specific insecticidal activity of this biocontrol agent [3]. Thus, metabolites of Bt with insecticidal activity have become the most important alternative to chemical insecticides. In spite of some variation in application of the term “biological control”, it seems appropriate to describe control which is based on products of living organisms or their metabolites as “ecologically safe insect pest control”. Primarily, this chapter will discuss the use of the most widespread natural insect control agents for plant health management including Bt, baculoviruses,

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entomopathogenic fungi and some microbial metabolites. No attempt will be made to review the entire spectrum of the literature. Reviews of past and current data provide a guide to the most promising research directions for the future. In this chapter, ecologically safe insect pest control is considered as the reduction or prevention of insect damage to plants with minimum risk to the host plant or the environment. The emphasis of this chapter will be to present ecologically safe control of insects important in Russia, with special emphasis on plants producing fresh fruits.

Bacillus thuringiensis as an important agent of ecologically safe insect pest control In nature, insects are susceptible to various bacterial diseases, which regulate insect population levels. Among them, Bt has emerged as the most common entomopathogenic bacterium used to control insects. Several Bt serovars based biological preparations have become successful bacterial formulations with worldwide application for plant protection in agriculture and forestry during last 40 years [4]. Bt is a spore-forming, crystalliferous bacterium very closely related to Bacillus cereus. Bt can only be distinguished from the latter by its ability to produce a parasporal crystalline protein during sporulation. This characteristic is essential for providing the insecticidal activity and environmental safety of Bt [5]. Bt is commonly considered to have been first isolated from the silkworm Bombyx mori L in Japan in 1901 [6], and later identified by German researcher Berliner in 1911 [7]. There is also some evidence that Pasteur first isolated this microorganism from silkworms in France, however he did not identify it [8]. Bt is a widespread agent naturally occurring in dead insects, soil and on foliage. In Russia, the first Bt preparations for insect control were developed in the middle of the 20th century. In 1949, Talalaev [9] isolated Bt subsp. dendrolimus (sotto) from larvae of the Siberian silkworm Dendrolimus superans sibiricus Tchetw. during epizootics in the taiga of Eastern Siberia. This Bt subspecies provided the basis of the first Russian bacterial preparation Dendrobacillin® for insect control. Initially, Dendrobacillin was used for plant protection against the Siberian silkworm in forestry. Artificial epizootics of this pest were produced through inoculation. Later, the same formulation was used for lepidopteran insect control on agricultural crops as well. Bt subsp. dendrolimus is a representative of pathotype A, pathogenic to lepidopterans in accordance with its Cry-toxins composition [10]. It should be noted that the strategy of its use in crop protection changed from inoculative (a single release of control agent with expectation of its proliferation) to inundative (two or more agent releases without expectation of progeny) [1]. This change was perhaps due to altered conditions in agricultural ecosystems, in which occurrences of Bt-induced

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epizootics are less likely. During the 1960s, 70s and 80s, further development of Russian formulations based on Bt subsp. galleriae and kurstaki, resulted in a sharp increase in the use of Bt for plant protection and led to the replacement of some chemicals with bacterial insecticides. In general, the majority of the work sought to achieve insect control in vegetable crops and large forested areas mainly in Siberia. Cabbage is the most common vegetable crop grown in Siberia,. Lepidopteran insect pests, such as beet webworm Pyrausta sticticalis L., cabbage moth Mamestra brassicae L., diamondback moth Plutella xyllostella L. and the large white butterfly Pieris brassicae L., are each able to completely eliminate yield. Fortunately, all of these pests are susceptible to formulations, and especially to Bt pathotype A. The larvae of M. brassicae are less susceptible to Bt than the other three species. For biological suppression of this insect, enhancement of Bt application techniques is required and will be discussed in a subsequent section. Complete replacement of chemical insecticides with Bt preparations (should it occur) would create favorable conditions for the beneficial insects that help control not only lepidopteran insects but also other insect pest species such as the cabbage aphid Brevicoryne brassicae L. Ecologically safe control of the cabbage pest, the flea beetle, has not been achieved to date; there is, however, some promise for controlling these pests using Bt subsp. darmstadiensis [11]. As for bacterial preparations used to control insects and mites in greenhouses, the possibilities are limited. Sucking insects predominate under these conditions, and these pests are not susceptible to commercial Bt preparations with the exception of β-exotoxin containing the preparation Bitoxibacillin®. This product is registered in Russia for use against spider mites, Tetranichus urticae Koch. However, there is some concern about possible harm of β-exotoxin to plants and non-target organisms. The application of other agents for ecologically safe control of mites is of great interest [12] (see later sections). For many years, Bt was considered to exclusively kill Lepidoptera species. However, in 1977, Goldberg and Margalit [13] discovered that Bt subsp. israelensis was pathogenic for Diptera. Later, Krieg et al. [14] reported the isolation of Bt subsp. tenebrionis, which showed activity against Coleoptera. Different compositions of Cry- and Cyt-toxins are basis for specificity of Bt pathotypes against insects of various orders [10]. The identification of Bt isolates, pathogenic to non-Lepidopteran insects justified the need for evaluation of selected Bt subspecies for safe and effective application on fresh market or medicinal crops. The raspberry cane midge Resseliella theobaldi Barnes may represent a good example of such a target (Table 1). This is a very serious pest in Russia and elsewhere. The insect invades canes of red raspberry which have split. Its damage to the raspberry plants is usually associated with mycoses caused by

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Table 1. Effect of Bacticide® on raspberry midge blight severity.

Didymella applanata (Niessl.) Sacc., Botrytis cinerea Pers. and some other plant pathogens. Because the cane midge is a member of the order Diptera, research was stimulated on field testing Russian commercial preparation Bacticide® based on Bt subsp. israelensis [15]. Under field conditions, it is not possible to differentiate damage due to the cane midge from that due to fungi. This syndrome is known as raspberry midge blight [16]. Thus, the efficacy of Bacticide® was assessed by estimating the severity of midge blight. The data presented in Table 1 show that the reduction of midge blight severity by Bt was comparable to the reduction by the chemical insecticide Actellic®, the product traditionally applied on raspberry. This study established the possibility of replacing a chemical insecticide with an ecologically safe bacterial formulation for control of midge blight in red raspberry. It should be noted that this association of an insect with plant pathogens does not represent an isolated case. For example, in Siberia blue stain fungi are associated with xylophagous insects on conifers [17].

Use of baculoviruses for ecologically safe insect control Baculoviruses are unique viruses that control insect populations in nature. These viruses consist of enveloped nucleocapsids (virions) embedded in a crystalline protein matrix. Two such viruses are Granuloviruses (GV) containing a single virion embedded in small protein granule and nucleopolyhedroviruses (NPV) containing several virions in a large matrix (polyhedra). These protein occlusion bodies are solubilized in the alkaline matrix of the lepidopteran midgut in the same manner as Bt crystals and virions are released that pass through the peritrophic membrane. Due to their

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obligate nature, baculoviruses are the most ecologically safe biological agents. Normally, they infect only a single lepidopteran species. Epizootics caused by baculoviruses are observed frequently, especially among insects damaging forests. Baculoviruses were first isolated in the middle of the 20th century in Russia. These include NPV of the satin moth, Leucoma salicis L.; nun moth, Lymantria monacha L.; gypsy moth, Lymantria dispar L.; cabbage moth, M. brassicae L. and GV of codling moth, Cydia pomonella L.; beet webworm, P. sticticalis L.; Siberian silkworm, D. superans sibiricus Tchetw. and turnip moth, Agrotis segetum Schiff. [18]. The first Russian virus-based formulations, Virin-ENSH and Virin–EKS were developed in the1960s [19] for control of gypsy moth and cabbage moth populations. The main advantages of viral preparations are high specificity and transovarial transmission across generations. The cabbage moth has been suppressed more efficiently with viral preparations than with Bt [20]. However, the advantage of high specificity of the virus was offset by a long disease latent period. Another serious pest on cabbage, P. sticticalis, was shown to be suppressed by specific GV and NPV [21,22]. The GV appeared to be more virulent to larvae of beet webworm than NPV. In Russia, the use of viral preparations is more important in forestry than in agriculture. The earliest Russian viral preparation has been applied against gypsy moth for 40 years. The initial strategy of inoculation employed for this preparation was treatment of insect eggs to create artificial epizootics [23]. Because the strategy of inoculation was not successful for the control of L. monacha, an approach of inundation was suggested [24]. It is also noteworthy that the cytoplasmic polyhedrosis virus of the Reoviridae, was less virulent against L. dispar than the NPV [25]. This finding attested to the need to develop viral preparations based solely on baculoviruses. The application of Virin-diprion®, registered in Russia for control of fox-colored sawfly Neodiprion sertifer Geoff, is considered to be very effective [26]. This effectiveness may be explained by the biological peculiarities of this pest. N. sertifer larvae are gregarious and their close contact promotes fast distribution of disease. In addition, the baculovirus development occurs in the N. sertifer gut only and causes rapid larval mortality, due to a short latent period compared with other baculoviruses which generally multiply in other insect tissues and organs. Despite their high ecological safety, baculovirus-based preparations are not widely used in Russia. The primary reason for this is difficulties in their production in living insects and the occurrence of long latent periods. Another factor is the preference of growers for a minimal number of applications per crop, therefore the advantage of the extreme specificity of viral preparations from an ecological point of view becomes a disadvantage for the farmer from an economic point of view.

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Use of entomopathogenic fungi in plant protection Microbial insect control with the pioneering work of the Russian scientist Mechnikov [27,28], at the end of 19th century. He isolated an entomopathogenic fungus from diseased larvae of the cereal beetle Anisoplia austriaca Hrbst. This fungus was identified as Metarhizium anisopliae (Metsch) Sorokin. However, most research on entomopathogenic fungi in Russia has been done since the middle of the 20th century [29-31]. The principal characteristic that differentiates fungi from bacteria and viruses for pest control lies in the fungal capability of infecting insects and mites not only per os but through the integument as well. This allows use of fungal preparations against important greenhouse pests. Typically, fungi require a high relative humidity for the infectious process, which is readily available under greenhouse conditions. The most common fungi used for insect control belong to the genera Beauveria, Metarhizium, Paecilomyces, Verticillium, Aschersonia, and Conidiobolus. Vegetables grown in greenhouses are the main crops protected with entomopathogenic fungi. Together with sucking insect pests, the spider mite is also an important target for control by fungi in greenhouses. In greenhouses located in regions with cold climates and short growing seasons such as Western Siberia, such species as the greenhouse whitefly, Trialeurodes vaporariorum Westwood and spider mite are especially damaging to cucumber and tomato. Effective control of aphids, whitefly and spider mites has been achieved with suspensions of Verticillium lecanii (Zimm.) Viegas and Beauveria bassiana. For effective mite control, a 5-10 fold increase in the concentration of fungal suspension is necessary. The preparations Verticillin® and Boverin®, based on V. lecanii and B. bassiana respectively, are registered in Russia. Western flower thrips, Frankliniella occidentalis Perg., recognized as a serious greenhouse pest, is a quarantined insect in Russia. A data on biological control of this thrips by V. lecanii [32] served as an incentive for comparison of the Novosibirsk strain with that of Berlin [33]. Significantly higher virulence of the Berlin strain was observed as compared to the original Novosibirsk strains towards the larvae of F. occidentalis. Repeated passage of both strains through F. occidentalis larvae increased the virulence of the Novosibirsk strain but not the Berlin strain. After a fifth passage and reisolation of the Novosibirsk strain, the virulence increased considerably and exhibited a similar level of virulence to the Berlin isolate. This established the Novosibirsk strain of V. lecanii as being capable of biological control of F. occidentalis. Representatives of the phylum Zygomycota are also useful for ecologically safe insect control. For example, Conidiobolus thromboides Drechsler isolated from diseased pea aphids in the Novosibirsk region [34] was tested for control of pests in greenhouses. The results demonstrated the potential of

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C. thromboides for control of T. vaporariorum, Myzus persicae Sulz., Aphis gossypie Glow., Macrosiphum euphorbia Thom., Aulacorthum solani Kalt., Brevicoryne brassicae L. [35]. Compared to V. lecanii and B. bassiana, this fungus has the advantage of a lower relative humidity requirement (60% instead of 80% RH). However, no preparation that is based on C. thromboides has yet been registered in Russia. In field crops and forestry, insect control by fungi occurs through both natural and artificial epizootics. For example, Conidiobolus obscurus Petch. in a population of pea aphids in St. Petersburg [36] or Entomophaga aulicia Batko in a population of gypsy moths in Russian Far East [37] were able to cause such epizootics following inoculation. It is evident that success of fungal preparations in the field is limited by a high humidity requirement. A case in point in which this limitation was overcome was one in which an oil-based formulation of M. anisopliae var. acridum was applied against locusts [38]. This finding led to the testing of Siberian strains of M. anisopliae and B. bassiana from other insects on larvae of the Italian locust Calliptamus italicus L., an outbreak of which was observed in 2000-2002 in the Novosibirsk region [39]. The efficacy of these strains did not exceed 50%. Fungal isolates from the host locust might have resulted in a more effective control. Biocontrol potential of entomopathogenic fungal agents against some insects damaging berry crops was demonstrated by introduction during the soil dwelling stages of the insects. For example, B. bassiana was observed to be useful against cryptocarpous insects, such as the sea-buckthorn fly Rhagoletes batava obscuriosa Kol. Sea-buckthorn is a valuable small fruit crop of dietary and pharmaceutical importance in Russia, especially in Siberia. Fly larvae penetrate the fruits, and cause significant yield loss. The introduction of B. bassiana into the soil beneath sea buckthorn bushes during the period of larval pupation was very effective in controlling this pest [40]. Different species of entomopathogenic fungi were tested against blackcurrant cane midge in model experiments on pupae found in soil near the blackcurrant bushes [41]. Similar experiments were conducted to control the raspberry cane midge [42]. In this case, three fungal isolates belonging to genus Beauveria from the “Collection of Microorganisms, Producers of Biopreparations” (CMPPB, Rosagroservice, Moscow) were used. In laboratory experiments, the efficacies of the treatments were estimated as percent of adults emerging from the soil. The results showed that only 1-3.5 % of adults emerged from the soil treated by all isolates studied, while 82% of adults emerged from the soil treated with water (control). Thus, introduction of entomopathogenic fungi to soil for control of the dwelling stages of insect pests is rather promising for ecologically safe plant protection. In the first section of this chapter, the association of raspberry cane midge with plant pathogens such as D. applanata was described [15]. The results of a recent experiment by the author and coworkers showed an inhibitory effect of

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the B. bassiana on growth of D. applanata. In an in vitro experiment, B. bassiana caused reduction in the diameter of D. applanata colonies by 1.72.1 times as compared to the controls. These data may indicate the production by Beauveria fungi of antibiotics capable of suppressing plant pathogens. The observation further suggests a dual property of Beauveria, for ecologically safe control of raspberry midge blight as an insect damage/disease complex of this small fruit crop.

Microbial secondary metabolites as natural agents of insect suppression It is well known that microbial secondary metabolites together with propagules constitute common entomopathogenic biological formulations. For instance, the most widely used appllied bacterial entomopathogenic preparations consist of Bt spores and δ-endotoxin crystal. As mentioned before, Cry- and Cyt- toxins are responsible for insecticidal properties of Bt. Other metabolites such as bacteriocins or vegetative insecticidal proteins have so far not been studied sufficiently. The production of antibiotics by some Bt strains could be useful for simultaneous protection of plants against insects and diseases. For example, Bt subsp. amagiensis, novosibirsk, wuhanensis and yunnanensis were shown to inhibit the growth of plant pathogen Erwinia carotovora under laboratory conditions [43]. More recently in Russia, there has been increased interest in the use of preparations from individual microbial toxins or mixture of the toxins. The preparations based on fungal toxins are discussed in this section. To obtain such metabolites, fungal biomass is separated from broth cultures, followed by extraction of the toxins from either broth cultures or mycelium and then concentrated into a natural product. This protocol was used to produce metabolite preparations based on fungal toxins from C. obscurus and V. lecanii that were called Mycoaphidin-T and Verticillin-M, respectively [44,45]. Longterm trials of the former preparation, demonstrated high efficacy against aphids, whitefly and thrips in different regions of Russia, including Siberia [12,44]. The creator of this preparation [45] established that this high activity of Mycoaphidin-T was due to a phospholipid compound. Interestingly, this toxin had no acetylcholinesterase activity like broad-spectrum organophosphate synthetic insecticides applied for control of such insects, and perhaps this fact explains the specificity and non-toxic effect of Mycoaphidin-T on beneficial insects. This effect was confirmed with direct experiments on beneficial insects such as ladybird, green lacewings and others [45], which caused no mortality. Secondary metabolites produced by V. lecanii were also studied in detail and it was shown that the toxins generally accumulated in mycelia [46]. The metabolite- based preparation Verticillin-M was developed and demonstrated

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high activity against sucking insect pests. However, neither Mycoaphidin–T, nor Verticillin-M has so far been registered. Several natural metabolic products of Streptomyces spp. (class Actinomycetes) have been of great interest for insect control. For example, Phytoverm® (Pharmbiomed, Russia), a metabolite-based preparation of the soil microorganism, S. avermitilis, was developed and registered recently [47]. This preparation contains a complex of natural metabolites which were extracted from biomass of actinomyces obtained by submerged culture and designated as Aversectin S. Unlike Phytoverm®, some Aversectin S preparations registered in other countries, such as Vertimec® (Syngenta, Switzerland) consist of semisynthetic avermectins, termed abamectins. In greenhouse trials, Phytoverm® was shown to be very effective against spider mites and several species of aphids, but not against whiteflies [47]. The mortality of spider mites 3 days after application was 99%, but rather low for the whiteflies [47]. In addition, Phytoverm® is considered to have potential for control of raspberry midge blight [15]. Experimental results showed significant reduction in midge blight severity by Phytoverm® (Table 2), as compared with the synthetic Actellic® pesticide which was probably due to increased resistance after repeated application. This observed efficacy of Phytoverm® prompted further studies of its mode of action against the plant pathogen D. applanata, a member of the “raspberry midge blight” complex [42]. Phytoverm® was shown to inhibit the radial growth of D. applanata in the lab (Table 3). The potential application of Aversectin was confirmed for ability to suppress insects and to inhibit the growth of some plant pathogens. The same effect has been shown for fungi of the genus Beauveria (see the previous section). Table 2. Effect of Phytoverm® on raspberry midge blight severity

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Table 3. Effect of Phytoverm® on Didymella applanata

Interestingly, Phytoverm® has shown efficacy against insects such as the cabbage aphid and the Italian locust, which are not susceptible to common microbial preparations. This preparation was highly effective against cabbage aphid on cauliflower plantations in both the Novosibirsk [48] and Krasnoyarsk [49] regions of Russia. In addition, laboratory experiments and field testing indicated the potential for application of Phytoverm® against larvae of Italian locust C. italicus [39]. A locust swarm treated with this product exhibited a 8798% mortality, which was equivalent to the effect of the traditional synthetic insecticide Adonis®. However, the action of the chemical insecticide was quicker than the metabolite-based preparation. At 2 days after treatment with Phytoverm® and Adonis®, locust mortality of 47 and 85% resulted, respectively. Similar Streptomyces metabolite- based preparations have been developed but are not yet registered in Russia. For example, Actinin was developed from S. globisporus metabolites in the1980s [50]. Actinin was initially recommended for control of the Colorado potato beetle, Leptinotarsa decemlineata Say. Some years later, greenhouse and field tests showed it to be very effective against spider mites [12,50] and wheat thrips Haplothrips tritici Kurd., respectively [51]. The microbial metabolite preparations have some advantages over living organism-based preparations. First, metabolites are less subject to variation in environmental factors such as temperature, humidity, UV-radiation, etc. Furthermore, shelf life is typically longer for metabolites than for propagules. Metabolite preparations also appear to have wider spectrum and quicker action, which is a valuable attribute for growers. As natural agents, metabolite-based preparations are environmentally safe and are not subject to accumulation in fruits as compared with synthetic insecticides. For example, the waiting period before harvest for Phytoverm® averaged 1-3 days depending on the crop, thereby allowing treatment of all stages of plant development for insect control. No evidence of plant toxicity has been observed. While these preparations are

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generally compatible with beneficial insects, there have been few negative impacts of Phytoverm® on entomophagous insects in greenhouse on reproduction [52]. Thus, microbial metabolites should be considered ecologically safe for control of insect pests.

Enhancement of insecticidal activity of ecologically safe control agents and preparations High ecological safety does not guarantee stability and high insecticidal activity of biopreparations because of the complexity of the interaction between the entomopathogen, host insect and the environment. Generally, insect mortality is not an instantaneous in response to a single agent, and requires a definite period for infection to develop, especially in the case of viruses. In addition, destruction of any insect control agent may occur under the influence of biotic and abiotic environmental factors. Consequently, conditions should be provided and maintained for survival and reliable activity of entomopathogens, in order to compete with synthetic insecticides. At least, two conditions should be fulfilled for the enhancement of microbial insect control. First, the choice of a virulent strain and its protection during storage and application is important. Secondly, knowledge of the mode of action of the pathogen toward the target is necessary for development of enhanced insecticidal activity. Variability is known to be inherent among microbial and viral agents. That phenomenon can be manifested as differences in shape, size of cells and particles, extent of enzyme production, etc. Virulence of baculoviruses depends, sometimes, on size of protein matrix [53]. Selection of Bt and fungal entomopathogens are assessed based on their virulence, potential to produce toxins, etc. Passage through a susceptible insect is a common way to enhance the virulence of a specific biocontrol agent. In this section, only natural (not genetically modified) strains of entomopathogens will be considered. Among the most harmful of abiotic factors which can cause undesirable changes in entomopathogens are UV-radiation exposure and even oxygen (through its free radical forms) [54, 55]. Thus, additives are usually applied to protect the biocontrol agents. [56]. Considering solar damage to the entomopathogens during field application as a free radical process, antioxidants have thus been suggested as protectants [54,57]. For Bt subsp. kurstaki, the antioxidant 2,4-dioxybenzophenon appeared to be the most appropriate for resistance to UV-radiation [58]. Interestingly, UV-protectors for B. bassiana were also derivatives of oxybenzophenon [59]. Oxybenzon, the best of the derivatives, provided good protection of Metarhizium conidia [60]. Antioxidants are also known as inhibitors of free radicals in biological processes of cell damage [61]. These substances were also suggested for

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protection of entomopathogens from some oxygen forms during storage [54]. Significantly prolonged shelf life was obtained for liquid formulation of viral and bacterial insecticides by introduction of antioxidants in low concentrations. It should be noted that antioxidants were also shown to inhibit free radical development in plant cells during the infection process of plant pathogens. Current available information in plant pathology indicates that there is an association of oxidative stress with free radicals formation [62]. Therefore, it would be expected that very low doses of antioxidants sprayed on plants could prevent or inhibit plant infection. There have been some attempts to use a mixture of different entomopathogens to enhance insecticidal activity [63-65]. The net effect depends on the nature of interaction between the organisms: additive, antagonistic or synergistic. Obviously, the last effect is preferable. In this case, the insecticidal activity is increased and the latent period is shortened. A mixture of Bt and GV was used successfully in ecologically safe control of Siberian silkworm, for example [64]. However, a mixture of baculovirus with Bt did not show consistent synergism [63,65]. The total effect depends on individual characteristics of both the insect and the entomopathogen. Valuable information about of the mode of action may lead to the development of techniques for enhancement of insecticidal activity. An influence on the mechanism of the agent’s biochemical activity can modify the effectiveness of insect control. For example, the well-documented mode of action of Bt δ-endotoxin in some insects is limited proteolysis by midgut enzyme under a pH of 9.5-10.5. [66-68]. Based on this phenomenon, addition to preparations of the ingredient that optimizes the pH during solubilization of Bt crystal was suggested [54,57]. This led to an acceleration of Cry-toxin action, and consequently, the enhancement of insecticidal activity against Lepidoptera. Some common biochemical responses such as intensification of lipid peroxidation of cell membranes has occurred in insects under the influence of both Bt and baculoviruses [69]. The products of lipid peroxidation have been shown to accumulate in midgut and fat body cells of some lepidopteran species after feeding of Bt or NPV treated plants [54, 69]. In order to strengthen the efficacy of this process through free radical formation, some initiators were added to bacterial and virus preparations. For example, the addition of low concentrations of FeSO4, a known initiator of free radical formation, to M. brassicae NPV resulted in significant enhancement of insect mortality 3 days after treatment [54]. Another common approach for enhancement of insecticidal activity of both bacterial and viral biocontrol agents is to increase the permeability of host insect cell membranes. This can be accomplished by the addition of dimethylsulfoxide (DMSO). DMSO is a well known non-toxic compound that is capable of penetrating cell membranes, and thereby facilitating the penetration and

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movement of other biologically active agents to their targets in living organisms [70]. It has been demonstrated that DMSO can serve as an enhancer of insecticidal activity of NPV and Bt [57,71]. Another approach for enhancing insecticidal activity is the exogenous application of enzymes, which are capable of damaging insect peritrophic membranes (PM). PM is a tube-like lining of the midgut lumen. Chitin and protein play important roles in maintaining the strength of the PM. In 1971, Smirnoff [72] used exogenous chitinase to enhance destruction of chitin of the PM and thereby increase the permeation of the Bt preparation. This approach was successful for ecologically safe control of Choristoneura fumiferana Clemens, in Canada. Two decades later, renewed attempts were made to test this approach on such biocontrol agents as Bt, baculoviruses and entomopathogenic fungi [73]. The results confirmed the findings of the earlier studies in that the influence of chitinase on Bt depended on its subspecies [72]. The addition of chitinase (0.5 mU/ml) to Bt subsp. sotto doubled the mortality of P. sticticslis. The insecticidal activity of two GV and one NPV was significantly increased by the addition of a chitinase (Table 4). These data agreed with findings by Shapiro et al. on control of the gypsy moth [74]. These results signify the possibility of reducing the doses of baculoviruses by the addition of a very low amount of exogenous chitinase, and this observation has served as the basis for development of improved viral formulations. Such low dosages were successful in field-tests against C. pomonella and M. brassicae in the Krasnodar and Novosibirsk regions of Russia. In addition to increased insecticidal activity, shortening of the latent period was observed. However, when B. bassiana and V. lecanii were tested, contradictory data were obtained that pointed to complexity in interactions between exogenous chitinase and an endogenous fungal one. Investigations showed inhibition of the fungal growth in the presence of chitinase. Thus, the specific effect chitinase depended on the nature of both the insect, and the biocontrol agent [73]. Table 4.The influence of chitinase on the baculoviruses biological activity

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In the last fifteen years, more data regarding the influence of chitinase on the efficacy of entomopathogens and their metabolites have emerged. The role of chitinase as an enhancer of insecticidal activity was demonstrated for the Bt toxin Cry 1C towards Spodoptera littoralis Boisd. larvae [75]. In the presence of the exogenous chitinase (Chi A), 3 µg/ml of Cry 1C had a toxic effect that was equivalent to the effect of 20 µg/ml of Cry 1C without the enzyme. Furthermore, Chi A was shown to induce formation of pores in the PM, which was associated with enhancement of insecticidal activity. The role of endogenous chitinases from Bt was also studied by Sampson and Gooday [76]. The enhancement of Bt activity against S. littoralis by these enzymes was demonstrated by the addition of a chitinase inhibitor [76]. It was shown that the structural component of PM, chitin, partly provides the resistance of insects to entomopathogens [77, 78]. Therefore, the addition of chitinase, which enhances penetration of the PM by entomopathogens, could also enhance their insecticidal effects by destroying the structure of the PM. This phenomenon may explain the universal action of chitinases in providing an enhancer effect on the activity of separate NPV [73, 74], Bt [72, 73] or cloned Cry protein [75]. This effect was expressed in the increased insect mortality without any toxic effect of the chitinase alone. At the same time, the prolonged period of entomopathogen activity was significantly reduced [73]. The above-mentioned data regarding synergism between Bt and NPV together with enhancers served as an incentive for development of a triple mixture for control of all lepidopteran insects on cabbage [79]. This development will be described in more detail below since it is the first time such a mixture has been proposed. Firstly, it is well established that damage of cabbage plants by insect pests is often very severe. Thus, the lack of pesticide treatment in Western Siberia allowed almost complete destruction of the crop by lepidopteran insects. As previously noted, the most harmful of these pests are the larvae of the cabbage moth, diamondback moth, and large white butterfly for cabbage. Moreover, all of these species are frequently found in fields simultaneously. As mentioned above, biological insecticides based on Bt are typically applied for the suppression of lepidopteran insect pests. The efficiency of such preparations depends in general on the composition of Bt subspecies, Cry proteins [3] and nature of the host [80]. In Siberia and other regions of the Russia, the populations of cabbage moth are less susceptible to the different Bt subspecies than the populations of large white butterfly or diamondback moth [81, 82]. At the same time, the Mamestra brassicae nuclear polyhedrosis virus (MbNPV) appeared to be an effective biological agent against the most harmful cabbage pest, the cabbage moth, but not for other Lepidoptera. Despite the merits of the NPV such as high specificity and transgenerational transmission, the infection by this entomopathogen requires a latent period of up to 10 days.

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This disadvantage could be overcome partly by the addition of Bt [81-83], or enhancers [82,84] to the NPV. Therefore, the development of a mixture containing Bt, MbNPV and certain enhancers for biocontrol of all lepidopteran pests of cabbage could increase control efficacy of these pests. This proposed new strategy for the improvement of microbial insect control was based on a similar approach with Bt and baculoviruses enabling the penetration of PM midgut. The addition of chitinase to the mixture of Bt and NPV was proposed in order to develop the optimum composition of these three ingredients for improved microbial control cabbage moth, diamond back moth and the large white butterfly. A crude preparation of the enzymes with strong chitinase activity from Streptomyces sp. was used in this work [79]. The crude chitinase preparation was characterized by neutral effects on larvae and entomopathogens, thus precluding possible contamination of the mixture with antibiotics, toxins and biologically active metabolites. Initially, some Bt subspecies, galleriae, sotto (dendrolimus) and kurstaki, were tested against cabbage moth in order to select the one most appropriate against M. brassicae. Due to the role of the cabbage moth as the most harmful cabbage pest and its prominent role in pest populations, larvae of this species were selected for the test. In laboratory experiments, Bt subsp. galleriae was shown to be the most effective against the larvae (Table 5). The addition of chitinase to the Bt subspecies generally increased mortality. The best result was observed with the combinations of Bt subsp. galleriae and the chitinase preparation and thus this subspecies was selected to be combined with MbNPV and chitinase (0.5 mU/ ml). In initial laboratory experiments, the influence of each of the three components was tested and their efficacies compared to that of the combined preparation on the second instar of M. brassicae larvae at the 3rd and 5th day after feeding (Table 6). The insecticidal activity of the combined preparation was high in comparison with any single component and showed a synergistic effect on larvae. It is necessary to emphasize that the addition of the chitinase to Table 5. LC50 of some Bt subspecies for control M. brassicae larvae

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Table 6. LT50 for MbNPV and its mixture with chitinase

the MbNPV also decreased latent period expressed as LT50. The fact that the same concentration of the chitinase increased Bt and MbNPV larval mortality is evidence for the role of chitinase as an enhancer of lethal effects of bacterial and viral entomopathogens by increasing the PM permeability. This data was used as a basis to formulate the original mixture of Bt subsp. galleriae, MbNPV, and chitinase to control lepidopteran pests on infested cabbage in the field. Field trials were conducted on cabbage plots infested with the three major species of lepidopteran pests simultaneously. The results demonstrated the efficacy of the mixture against such a complex of lepidopteran pests. The mortality of the larvae varied from 74.1 to 80.4 percent depending on the insect species. Furthermore, the addition of the chitinase to the Bt and NPV composition provided the acceleration of insect mortality at a considerably reduced concentration of entomopathogens. The composition consisted of a 5fold lower amount of Bt and 10-fold lower amount of polyhedra per ml compared with the values recommended for the standard commercial formulations. Thus, the data obtained in this study indicated the advantage of supplementing the original composition of Bt, MbNPV with the chitinase for ecologically safe insect control on cabbage. In addition, chitinase also plays an important role in the suppression of phytopathogenic fungi that contain chitin in their cell walls [85, 86]. For example, it was shown that the chitinase alone (in more concentrated solution) was successfully used for protection of raspberry against spur blight caused by D. applanata [15]. Improvement of insect control may be accomplished in two ways: either as tank mixtures of biopreparation, enhancers and protectors immediately before application, or by developing a formulation containing all these components that facilitate longer storage. In 1950s, tank mixtures of commercial biological preparations with sublethal dosages of chemical insecticides were suggested by Telenga [87] in the former Soviet Union. This concept eventually gained universal acceptance and was widely adopted [88]. Though these mixtures were proposed for enhanced activity of microbial agents, the desired effect was not always observed [88]. Moreover, even sublethal dosages of synthetic

18


insecticides can be accumulated in the environment and adversely impact beneficial insects. As Franz and Krieg [89] pointed out, this approach eliminated the selectivity of biocontrol agents, which is important for environmental health. Acquired resistance to by insect pests may be accelerated by replacement of lethal with sublethal doses. Because of these demerits, tank mixtures of biopreparations may be improved with non-toxic enhancers which ecologically safe. For example, addition of the DMSO at low concentrations, 2-5% vol, to commercial preparation Entobacterin® based on Bt subsp. galleriae, caused a significant increase in P. sticticalis mortality on cabbage [54]. The same effect was observed with a viral formulation against M. brassicae [20]. As previously mentioned, a tank mixture preparation with two diverse microbial agents should be limited to a mixture with proven synergism. Synergistic effects allow reduced dosages of preparations. In the case of a mixture of Bt and baculoviruses, the possible decline in feeding by the larvae under the influence of Bt can prevent viral infection [23]. However, this obstacle may be overcome by a triple combination of Bt, NPV and enhancers that decrease the application rate of Bt as described above. An efficient means to reduce Bt dosage was shown for the combination of Thuricide® and M. anisopliae mycotoxin (destruxin) which revealed the synergistic effect [90]. It should be noted that preparations based on inhibitors of insect chitin synthesis such as Dimilin®, also were shown to enhance the effectiveness of a Bt activity. Synergism causing increased mortality of larvae was observed when a mixture of Dendrobacillin® and Dimilin® was applied against P. xylostella [91]. Thus, there are at present few prospects to mix entomopathogens with ecologically safe components rather than sublethal dosages of synthetic insecticides to enhance larval mortality. Generally, plant growers prefer formulations which include all necessary constituents instead of tank mixtures. The peculiarities of biological formulation technology have been described in detail [54, 56, 92]. In this section, limited consideration has been given to the details of formulations, which are designed in accordance with principles of enhancement of ecologically safe formulations. At least two Bt formulations were developed for this purpose [54]. A liquid formulation containing DMSO and an antioxidant was characterized by high insecticidal activity together with an increased shelf life. A solid formulation based on Bt subsp. kurstaki was registered in Russia in 1980s and designated as LEST®. This formulation also contained an enhancer and an antioxidant that facilitated a reduced dose of Bt, and better persistence during storage and application.

Conclusion: The future challenges Ecologically safe control of insect pests may be accomplished by application of a wide range of entomopathogens and their secondary

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metabolites. Their application provides protection for plants and additionally improves both plants and environmental health. The use of ecologically safe agents for insect pest control promotes the conservation of other natural enemies such as beneficial insects, birds, and also antagonistic microorganisms useful for plant disease control. Therefore, the inoculative and inundative reintroduction of these agents into both fields and forests provides opportunities for the development of one or more strategies for biocontrol (protection and enhancement of specific natural enemies) [1]. Some observed disadvantages in the practice of ecologically safe control could be reduced by enhancing their potency with one or more additives. However, to provide environmental safety, the mixture of microbial agents with synthetic insecticides should be avoided, even at sublethal dosages of the latter. Eventually, accumulation of the synthetic compounds in the environment will ultimately lead to harm of non-target beneficial organisms and increase the probability of development of insect resistance to these compounds. In place of such mixtures, other techniques such as combining biocontrol agents with low concentrations of ecologically friendly components are available to enhance biocontrol activity. These components, designated as enhancers may include other entomopathogens, microbial metabolites, and inhibitors of insect chitin. Preliminary study of the compatibility of additives in various proportions is necessary to optimize enhancement of insecticidal activity. Furthermore, additional research on understanding the mode of action of entomopathogens and their metabolites on target insects may be required to find biologically active substances that accelerate the mechanisms of certain interactions which may lead to high insect mortality. Such compounds could be useful as in dual mixtures with a pathogen or in triple mixture with two synergistic entomopathogens. Thus, a preliminary study to understand the effect of the enhancer on each biocontrol agent will be required. Occasionally, it may be beneficial to replace preparations which are based on living microorganisms with preparations from microbial natural metabolites. The latter are less dependent on ecological factors as compared with living microorganisms and often provide better insecticidal activity with a wider spectrum. In addition to insecticidal properties, these products can occasionally suppress the development of plant pathogens. These dual properties are rather important for plant health management. Some fungal and bacterial entomopathogens are also capable of controlling plant pathogens through the production of secondary metabolites. It seems therefore reasonable to explore and exploit the dual properties of these agents much more in the future. Another aspect of enhancement of insecticidal activity is protection of control agents from the negative effect of some environmental factors such as UV-radiation, rainfall etc. Obviously, the selection of a protectant must be compatible with effectiveness and ecological safety.

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Enhancement for insect control may be realized in two major ways: first, as a mixture which is produced just before spraying; and secondly, as an improved formulation with enhancers. It seems at first that the use of the ready to spray formulations would be promising. Since variable environmental conditions may affect their efficacy, however, different tank mixtures appear to be preferable. Recently, concepts of “precision agriculture”, including precision integrated pest management (IPM) have emerged. Presently, these concepts in IPM have been principally applied to chemical weed control [93]. The initial step in the development of an insect control program should involve the creation of a precision support system. This support system is a database of performance under various environmental conditions. Such an expert system could include information on the influence of variable factors on insect control agents. Thus, the essential components and proportions of a desired mixture for plant health could be linked to the precision support system to provide information on the technology of ecologically safe insect control. For instance, it could be possible to take into account both natural insect infection, and the contribution of beneficial insects for pest control. In plant health management systems, the key component is the plant itself. Thus, consideration of ecologically safe control of insect pests cannot be comprehensive without understanding the influence of plant compounds on the insect control agents on plant foliage. Certainly, if an insect control agent is introduced into the soil, the influence of plant root secondary metabolites should be considered. Current knowledge on how insect control agents interact with plant metabolites is limited to a few studies, which suggest that the nature of the host plant can affect the insecticidal activity. It should be noted that the efficacy of insect control agents may not depend only on phytochemicals directly but be mediated by the insect midgut as well. For instance, it was reported that the biological mechanism for reduced susceptibility to NPV by larvae feeding on different plants could be midgut based, depending on rates of sloughing of infected midgut cells [94]. It is therefore reasonable to broaden the study on a tritrophic level (insect- plant - control agent). Research development in this direction will determine the most relevant agent and its exact dosage depending on the nature of insect, plant and environmental factors. Increased attention should be paid to the compatibility of ecologically safe insect control agents with antagonistic or hyperparasitic microorganisms for plant disease control. This compatibility will help to solve the dual problems of insects and diseases in plant protection simultaneously. Gene manipulation for control of insect pests is beyond the scope of this chapter. Nevertheless, the notion of involving the above-described data in gene modification for both entomopathogens and plants should not be precluded. This view is supported by demonstration of enhanced insecticidal activity by the cloning of chitinase gene into Bt subsp. aizawa [95] or expression of Bt toxin

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gene in an insect baculovirus [96]. However, because of high initial cost for genetic modification, techniques that use both traditional mixtures and application of biotechnology will be developed simultaneously in the future. Successful development of ecologically safe insect control requires a broad understanding of the mode of action of any agent at the molecular and physiological levels. Interdisciplinary communications among researchers will offer progress towards development of environmentally safe insect pest control in order to manage plant health.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Eilenberg, J., Hajek, A.E., and Lomer, C.J. 2001, BioControl, 46,387. Regulation of the Eastern Palearctic Regional section of The International Organization for Biological Control of Noxious Animals and Plants, Moscow, 1995. Schnepf, E. Crickmore, N. Van Rie, I., Lerecluse, D., Baum, I., Fietesson, I., Zeigler, D., and Dean, D. 1998, Microbiology and Molecular Biology Reviews, 62, 775. Yamamoto, T., and Dean, D. 2000, Entomopathogenic Bacteria: From Laboratory to Field Application, J.-F. Charles, A.R.M. Deleclus, and C.A. Nielsen-Le Roux (Eds.), Kluwer Academic Press, The Netherlands, 81. Siegel, J.P. 2001, Journal of Invertebrate Pathology, 77,13. Ishawata, S. 1901, Dainihon Sanshi Kaiho, 114, 1. Berliner, E. 1915, Zeitschrift fur Angewandte Entomologie, 2, 29. Weiser, J. 1972, Microbiologicheskie metody borby s vrednymi nasekomymi (Microbial Insect Pest Control), Kolos, Moscow. Talalaev, E.V. 1959, Transactions of the First International Conference on Insect Pathology and Biological Control, Prague, 1958, 51. Hofte, Н. and Whiteley, H.R. 1989, Microbiological Reviews, 53, 242. Smirnov, O.V. 2002, Informazionnyi bulleten VPRS MOBB (Information Bulletin EPRS/ IOBC), 33,126. Andreeva I.V. and Shternshis, M.V. 1995, Zashchita rastenii (Plant Protection), Moscow, 11, 41. Goldberg, L.G., and Margalit,G. 1977, Mosquito News, 37,355. Krieg, A., Huger, A.M., Langenbruch, G.A., and Schnetter, W. 1983, Zeitschrift fur Angewandte Entomologie, 96, 500. Shternshis, M.V., Beljaev, A.A.,Shpatova, T.V., Bokova, J.V., and Duzhak, A.B. 2002, BioControl, 47, 697. Pitcher, R., S., and Webb, P.S. 1952, Journal of Horticultural Sciences. 2, 95. Pashenova, N.V., Vetrova, V.P., Matrenina, R.M., and Aphanasova, E.N. 1995, Bark Beetles, Blue-stain Fungi and Conifer Defense Systems, International Symposium, Norway, 40. Gouli, V.V., Shternshis, M.V., and Koltunov, E.V. 1976, Sibirskii Vestnik Selskokhozjastvennoi Nauki (Siberian Bulletin of Agricultural Science), 5, 36. Orlovskaya, E.V. 1962, Voprosy Ecologii (Problems of Ecology), Kiev, 87. Shternshis, M.V. 1995, Zashchita rastenii (Plant Protection). 9,29.

22


21. Ermakova, N.I., and Shternshis, M.V. 1987, Selskokhozjastvennaja Biologia (Agricultural Biology), 4, 34. 22. Shternshis, M.V., Ermakova, N.I., and Tsvetkova, V.P.1997, Sibirskii ecologicheskii zhurnal (Siberian Journal of Ecology), 6, 585. 23. Orlovskaya, E.V. 2002, Informazionnyi bulleten VPRS MOBB (Information Bulletin VPRS /IOBC), 33, 141. 24. Bakhvalov, S.A. 1995, Sibirskii ecologicheskii zhurnal (Siberian Journal of Ecology), 5, 466. 25. Shternshis, M.V., and Severina N.I. 1976, Sibirskii Vestnik Selskokhozjastvennoi Nauki (Siberian Bulletin of Agricultural Science), 2, 61. 26. Gouli, V.V., and Golosova, M.A. 1975, Virusy nasecomyh v zashchite lesa (Viruses in Forest Insect Control), Forest Industry, Moscow. 27. Mechnikov, I.I.1879, Bolesni khlebnogo zhuka (The Diseases of Cereal Beetle), Odessa. 28. Shternshis, M.V., Issi I.V., Voronina, E.G., and Glupov, V.V. 2001, Patogeny nasekomykh: structurnii i funczionalnii aspecty (Insect Pathogens: Structural and Functional Aspects), V.V. Glupov (Ed.), Publishing House “All The Year Round”, Moscow, 5. 29. Evlakhova, A.A.1974, Entomopatogennie griby (Entomopathogenic Fungi), Leningrad, Nauka. 30. Ogarkov, B.N., and Ogarkova, G.R. 2000, Entomopatogennie griby Vostochnoi Sibiri (Entomopathogenic Fungi of Eastern Siberia), State University, Irkutsk. 31. Borisov, B.A., Serebrov, V.A., Novikova, I.I., and Boikova, I.V. 2001, Patogeny nasekomykh: structurnii i funczionalnii aspecty (Insect Pathogens: Structural and Functional Aspects), V.V. Glupov (Ed.), Publishing House “All The Year Round”, Moscow, 352. 32. Sermann H., Kastner, U., Hohner, S., and Hirte, W. 1994, Mitteilungen aus der Biologischen Bundesanstald fur Land- und Forstwirtschaft, 301, 351. 33. Hetsch, N., Sermann H., and Shternshis, M.V. 2002, Micologia i Phytopathologia (Mycology and Phytopathology), St.-Petersburg, 5, 91. 34. Makhova, N.M., and Rakshaina, M.T. 1980, Izvestija Sibirskogo otdelenija Academii nauk SSSR (Bulletin of Siberian Branch of the Academy of Sciences of the USSR), 3, 107. 35. Lobanova, N.V., Rakshaina, M.T., and Shternshis M.V. 1989, Proizvodstvo i primenenie piryformina (Production and Application of Pyriformin), Siberian Branch of Academy of Agricultural Sciences, Novosibirsk. 36. Voronina, E.G., Lednev, G.R., and Mukamolova, T.U. 2001, Patogeny nasekomykh: structurnii i funczionalnii aspecty (Insect Pathogens: Structural and Functional Aspects), V.V.Glupov (Ed.), Publishing House “All The Year Round”, Moscow, 271. 37. T.U. Jurchenko, U.B., Turova, and Chelysheva, Z.P. 2000, Zashchita i karantin rastenii (Plant Protection and Quarantine (Moscow), 8, 12. 38. Lomer, C.J., Bateman R.P., Johnson, D.L., Langewald, J., and Thomas, M. 2001, Annual Review of Entomology, 46, 667. 39. Shternshis, M.V., and Tsvetkova, V.P. 2002, Zashchita i karantin rastenii (Plant Protection and Quarantine), Moscow, 6,26.

Insect pest control

23

40. Kalvysh, T.K. 1980, Izvestija Sibirskogo otdelenija Academii nauk SSSR (Bulletin of Siberian Branch of the Academy of Sciences of the USSR), 3, 8. 41. Borisov, B.A., and Goncharova, N.G. 1994, The Ecologically Safe and PesticideFree Technologies of Plant Growing Production Obtaining, Proceedings of AllRussian Conference, Pushchino, 103. 42. Shternshis, M.V., Shpatova, T.V., Borisov, B.A., and Beljaev, A.A. 2003, Micologia i Phytopathologia Mycology and Phytopathology (submitted). 43. Judina, T., and Burtseva, L. 1997, Microbiologia (Microbiology, Moscow). 66, 25. 44. Voronina, E.G. 1994, The Ecologically Safe and Pesticide-free Technologies of Plant-growing, Proceedings of All Russian Conference, Pushchino, 189. 45. Oleinikova, G.A., Novikova, I..I., Voronina E.G. and Ginak V.I. 1995, Sovremennaja biotehologia v reshenii sadach zashchity rastenii (The Modern Biotechnology in Solving Problems of Plant Protection), All-Rissian Institute of Plant Protection, St.-Petersburg, 175. 46. Mitina, G.V., Sokornova S.V., and Pavljushin V.A. 2002, Informazionnyi bulleten VPRS MOBB (Information Bulletin VPRS/ IOBC), 33, 96. 47. Beresina, N.V., Chizhov, V.N., Drinyaev, V.A., and Jurkiv, V.A. 1997, Gavrish (Moscow). 2, 10. 48. Shternshis, M.V., Andreeva, I.V., Tomilova, O.G., and Shpatova, T.V. 2002, XII Congress of Russian Entomological Society, St-Petersburg, August 19-24,2002, 386. 49. Kuznetsova, I.A., and Shternshis, M.V. 2000, Sibirskii Vestnik Selskokhozjastvennoi Nauki (Siberian Bulletin of Agricultural Sciences), 3, 50. 50. Kandybin, N.V., Samoukina, G.V., and Sergeev, M.V. 1995. Pervii Vserossiiskii congress po zashchite rastenii (First All-Russian Congress on Plant Protection, StPetersburg), 318. 51. Shternshis, M.V., Galaktionov, K.V., and Giljov, S.D. 1997, Sibirskii Vestnik Selskokhozjastvennoi Nauki (Siberian Bulletin of Agricultural Sciences), 1, 123. 52. Dobrokhotov, S.A. 1997, Zashchita rastenii (Plant Protection), Moscow, 5, 13. 53. Shternshis, M.V., Severina N.I., and Gouli V.V. 1975, Izvestija Sibirskogo otdelenija Academii nauk SSSR (Bulletin of Siberian Branch of the Academy of Sciences of the USSR), 3, 51. 54. Shternshis, M.V. Povyshenie Effectivnosti Mikrobiologicheskoi Borby s vrediteljami (Enhancement of Microbial Insect Control). 1995, State Agrarian University, Novosibirsk. 55. Shternshis, M.V., and Gouli V.V. 1999, Society for Invertebrate Pathology, XXXII Meeting , University California, Irvine, August 22-27, 1999, 69. 56. Couch, T.L. 2000, Entomopathogenic Bacteria: From Laboratory to Field Application, J.-F. Charles, A.R.M. Delecluse, and C.A. Nielsen-LeRoux (Eds.), Kluwer Acad. Publisher, The Netherlands., 297. 57. Shternshis, M.V. 2001, Patogeny nasekomykh: structurnii i funczionalnii aspecty (Insect Pathogens: Structural and Functional Aspects), V.V. Glupov (Ed.), Publishing House All The Year Round, Moscow, 562. 58. Shternshis, M.V., and Solodova, K.V. 1989, All-Union Conference on Bioantioxidant, Institute of Chemical Physics, Moscow, 100. 59. Inglis G D, Goettel M.S. and Johnson D.L. 1995, Biological Control, 5, 581.

24


60. Moore, D, Brigda, P.D., Higgins, P.M., Bateman, R P, and Prior C. 1993, Annals of Applied Biology, 122, 605. 61. Free Radicals in Biology. 1976,W.A, Pryor (Ed.), Academic Press, New York, San Francisco, London 62. Lamb, C. and Dixon R A. 1997, Annual Reviews of Plant Physiology and Plant Molecular Biology, 48, 251. 63. Jaques, R.P., and Morris, O.N. 1981, Microbial Control of Pests and Plant Diseases 1970-1980, Academic Press, London and New York, 695. 64. Larionov, G V. 1995, Sibirskii ecologicheskii zhurnal (Siberian Journal of Ecology), 5, 438. 65. Geervliet, G.B.F., Vlak, J.M. and Smith, P.H. 1991, Mededelingen Faculteit Landbouwwetenschappen Rijks Universiteit Gent, 56,305. 66. Faust, R.M., Adams, J.R., and Heimpel, A.M. 1967, Journal of Invertebrate Pathology, 9, 488. 67. Tojo, A., and Aizawa, K. 1983, Applied and Environmental Microbiology, 45, 576. 68. Oppert, B. 1999. Archieves of Insect Biochemistry and Physiology, 42, 1. 69. Shternshis, M.V. 1987, Mededelingen Faculteit Landbouwwetenschappen RijksUniversiteit Gent, 52, 395. 70. Knubovez, T.L., Zinchenko, V.D., and Sabeldina, L.A. 1987, Biologicheskie membrany (Biological Membranes), Moscow, 4, 84. 71. Shternshis, M.V., and Gouli V.V. 2000, XXXIII Society for Invertebrate Pathology. Annual Meeting, University Guanajuato, Mexico, 89. 72. Smirnoff, W. A. 1971, Canadian Entomologist, 19, 1829. 73. Duzhak A.B., Salganik, R.I., Shternshis, M.V., Ermakova, N.I., Tsvetkova, V.P., Andreeva I.V., and Kushnikova T.A. 1995, Sibirskii ecologicheskii zhurnal (Siberian Journal of Ecology), 5, 457. 74. Shapiro M., Preisler, H.K., and Robertson, I.L. 1987, Journal of Economic Entomology, 80, 1113. 75. Regev, A.,Keller, M., Strizhov, N., Sneh, B., Prudovsky, E., Chet, I., Ginzberg, I., Koncz-Kalman, Z., Koncz, C.,Schell, I., and Zilberstein, A. 1996, Applied and Environmental Microbiology, 62, 3581. 76. Sampson, M.N., and Gooday, G.W.1998, Microbiology, 144, 2189. 77. Lehane, M.I. 1997, Annual Review of Entomology, 42, 525. 78. Wang, P., and Granados, R.R. 1998, Journal of Invertebrate Pathology, 72, 57. 79. Shternshis, M.V., Ovchinnikova L.A., Duzhak, A.B., and Tomilova, O.G. 2002, Archives of Phytopathology and Plant Protection, 35, 161. 80. Federici B. 1996, BioScience, 46, 410. 81. Spichenko, L.A., Gouli V.V., and Krupko, L.A. 1980, Biologicheskie metody borby s vrednymi organismami (Biological Control of Pest Organisms, V.V.Gouli (Ed.), Siberian Branch of the Academy of Agricultural Sciences), Novosibirsk, 16. 82. Shternshis, M.V. 1987, Vestnik selskokhozjastvennoi nauki (Bulletin of Agricultural Science, Moscow), 9, 54. 83. Krieg, A. 1971, Microbial Control of Insects and Mites, H.D.Burges, and N.W. Hussey (Ed.), Academic Press, London-New York, 365. 84. Shapiro, M., and Robertson, I.L.1992, Journal of Economic Entomology, 85, 1120. 85. Ordentlich, A., Elad Y., and Chet, J. 1998, Phytopathology, 78, 84.

Insect pest control

25

86. Cohen-Kupiec, R., and Chet, I. 1998, Current Opinion in Biotechnology, 9, 270. 87. Telenga, N.A. 1956, Doklady Academii nauk SSSR (Proceedings of the Academy of Sciences of the USSR), 109, 665. 88. Benz, G. 1971, Microbial Control of Insects and Mites, H.D.Burges, and N.W. Hussey (Eds.), Academic Press, London and New York, 327. 89. Franz, J., and Krieg, A. 1982, Biologische Schadlings-bekampfung, Verlag Paul Parey, Berlin und Hamburg. 90. Brousseau, C., Charpentier, G., and Belloncik, K. 1998, Journal of Invertebrate Pathology, 72, 262. 91. Shternshis, M.V., and Spichenko, L.N. 1995, Pervyi Vserossiiskii congress po zashchite rastenii (First All-Russian Congress on Plant Protection, St-Petersburg), 381. 92. Couch, T.L., and Ignoffo, C.M. 1981, Microbial Control of Pests and Plant Diseases 1970-1980, Academic Press, London and New York, 621. 93. Dille, J.A., Mortensen, D.A., and Young, L.J. 2002, Precision Agriculture, 3, 193. 94. Hoover, K., Washburn, J.O., and Volkman, L.E. 2000, Journal of Insect Physiology, 46, 999. 95. Tantimavanich, S., Pantuwatana, S., Bhumiratana, A., and Panbangred, W. 1997, Journal of General and Applied Microbiology, 43, 341. 96. Merryweather, A.T., Weyer, U., Harris, M.P.G., Hirst, M., Booth, T., and Posee, R.D. 1990, Journal of General Virology, 71, 1535.