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We propose a treatment strategy combining an initial disinfestation treatment with one of three protective treatments as an alternative for chemical fumigation of almonds and raisins for control of postharvest insect populations. Initial disinfestation treatments using low oxygen controlled atmosphere (0.4% O2) were designed to disinfest product of field populations of pyralid moths; navel orangeworm, Amyelois transitella (Walker), in almonds and raisin moth, Cadra figulilella (Gregson), in raisins. The protective treatments were cold storage (10 degrees C), controlled atmosphere (5% O2) storage, and application of the Indianmeal moth granulosis virus, and were designed to prevent establishment of Indianmeal moth, Plodia interpunctella (Hübner). The initial disinfestation treatment was effective against laboratory populations of navel orangeworm and raisin moth. Efficacy of protective treatments was determined by exposure of commodities to laboratory Indianmeal moth populations at levels far higher than those found in commercial storage facilities. All three protective treatments prevented development of damaging Indianmeal moth populations as measured by pheromone trap catches and evaluation of product samples. Quality analysis by commercial laboratories showed that overall product quality for all protective treatments was maintained at levels acceptable by industry standards.

A variety of plant species are being considered for the phytoremediation of selenium (Se) contaminated soils in agricultural regions of central California. Use of this plant-based technology may also attract a wide range of insects to these Se-accumulating plants. The first field study surveyed the diversity of insects attracted to tall fescue, birdsfoot trefoil, kenaf, and Indian mustard. Over 7500 specimens were collected by a sweep net collection technique for one complete growing season. Most of the 84 families identified were associated with beneficial insects, although pestiferous insects, for example, thrips, aphids, lygus, and leafhoppers, were also found. In the second study the bioaccumulation of Se in the cabbage looper [Trichoplusia ni (Hubner)] was investigated on Indian mustard grown in Se-rich water culture solution. Neonate larvae were transferred to plants and fed on Se-treated and no Se treated plants (controls) for 14 days, respectively. Pupae were collected from each treatment...

A number of the members of the Noctuidae (subfamily Plusiinae) are polyphagous insects that infest a variety of economically important crops world-wide. These hosts include but are not limited to cotton, vegetable and field crops and also such specialty crops as mint. Many of these crops require multiple applications of chemical pesticides to protect them from various loopers and other economic pests. One proposed alternative to the use of chemical pesticides to protect these crops is the development of insect-specific viruses of the family Baculoviridae. Hundreds of these viruses of the genus Nucleopolyhedrovirus have been isolated, many from economically important Lepidoptera. The advent of the discovery that some of these viruses may have a broad host range increased the interest in the development of them as microbial agents throughout the world. The broad host range (over 30 species in some cases) provided the possibility to control not only one species, but a complex of lepidopterous pests infesting specific or multiple crops. From the 1960s to the present, extensive basic and applied research has been conducted on the multi-nucleocapsid (MNPV) forms within which a broad host range appears to be characteristic. Of these, the virus isolated from the alfalfa looper, Autographa californica (Speyer) (AcMNPV) has been the most studied, particularly as related to microbial control. Two of the viruses isolated from loopers have been registered in the USA. Although efficacy has been demonstrated for loopers, there is a paucity of data on the other important species that may simultaneously infest looper hosts. Because of their relatively slow action, the viruses will probably be used in integrated programs with chemical pesticides applied during critical periods of high population pressure. Further research needs to be conducted to establish efficacy for other hosts, improved production methods either in vivo or in vitro, increased field persistence as related to timing and frequency of applications, and finally the utility of genetically engineered MNPVs as microbial pesticides. The potential of these organisms has been demonstrated and new developments are being made to increase the overall efficiency and economics of their use. These improvements can only increase interest in use of the baculoviruses in pest management systems for loopers and other pests.

A full-length synthetic version of the cry IA(c) gene, expressing a Bacillus thuringiensis (Bt) insecticidal crystal protein (ICP), was transferred into walnut somatic embryos. Sixty one transgenic embryo lines or clones were obtained and bioassayed with first instar codling moth larvae. In 34% of these lines, designated as ‘class A’, expression was high enough to obtain 80–100% mortality. A total of 20% were designated ‘class B’, which produced a mortality between 25 and 70% and also caused a decreased rate of larval development. Insect mortality and development from the remaining 46% of the lines were indistinguishable from that of the control. Expression of a chimeric gene encoding β -glucuronidase (GUS) was evaluated to serve as a linked but unselected marker gene. About 62% of the class A embryo lines showed correspondingly high activity for the GUS gene. However, 38% of the class A would have been missed if the level of GUS activity was used as the sole indicator. Detectable levels of protein corresponding to cry IA(c) could be found only in class A, but not in class B or C clones. Southern analysis of border regions revealed single inserts for class A clones and multiple inserts for classes B and C.

Japanese persimmon (Diospyros kaki L. `Jiro') was transformed using a disarmed strain of Agrobacterium tumefaciens, EHA101, carrying the binary plasmid vector, pDU92.710. The T-DNA region of pDU92.710 contained the kanamycin resistance gene (nptII), the β-glucuronidase gene (uidA), and a synthetic reconstruct of cryIA(c) encoding the insecticidal crystal protein fragment of Bacillus thuringiensis subsp. kurstaki HD-73. Leaf discs made from leaves of shoot cultures were cocultivated with Agrobacterium and cultured on a callus-induction medium containing kanamycin and cefotaxime. Among 720 infected leaf discs, 17 putative transformed callus lines showing kanamycin resistance were obtained after 8 weeks of culture. When these were cultured on a regeneration medium containing kanamycin, 15 formed adventitious buds. Of the 15 shoot lines, 11 grew well on a shoot-proliferation medium containing kanamycin, while 4 lines did not grow well. Of the 11 shoot lines, 10 showed GUS activities by fluorometric assay and were subjected to polymerase chain reaction (PCR) and Southern analyses. Except for two lines, all results were consistent with a stable integration of T-DNA into the persimmon genome. The production of CryIA(c) protein in transformed shoot lines was confirmed with Western analysis using anti-CryIA(c) serum. Insect bioassays were conducted with 10 lines showing GUS activity. Many of these lines showed high significant mortality of the test insects, Plodia interpunctella Hüber and Monema flavescens Walker, when compared to nontransformed controls.

The multiple nucleocapsid polyhedrosis virus ( M NPV) isolated from the celery looper, Anagrapha falcifera (Kirby), has a relatively broad host range within the order Lepidoptera. Preliminary tests indicated that Af M NPV was infectious to the navel orangeworm, Amyelois transitella (Walker). The pathogenicity of Af M NPV to a laboratory colony of A. transitella was established. Af M NPV was infectious to A. transitella with an LC50 of 36 polyhedral inclusion bodies (PIB)/mm2 of diet surface. Histopathology showed that PIBs were concentrated in the midgut but also were found in the hypodermis, fat body, and tracheal matrix. Malpighian tubules and ganglia also showed evidence of infection. The virus did not cause any damaging effects to the survivors of a sublethal dose (LC50).

The influence of Calcofluor white M2R (=Tinopal LPW = fluorescent brightener 28), a fluorescent brightener, on the laboratory activity of a nuclear polyhedrosis virus (Af M NPV) isolated from the celery looper Anagrapha falcifera (Kirby) was determined. The species challenged with and without M2R included Heliothis virescens (Fabricius), Helicoverpa zea (Boddie), Spodoptera exigua (Hubner), and Trichoplusia ni (Hubner), all economically important pest species of a number of food and fiber crops. Levels of enhancement as determined by LC 50 values varied from 2.9- to 13.6-fold, whereas levels of LC 95 values ranged from 3.7- to 16-fold, depending on the species. The greatest enhancement occurred among T. ni larvae. LT 50 values were significantly affected particularly when lower concentrations of polyhedra were fed. The presence of M2R reduced LT 50 values up to 2.1-fold among the species tested. The optimum concentrations for M2R enhancement were between 0.25 and 1% (wt/vol). Choice tests between water- and M2R-treated diet showed that larvae preferred non-M2R-treated diet. The implications of these data for use of M2R as an adjuvant for Af M NPV and other baculoviruses are discussed.

Insecticidal crystal protein fragments (ICPFs) of Bacillus thuringiensis encoded by cry IA (c) and cry IA (b) were previously shown to be lethal to key target walnut insect pests (codling moth, navel orangeworm and Indianmeal moth). One of these genes, cry IA (c), was used to transform walnut somatic embryos using a binary vector (pWB139) in which this gene was expressed as a protein fusion with the kanamycin resistance gene from bacteria. Transgenic embryos representing individual transformation events were germinated to produce shoots that were maintained as micropropagated shoot lines in the laboratory. Transgenic shoots were then grafted onto seedling rootstocks and transplanted into the field. DNA analysis (Southern blotting) demonstrated that these shoots were transformed by pWB139 or by the control construct pWB149 (which does not contain the cry IA (c) gene). Insect feeding trials of walnut embryos grown in vitro and infested with neonatal Indianmeal moth, codling moth or navel orangeworm larvae showed non-significant mortality; confirming insufficient or incomplete expression of ICPs in the transgenic embryos. Therefore, transformation of somatic walnut embryos with the vector pWB139 was ineffective in protecting the embryos from damage by lepidopteran insect larvae.

Infestation rates of codling moth, Cydia pomonella (L.), in commercially produced walnuts were determined over a 3-yr period. Infestation rate averaged 0.245 (0.11-0.46) codling moth per kilogram of walnuts. Only the larval stage (fourth [4%] or fifth instars [96%]) was found infesting walnuts at harvest, indicating the potential for diapausing codling moth larvae to occur in stored walnuts. In laboratory tests in which dehydrated walnuts were infested with nondiapausing larvae, < 1% of first, second, or third instars completed development to adult. Significant survival was observed for fourth and fifth instars. Laboratory trials with diapausing larvae in walnuts stored for 30, 60, or 90 d showed that survival was reduced to an average of only 35%. Tests designed to simulate very high levels of infestation of nondiapausing fifth instars inside bags of walnuts showed that 99% of the larvae remained inside the bag and pupated inside or among the walnuts. Most of these larvae survived to adult, but only 18% of the female moths successfully mated. In further laboratory investigations in which we controlled distances of separation between male and female moths in walnut masses, mating was reduced to 9 or 3% when single pairs of moths were separated by only 0.17 or 0.33 m, respectively. No mating occurred when moths were separated by distances of greater than or equal to 0.67 m. We discuss implications of these data in relationship to biological approaches to quarantine treatments for codling moth in in-shell walnuts.

The nuclear polyhedrosis virus (Af M NPV)isolated from the celery looper, Anagrapha falcifera (Kirby), was tested against six species of production and postharvest lepidopteran pests and four insect cell lines. LC50s in PIB/mm2 were 23.9, 42.2, 47.3, and 510.4, respectively, for raisin moth, Cadra figulilella (Gregson), navel orangeworm, Amyelois transitella (Walker), codling moth, Cydia pomonella (L.), and Indianmeal moth, Plodia interpunctella (Hubner). The almond moth, Cadra cautella (Walker), and the tobacco moth, Ephestia elutella (Hubner), were not susceptible. Histological examination revealed that hypodermis, fat body, tracheal matrix, midgut epithelium, and Malpighian tubules in all four susceptible species were infected. The Trichoplusia ni cell line TN368 and the P. interpunctella cell line PID2 were permissive to Af M NPV as well as to a contaminant cytoplasmic polyhedrosis virus (CPV). However, two navel orangeworm celllines, ATIO and AT20, were permissive to the CPV only. Restriction endonuclease analysis showed that the NPV was unchanged after passage through all of these hosts. Probably of greatest significance is that Af M NPV is the first baculovirus reported to be infectious to the navel orangeworm, a major pest of almonds.

Healthy adult males of Plodia interpunctella (Hubner) were contaminated with a freeze-dried powder formulation of a homologous granulosis virus (IMMGV) when attracted to a source by a pheromone lure. Surface contamination of males was transferred to adult females during copulation and larval food was subsequently contaminated by females during oviposition as well as by other adult activities. Both the F1 and F2 generations of larvae acquired patent infections of IMMGV, resulting in 60% and 50% mortality, respectively. Concurrent introduction of both sexes into the exposure room compared unfavorably with trials in which males were exposed for 24 hr before females were added. Only 16% mortality resulted in the F1 progeny when males and females were exposed simultaneously. Cadaver feeding was found not to be significant. Although incidence of infection was not high when both sexes were introduced simulatenously, the results confirm previous indications that autodissemination may have potential for limited control of P. interpunctella.

The current criterion for insect quarantine treatments in many countries is the probit 9 (i.e., 99.9968% mortality). This concept is used without due consideration for commodity infestation levels or types (multiple or solitary) or survival on the harvested commodity. Historically, probit 9 was based on security levels required for tropical fruit fly species. However, other quarantined insects such as codling moth, Cydia pomonella (L.), infest commodities at very low levels; in addition, inherent survival and reproductive potential on these commodities after harvest is quite low. When infestation levels at harvest alone are considered, several of the currently approved quarantine treatments appear to be extremely conservative based on potential for survival and reproduction. Tables are provided showing the probability of a potential mating pair of codling moths occurring in a shipment based on infestation levels, volumes shipped, as well as mortality required, if any, to maintain survival below this level. Systems approaches to quarantine include development of more quantitative biology data, modification of shipment volume, arrival times, and the distribution of the commodity upon arrival. All of these data suggest that quarantine treatment should be based on survival and that, in a number of situations, treatment is not needed at all.

In laboratory tests, larvae of the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), and the sugarbeet wireworm (SBW), Limonius californicus (Mannerheim), were exposed to the nematodes Steinernema feltiae Filipjev (Mexican strain) (= Neoaplectana carpocapsae) and S. glaseri Steiner in soil. S. feltiae caused significantly higher mortality in SBW larvae than did S. glaseri, but both nematode species were equally effective against CPB larvae. The minimum concentration of S. feltiae for 100% mortality of CPB larvae after 13 days was 157 nematodes/cm(2) of soil, and the LC based on 6-day mortality was 47.5 nematodes/cm(2); in contrast, 100% mortality of SBW larvae was not achieved with even the highest concentration tested, 393 nematodes/cm(2). CPB adults emerging from nematode-contaminated soil were not infected. In field cage tests, S. feltiae applied to the soil surface at the rates of 155 and 310 nematodes/cm(2) soil caused 59% and 71% mortality, respectively, of late-fourth-instar spring-generation CPB, and 28% and 29% mortality, respectively, of SBW. No infection was obtained when larvae of summer generation CPB and SBW were placed in the same cages approximately 6 weeks after nematodes were applied to the soil. Inundative soil applications of S. feltiae, though cost prohibitive at present, were effective in reducing caged CPB and SBW field populations.

Recent advances in genetic engineering have provided the opportunity to induce walnut plants to produce Bacillus thuringiensis Berliner insecticidal crystal protein fragments (ICPFs) for insect control. We studied the effects of two ICPFs CryIA(b) and CrylA(c) previously shown to be encoded by the cryIA (b) and cryIA (c) genes in the B. thuringiensis strains HD-1 and HD-73, respectively. The lethal effects on larvae of codling moth, Cydia pomonella (L.), navel orangeworm, Amyelois transitella (Walker), and the major postharvest pest Indianmeal moth, Plodia interpunctella (Hubner), were investigated. Both proteins were toxic to the three species tested. Indianmeal moth larvae were the most susceptible and navel orangeworm the least; CryIA(b) was generally more toxic to navel orangeworm. Similar relationships resulted when ICPFs were incorporated into the diet. Both ICPFs caused decreased rate of development of navel orangeworm. Effects on pupal weight occurred only at the highest concentration (100 ng/cm 2 ). Neither ICPF affected frequency of mating or fecundity. In addition to the lethal effects, the extended development times observed could have considerable effects on the population dynamics of the navel orangeworm and possibly other species.

Commercially produced, experimental lots of codling moth, Cydia pomonella (L.), granulosis virus (CMGV) formulations were field-tested for control of codling moth larvae on commercial walnuts. Because it has little or no effect on beneficial species and probably would not disturb the control of the walnut aphid, Chromaphis juglandicola (Kaltenbach), by the parasite Trioxys pallidus (Haliday), CMGV was evaluated in 1980-1983 in Kings, Tulare, and Yolo counties in California. In 1980, control of codling moth by CMGV was similar to that of conventional insecticides (e.g., 80%). However, a maximum of 60% control was obtained in 1982. The level of control obtained in 1982 was lower than that suggested by the residue studies. In 1983, control was equivalent to that of a chemical insecticide standard, but control depended upon application timing. Field persistence of the virus was sufficient, to cause 90% larval mortality immediately after application and 80% mortality 5 d later. The addition of several concentrations of skim milk did not increase persistence. It was concluded that timing of CMGV applications to coincide with egg hatch and field persistence of formulations are critical to control of codling moth on walnuts.

Unshelled walnuts were artificially infested with diapausing fifth-instar codling moth, Cydia pomonella (L.), and fumigated with 56 g/m3 methyl bromide for 4 hat 15.6 under a reduced pressure of 100 mm Hg. When 34,959 were treated, 1 larva survived, indicating a survival rate of 2.91larvae/100,000 treated. The upper 95% CL for this survival rate was 13.8 larvae/100,000 treated. When larval survival rates of methyl bromide fumigation tests of a normally applied domestic treatment were combined with survival rates from quarantine vacuum fumigation tests, the 95% CL survival rates were ≤0.4 larva/100,000 treated. No significant differences were found in mortality of larvae among the four walnut cultivars tested, nor was variation in the size of walnuts of each cultivar a significant factor. Because whole walnuts sorbed 79.6% of the methyl bromide applied, a relatively high rate of methyl bromide was required to obtain an efficacious treatment. The treatment was accepted by the Japanese Ministry of Agriculture, Forestry and Fisheries in 1986.

California walnuts were fumigated unshelled with a quarantine treatment to control codling moth, Cydia pomonella (L.). The treatment was done with 56 g/m3 methyl bromide for 4 h at 15.6°C and a chamber pressure of 100 mm Hg. There were no significant differences in organic or inorganic bromide residues regardless of walnut cultivar or size. Inorganic residues were below the established tolerance level of 200 ppm. The ‘Eureka’ cultivar, although not significantly different in its desorption rate of residual methyl bromide, had higher organic residues than the other cultivars tested. Residue levels in treated nut meats showed no significant change in inorganic bromide content over a 25-d period. Accumulated inorganic bromide residues in nut meats fumigated once or twice with a domestic methyl bromide schedule (56 g/m3 for 24 h at 15.6°C) to control field infestation and stored-product insects followed by fumigation with the quarantine treatment did not exceed the established tolerance level. Residual methyl bromide in treated nut meats stored unshelled at 1.7 or 10°C was ≤10 ppb after 70 or 53 d, respectively, whereas those stored at 21 or 32°C had ≤10 ppb after 20 or 14 d, respectively.

LC50 values were determined for six populations (two laboratory and four wild-type) of Indianmeal moth, Plodia interpunctella (Hubner), to peroral inoculations of a granulosis virus. Various biological parameters of the populations were measured to determine similarities or differences among the moth populations tested. A significantly (P 0.05) different among them, but showed them to be more susceptible to the virus and with shorter developmental times compared with the two wild-types discussed above.

Insects are a major cause of post-harvest losses of stored foods and food products the world over. Stored-product insect infestation can occur beginning at harvest and continuing through bulk storage; conversion into processed commodities in food processing facilities; storage in warehouses; transportation in trucks, railcars, and ships; presentation on retail shelves; and ultimately storage in consumer pantries. At each of the locations in the processing and distribution channel there are differences in the nature of the environment, the pest species complex, and the management strategies and tactics used.

The Indian meal moth (IMM, Plodia interpunctella) granulovirus (GV) was first isolated and characterized in 1968 by Arnott and Smith. As a group, the Baculoviridae are considered to be safe. A number have been registered worldwide for control of production pests. In the late 1960s, interest increased in the use of microbial agents to control stored-product pests in durable commodities. Numerous studies have demonstrated efficacy against IMM infestations in raisins, almonds, and walnuts. A production/formulation method for IMM-GV was later patented. The GV was combined with controlled atmospheres for initial disinfestation of IMM populations in unshelled walnuts followed by treatment with the GV for long-term storage. The IMM-GV provided excellent protection of walnuts even with severe IMM pressure for a period of four months. Similar tests were conducted with almonds. After the tests described above were complete, we determined the persistence/efficacy of GV on treated nuts through a period of two years. Immediately after the initial 3-4 months, treated and control walnuts and almonds were frozen to kill any IMM remaining in the commodities. Thereafter, walnuts or almonds were removed and placed in sealed storage at 26.7°C. Samples were removed bimonthly, placed in 3.8-litre jars, infested with approximately 1000 IMM eggs, and incubated at 26.7°C for six weeks. The tests were replicated twice in time. Following incubation the nuts were observed for numbers of IMM adults and damage (pin hole, moderate, and severe). Although efficacy was determined, the primary objective of this study was to determine persistence. Results of these studies showed that IMM-GV would persist for at least two years. Survival and damage to walnuts exceeded that of almonds. Infestation rates used in these tests are unreasonably high and bias the data towards more damage than would likely occur commercially. Our grading procedures are also likely to be more rigid than would be used commercially. The virus is now registered with the United States Environmental Protection Agency and California Environmental Protection Agency.

The granulovirus of Cydia pomonella (L.) (CpGV) offers potential for selective control of codling moth. Two major limitations of CpGV are its narrow host range and lack of persistence in the orchard agroecosystem. The nucleopolyhedroviruses of the alfalfa looper Autographa californica (Speyer) (AcMNPV) and those of the celery looper Anagrapha falcifera (Kirby) (AfMNPV) have broad host ranges. Comparative assays of CpGV, AcMNPV, and AfMNPV against codling moth neonate larvae revealed a 54–93-fold greater susceptibility of codling moth to the granulovirus than to the two nucleopolyhedroviruses based on the LC50 values for each virus. The LC50s for CpGV, AfMNPV, and AcMNPV were 32.7 capsules/mm2, 1.77×103 occlusion bodies (OBs)/mm2, and 3.05×10 3 OBs/mm 2 , respectively. The LT50 determined for AfMNPV using an approximate LC95 of the virus against neonate larvae was 3.6 days. Histological examination of tissues in moribund codling moth larvae that had been treated with AfMNPV revealed the presence of nonoccluded and unenveloped virus rods in midgut tissue. Neither OBs nor signs of infection were detected in other tissues. The activity of AfMNPV was also evaluated in three other tortricid apple pests (obliquebanded leafroller, Choristoneura rosaceana (Harris); Pandemis leafroller, Pandemis pyrusana Kearfott; and the oriental fruit moth, Grapholitha molesta (Busck)). Codling and Oriental fruit moths were significantly more susceptible to AfMNPV than were the two leafroller species.

Naturally occurring entomopathogens are important regulatory factors in insect populations. Many species are employed as biological control agents of insect pests in row and glasshouse crops, orchards, ornamentals, range, turf and lawn, stored products, and forestry and for abatement of pest and vector insects of veterinary and medical importance. The comparison of entomopathogens with conventional chemical pesticides is usually solely from the perspective of their efficacy and cost. In addition to efficacy, the advantages of use of microbial control agents are numerous. These include safety for humans and other nontarget organisms, reduction of pesticide residues in food, preservation of other natural enemies, and increased biodiversity in managed ecosystems. As with predators and parasitoids, there are three basic approaches for use of entomopathogens as microbial control agents: classical biological control, augmentation, and conservation. The use of a virus (Oryctes nonoccluded virus), a fungus (Entomophaga maimaiga), and a nematode (Deladenus siricidicola )a s innoculatively applied biological control agents for the long-term suppression of palm rhinoceros beetle (Oryctes rhinoceros), gypsy moth (Lymantria dispar), and woodwasp (Sirex noctilio), respectively, has been successful. Most examples of microbial control involve inundative application of entomopathogens. The most widely used microbial control agent is the bacterium Bacillus thuringiensis. The discovery of new varieties with activity against Lepidoptera, Coleoptera, and Diptera and their genetic improvement has enhanced the utility of this species. Recent developments in its molecular biology, mode of action, and resistance management are reviewed. Examples of the use, benefits, and limitations of entomopathogenic viruses, bacteria, fungi, nematodes, and protozoa as inundatively applied microbial control agents are presented. Microbial control agents can be effective and serve as alternatives to broad-spectrum chemical insecticides. However, their increased utilization will require (1) increased pathogen virulence and speed of kill; (2) improved pathogen performance under challenging environmental conditions (cool weather, dry conditions, etc.); (3) greater efficiency in their production; (4) improvements in formulation that enable ease of application, increased environmental persistence, and longer shelf life; (5) better understanding of how they will fit into integrated systems and their interaction with the environment and other integrated pest management (IPM) components; (6) greater appreciation of their environmental advantages; and (7) acceptance by growers and the general public. We envision a broader appreciation for the attributes of entomopathogens in the near to distant future and expect to see synergistic combinations of microbial control agents with other technologies. However, if future development is only market driven, there will be considerable delays in the implementation of several microbial control agents that have excellent potential for use in IPM programs.

Insects are a major cause of post-harvest losses of stored foods and food products the world over. They not only consume these commodities but may also damage or contaminate them with insect fragments or whole bodies, feces, webbing, foul smelling metabolic products, and promote growth of a variety of microflora some of which pose serious health hazards (i.e., mycotoxins) to humans and livestock. The pest insects most often associated with stored products are in the orders Coleoptera (beetles), Lepidoptera (i.e., moths), and Psocoptera (book lice). Beetles comprise about 90% of the total number of stored product insect pest species, while moths comprise around 6.4% and book lice 3.6%. We will focus on the moths as the group of pests of interest in this chapter because they are the most amenable to microbial control tactics.

Codling moth, Cydia pomonella (L.), eggs on 'd'Agen' cultivar of plums, Prunus salicina Lindl., were treated with methyl bromide to determine if a quarantine treatment could be developed so that the plums could be exported to Japan from the United States. Small-scale tests consisted of treating codling moth eggs on plums with various doses of methyl bromide at 20 degrees C for 2 h. Small-scale tests showed that 0- to 24-h-old eggs of codling moth on the plums were controlled by doses22.5 g/m3. Because testing showed that 48 g/m3 had no adverse effect on the quality of plums, this dose was chosen for large-scale testing to establish the quarantine dose. Large-scale tests consisted of treating plums at 18.5 degrees C for 2 h using methyl bromide at a dose of 48 mg/liter. Large-scale tests showed that the dose of 48 g/m3 killed all 0- to 24-h-old codling moth eggs exposed on plums in packing cartons without affecting the quality of the plums.

Insecticidal crystal protein fragments (ICPFs) of Bacillus thuringiensis (Bt) encoded by cryIA(c) gene were shown in diet incorporation studies to be lethal to codling moth (CM; Cydia pomonella) the key insect pest for walnut. However transformed walnut tissues expressing cryIA(c) with Bt codon usage patterns and native DNA sequence revealed very low levels of expression in planta. To correct this problem synthetic versions of one of these genes, cryIA(c) was used to transform walnut tissue. A total of 61 individual transgenic embryo lines were obtained. 34% of these lines (21/61) were high expressors (“class A”) demonstrating 80 to 100% mortality of first in star CM larvae and displaying no further larval development. Twelve clones (20%) were designated “class B” and these showed a marked retardation of larval development and a mortality between 40 to 79%. Embryos from the remaining 28 lines designated “class C” (46%). although transformed, were indistinguishable from the control (untransformed embryos) and showed a mortality of 0 to 39%.

When rearing insects for experimental purposes, it is routine practice to treat eggs to assure that microbial pathogens and fungal spores are eliminated. Forty (40) ml of 37% (v/v) formalin (CH20) was placed into each of several one-quart, wide mouth canning jars. CM eggs (12 ± 12-h-old), oviposited on waxed paper, were exposed to CH20 by suspending them inside the jar and then sealing the jar with a ring and lid. Exposure times were 15, 30, 45, 60, 90, or 120 min at 21 ± 1°C. A control group which consisted of eggs suspended over 40 ml of water for 120 min at 21 ± 1°C was included. This test was replicated 3 times with 100 to 200 eggs per replication per time of exposure. To determine the effect on infectivity of CM granulosis virus (CMGV), we also fumigated, in the same manner, small vials (2.5 ml) containing 1 ml of CM agar diet (Bioserv #9370; surface area = 1 cm2) that had been surface-inoculated with 2 × 10s CMGV capsules (dose = 2 × 10" capsules/cm2). A control group of diet cups without CMGV was also fumigated. After fumigation each cup was infested with one CM larva (12 ± 12-h-old) and then held at 27 ± 1°C. Larval mortality was determined after 7 days. The test was replicated 2 times with 25 and 30 larvae, respectively.

We compared three formulations of B. thuringiensis var. kurstaki (Btk) for their insecticidal activity against first-instar OLR. Products tested included: (1) NOVO Laboratories Biobit, a flowable concentrate containing live bacteria with spores and parasporal crystal protein bodies; (2) Abbott Laboratories Dipel 2X, a wettable powder also containing active bacteria with spores and parasporal crystal protein bodies; and (3) Mycogen Corporations MVP Bioinsecticide, a flowable concentrate containing dead bacterial cells which bioencapsulate the Btk proteins. The AI in each of these products is the insecticidal protein, or delta-endotoxin, of Btk. Dose-response was determined by feeding trials exposing first-instar OLR to each product. Serial dilutions of each formulation were made and 10 μ1 of each dilution was applied to the surface of an agar-based diet (Bioserv No. 9370) and then allowed to air dry. One larva was placed in each vial containing ca. 1 ml of surface-contaminated diet (surface area = 1 cm2). Thirty to 60 larvae were observed for each dilution and formulation tested. Doses were expressed in mg AI per sq. cm. Mortality evaluations were made after 3, 5, and 7 days. Response data were analyzed by the POLO-PC PROBIT procedure (LeOra Software, 1987).

Codling moth eggs (12 ± 12-h-old), oviposited on waxed paper, were submerged in water at 50°C or in a 5% solution of ethanol at 50°C for 0, 1, 3, or 5 minutes. The treated eggs were incubated at 27 ± 1°C for 7 days and then evaluated to determine the degree of development obtained. The number of eggs hatched, the number not hatched but showing development (red-ring and black-head stages), and the number not hatched and also undeveloped (white or yellow stages) were recorded.

Studies were conducted to determine the relative increase of nonoccluded virus in two hosts of the nuclear polyhedrosis virus (Ac M NPV) isolated from the alfalfa looper, Autographa californica (Speyer). One host, Heliothis virescens (F.), is highly susceptible, but the other, Heliothis zea (Boddie), is a poor host. Replication of the virus after per os infection was determined by removal and end-point titration of the following tissues: hemolymph, fat body, hypodermis, midgut, Malpighian tubules, and silk glands. We confirmed that replication of the virus in all tissues of H. zea was very low and often difficult to detect. H. virescens tissues, on the other hand, readily supported replication. These data provide information about the ability of Ac M NPV to replicate in the two species and also may provide another basis to discriminate differences in virulence of isolates, variants, and recombinants.

Fumigation of inshell pistachio nuts, Pistacia vera L. ‘Kerman’, with 32 g/m3 methyl bromide (MB) at 15.5°C for 24 h and 16 or 24g/m3 MB at 26.6°C for 24 h resulted in >98.5% mortality of the egg, larval, and pupal stages of the navel orangeworm, Amyelois transitella (Walker). Inshell pistachio nuts fumigated three times with MB using the approved schedule of 56g/m3 for 24 h at ≥ 15.5°C had inorganic bromide residues above the United States tolerance of 200 ppm. Pistachios fumigated repeatedly with 16, 24, or 32 g/m3 MB for 24 h at 15.5°C or 8, 16, or 24g/m3 MB for 24 h at 26.6°C had residues well below the tolerance. Organic and inorganic residues were higher in nuts fumigated at 15.5°C than at 26.6°C. Sorption of MB by the nuts was not significantly different at the two temperatures. By the end of 13 days, desorption of MB by the nuts caused organic bromide residues to decrease below 0.50 ppm for all treatments used.

A total of eight isolates, including variants and natural recombinants of Autographa californica M NPV (Ac M NPY) for which genetic markers or physical maps are known were tested for in vivo virulence, and maintenance of genetic characteristics after infection of Trichoplusia ni (Hubner) (Lepidoptera: Noctuidae) and Heliothis virescens (F.) (Lepidoptera: Noctuidae) larvae. Only one of the isolates was more virulent to T. ni than the wild type; however it was less virulent to H. virescens . One recombinant (AR-66) exhibited low production of polyhedra in TN368 cells, which were used for production of the polyhedra. One variant (Ac M NPY-S1) with an insertion of DNA when compared with the Ac M NPVE2 variant was at least 9.2 times less virulent than the wild isolate. After one in vivo passage of Ac M NPY-S1, a new variant (Ac M NPY-S1a) was detected with a new Eco-R1 fragment of 2.17 Mdal and a corresponding loss of a 1.55-Mdal Eco-R1 fragment found in the parental type.

Damage to raisins, almonds, and walnuts due to feeding by irradiated larvae of the Indianmeal moth, Plodia interpunctella (Hubner), and the navel orangeworm, Amyelois transitella (Walker), was determined. Larvae of both species were reared on bran diet. Age of the larvae when irradiated was 8 d for Indianmeal moth and 13 d for navel orangeworm. Larvae were irradiated with doses ranging from 337 to 497 Gy (gray) before transfer to almonds, walnuts, and raisins. Adult emergence was prevented by all doses. In products containing irradiated larvae, damage was consistently reduced. The percentage of weight losses of almonds infested with untreated Indianmeal moth and navel orangeworm larvae and of walnuts infested with untreated navel orangeworm larvae was significantly higher than the percentage of weight loss of those infested with irradiated larvae. Radiation also improved the overall appearance of the product by reducing webbing and frass. These results indicate that although radiation-induced mortality may be delayed, damage to product quality due to infestation by larvae would be significantly reduced.

Field and laboratory tests were conducted to determine the relationships between a parasitoid, Voria ruralis (Fallen), its host, Trichoplusia ni (Hubner), and a nuclear polyhedrosis virus. By dissection it was observed that up to 100% of the parasitoidism of field-collected T. ni larvae was in hosts infected with nuclear polyhedrosis virus. Thus, accounting for parasitoidism in diseased hosts can significantly alter apparent parasitoidism rates. Laboratory tests confirmed that the parasitoid can survive, develop in, and emerge from diseased larvae. Although histological sections revealed nuclear polyhedrosis virus in the gut lumen of V. ruralis puparia that had fed on diseased hosts, the virus was voided from adults soon after emergence and before oviposition. Tests revealed that V. ruralis does not act as a biological vector, and it acts as a mechanical vector only under very restricted conditions.

Field tests on fall lettuce in southern Arizona during 1970–71 demonstrated the efficacy of a nuclear polyhedrosis Virus isolated from Autographa californica (ACNPV) for control of Trichoplusia ni (Hubner). An application rate of 7.4 × 1011 polyhedra/ha provided adequate control when compared to Bacillus thuringiensis Berliner or chemical insecticides. IMC Shade®, a protectant, did not enhance the effectiveness of the virus as a control agent. Combinations of the ACNPV with reduced application rates of chemical insecticides provided as good a control as chemicals alone. Cabbage looper damage to lettuce heads and weights at harvest was markedly reduced in virus and insecticide-treated plots.

We conducted bioassays and histological and restriction endonuclease studies to determine the susceptibility of Cactoblastis cactorum (Berg) to the nuclear polyhedrosis virus (Ac M NPV) isolated from Autographa californica (Speyer). C. cactorum was found to be at least moderately susceptible to the virus. The virus, reisolated from C. cactorum , was grown in tissue culture cells, and characterized as AcMNPV via gel electrophoresis of restriction endonuclease fragments.

Populations of Trichoplusia ni (Hubner) and Autographa californica (Speyer) were monitored in alfalfa, cotton, and lettuce in the Imperial Valley of California. Both species were found in all three crops with T. ni predominant in cotton and lettuce. In alfalfa T. ni was predominant during most of the year, but A. californica was more abundant during March and April. Populations of T. ni were highest in spring, early summer, and late fall; A. californica populations were highest during spring.

Applications of plant growth regulators in late August effectively terminated the fruiting of cotton and significantly reduced populations of diapausing larvae of Pectinophora gossypiella (Saunders) (by 90% or more) in Arizona during 1972–74. As a result, fewer overwintered moths (39%) emerged the following spring from terminated plots. Applications in mid-, or late August were more effective than applications in early September. Yields were not significantly reduced by most treatments. The most effective materials tested were 2.4-D (amine salt of 2,4-dichlorophelloxy) acetic acid + chlorflurenol (methyl 2-chloro-9-hydroxyfluorene-9-carboxylate) and 2, 4-D + chlormequat ((2-chloroethyl) trimethylammonium chloride).

Dose/responses of certain stages of the codling moth, Cydia pomonella (L.), to methyl bromide (MB) when fumigated at different temperatures (10, 20, or 30°C)and at normal atmospheric (NAP) or reduced (VAC) pressures were estimated. Stages and parameters tested were separated into two distinct groups. Stages in group 1 were less tolerant of MB; this group consisted of nondiapausing fifth in stars fumigated at 20 or 30°C and NAP and diapausing larvae fumigated at 20°C and VAG Stages in group 2 were more tolerant to MB. Group 2 consisted of 1- to 3-day-old eggs fumigated at 20°C and NAP, nondiapausing fifth instars fumigated at 10°Cand NAP, and diapausing larvae fumigated at 20°C and NAP. Lethal doses decreased at higher temperatures. For the same treatment and handling conditions, diapausing larvae (disturbed) were 2.3-fold more tolerant at LD50 or LD50 to MB than nondiapausing larvae. However, fumigation at a reduced pressure of 100 mm Hg significantly increased toxicity of MB. When VAC-fumigated, the response of diapausing larvae (disturbed) at LD50 or LD50 to MB was not significantly different compared with nondiapausing larvae fumigated at NAP. Handling diapausing larvae before fumigation significantly increased their susceptibility to MB, probably due to an increased rate of respiration as a result of the disturbance. Undisturbed diapausing larvae were 1.7-fold more tolerant at LD50 of MB than those disturbed due to handling within 1 day before treatment. At LD50, undisturbed diapausing larvae were 1.7-fold more tolerant than the stages in group 2 and 4.0-fold more tolerant than the stages in group 1.

Larvae of the Indianmeal moth, Plodia interpunctella (Hubner), and the navel orangeworm, Amyelois transitella (Walker), reared individually in small beakers containing artificial diet, were irradiated at doses ranging from 149 to 627 Gy. Adult emergence was prevented by all doses. Only 13% of the oldest lndianmeal moth larvae irradiated at the lowest dose were able to pupate. No pupation occurred at any other treatment level. Higher radiation doses decreased the time required for 100% larval mortality. Longevity of young larvae was reduced compared with that of controls, whereas longevity of older larvae was increased. Weight gain and food consumption of irradiated larvae were reduced, indicating that while radiation-induced larval mortality may be delayed, damage from feeding would be reduced.

Raisins, almonds, and walnuts infested with pupae of the Indianmeal moth, Plodia interpunctella (Hiibner), were treated with gamma radiation (14.4-92.1 krad). Effects of radiation on adult emergence and reproduction were similar in all commodities. Adult emergence was reduced or eliminated at the two highest doses;adults that did emerge were deformed and unable to mate successfully. Females from pupae irradiated with 26.9- 31.9 krad were completely sterile, whereas this dose only partially sterilized males. F, progeny of irradiated parents showed a high degree of sterility; female progeny were lessaffected than males. Presence of partially sterile males after irradiation with 26.9-31.9 krad does not reduce the potential of this dose for eliminating infestations in dried fruits and nuts because irradiated males are unlikely to mate with untreated unmated females within irradiated products.

A freeze-dried preparation of granulosis virus-infected Plodia interpunctella (Hubner) larvae was tested for viral capsule activity, storage life, and the potential of nonviral components for promoting P. interpunctella infestation and development. The LC50 of the formulation was 7.9 × 104 capsules per gram of diet, and loss of activity under typical warehouse conditions appeared to be insignificant commercially. Larval and medium components in the formulation promoted P. interpunctella development in almond meats, almonds within the hull, and processed and unprocessed raisins. This effect was counteracted, however, by a sufficient dose of virus. Formulation components did not preferentially attract ovipositing moths when tested on shelled almonds.

Seasonal population densities of Heliothis virescens (F.) and H. zea (Boddie) in the Imperial Valley of California were determined by pheromone-baited trap catches and by egg and larval densities in alfalfa, cotton, and lettuce. Moth populations were present from March to December; low densities during winter and early summer corresponded to conditions favoring winter diapause and summer estivation, respectively. The relative importance of each host in supporting moth populations varied seasonally. The dominant species in alfalfa was H. zea , whereas H. virescens was dominant in cotton and lettuce. The highest population of H. zea in alfalfa occurred in late spring and late summer, in cotton during early summer, and in lettuce during October. The highest populations of H. virescens in alfalfa were found during October, in cotton from July through October, and in lettuce from October through December. Populations in alfalfa and cotton were greater in commercial fields than pesticide-free untreated fields, whereas lettuce populations were greater in untreated fields. Larval mortality from parasitoids and viruses was infrequent except in alfalfa during late spring and early summer.

Inclusion bodies of a nuclear polyhedrosis virus isolated from the alfalfa looper, Autographa californica (Speyer), applied to cotton in Arizona, decreased the survival of cabbage loopers, Trichoplusia ni (Hubner), 40% throughout the season compared with chemical treatment or no treatment. Moreover, lettuce planted adjacent to the treated cotton had less than 50% fewer cabbage looper eggs than lettuce fields adjacent to chemically treated or untreated cotton fields. No differences in mortality were observed of cabbage looper larvae collected from monocrotophos treated or untreated cotton fields. When a chemical defoliant was applied to cotton, the number of cabbage looper eggs on adjacent lettuce fields was 4 times higher than that next to nondefoliated cotton, probably because the chemically defoliated cotton became unattractive as a host plant or oviposition substrate. Application of monocrotophos to cotton reduced the total number of predators on cotton compared to Moth collections in pheromone baited light traps were extremely high but densities could not be related to a specific treatment.

The trace element rubidium was used to mark adults and ova of Spodoptera exigua (Hubner). Marked ova were produced by moths reared on rubidium-treated alfalfa and cotton and on an artificial diet containing rubidium, by females mated with rubidium-marked males, and by females exposed to plants sprayed with rubidium chloride. Decreasing levels of rubidium were found in ova produced by rubidium-marked females fed a sucrose diet containing potassium in a concentration similar to that found in cotton nectar. Increasing levels of rubidium were found in ova produced by previously unmarked female moths after mating with marked males or after exposure to cotton sprayed with rubidium.

The histopathology was studied and mortality, polyhedra production, total virus production, and nonoccluded virus production were measured in Heliothis zea (Boddie) and H. virescens (F.) (Lepidoptera: Noctuidae) larvae infected with the nuclear polyhedrosis virus isolated from Autographa californica (Speyer) (Lepidoptera: Noctuidae). Both per os and injection routes of inoculation were used, and most tests were purposely biased toward demonstrating viral replication in H. zea. In addition to the great difference in the histopathological responses of the two hosts, large differences in susceptibility via both routes of inoculation were observed. Polyhedra formation, total virus production, and nonoccluded virus production were severely reduced in H. zea as compared with H. virescens larvae. In some cases, these differences exceeded 5,992-fold, even with the tests biased toward obtaining increased responses from H. zea larvae.

Twenty-one plant families (44 species) were tested as suitable hosts for development of the cabbage looper, Trichoplusia ni (Hubner). Of these, 32 species supported development from fourth instar to adult. However, complete development (first instar to adult) occurred in only 15 species. Development from first or fourth instar to adult occurred on 20 hosts not previously reported; thus indicating survival on substitute hosts which could take place in the loss of their preferred cultivated hosts or if these host plants became undesirable.

Heliothis virescens (F.) was more susceptible to a nuclear polyhedrosis Virus (NPV) isolated from Autographa californica (Speyer) than to a commercial formulation of an NPV isolated from Heliothis species . H. zea (Boddie) was less susceptible to the A. californica NPV than H. virescens (ca. 10-fold difference), and therefore, the LD50 and LT50 for H. zea were higher. Viral mortality in the pupal stage was higher among treated insects but did not follow a dose response.