Effect of white grub developmental stage on susceptibility to entomopathogenic nematodes

The pathogenicity of the entomopathogenic nematodes Heterorhabditis bacteriophora Poinar and Steinernema scarabaei Stock & Koppenh?fer against different developmental stages of the Japanese beetle, Popillia japonica Newman, and the oriental beetle, Anomala (=Exomala) orientalis Waterhouse, were studied under laboratory conditions. The efficacy of S. scarabaei did not differ between second and third instars in P. japonica or A. orientalis or between small (young) and large (older) third instars in A. orientalis. However, H. bacteriophora efficacy decreased from first over second to third instar and also from small third instars to large third instars in A. orientalis but did not differ significantly between P. japonica larval stages. Once A. orientalis third instars had purged their intestines in preparation for pupation, no significant mortality by S. scarabaei and H. bacteriophora was observed. In contrast, P. japonica susceptibility to both nematode species gradually decreased from stage to stage from actively feeding third instars to pupae. In two additional experiments, we found no difference in Steinernema glaseri (Steiner) susceptibility between second and third instars of A. orientalis but an increase in S. scarabaei susceptibility from the second to third instar of Asiatic garden beetle, Maladera castanea (Arrow). Our observations combined with those of previous studies with other nematode and white grub species show that nematode efficacy against white grub developmental stages varies with white grub and nematodes species, and no generalization can be made.     

Enhanced toxicity of Bacillus thuringiensis japonensis strain Buibui toxin to oriental beetle and northern masked chafer (Coleoptera: Scarabaeidae) larvae with Bacillus sp. NFD2

Bacillus thuringiensisjaponensis strain Buibui (Btj) has the potential to be an important control agent for pest scarabs. Bioassays using autoclaved and nonautoclaved soil showed there were always lower LC, values associated with nonautoclaved soil. We identified five other bacteria found in the hemolymph of insects killed by Btj and used them in bioassays to see whether we could enhance the control achieved with Btj alone. One bacterium, designated NFD2 and later identified as a Bacillus sp., showed the greatest enhancement of Btj in preliminary experiments and was used in bioassays with Btj versus oriental beetle, Anomala orientalis (Waterhouse), and northern masked chafer, Cyclocephala borealis Arrow (Coleoptera: Scarabaeidae), larvae. This bacterium alone was nontoxic to grubs in bioassays. A combination of this bacterium with Btj in nonautoclaved soil resulted in a significantly lower LC50 value (0.23 microg toxin per g soil) from all other treatments for A. orientalis with one exception; the LC50 where NFD2 was added back into autoclaved soil (0.29 microg toxin per g soil). A combination of this bacterium with Btj in nonautoclaved soil resulted in a significantly lower LC50 value (48.29 microg toxin per g soil) from all other treatments for C. borealis with the exception of the treatment where Bacillus sp. NFD2 was added back to autoclaved soil (96.87 microg toxin per g soil) with Btj. This research shows that other soil bacteria can be used to enhance the toxicity of Btj and possibly other Bts.     

Relationship between the successful infection by entomopathogenic nematodes and the host immune response

Reproduction of entomopathogenic nematodes requires that they escape recognition by a host's immune system or that they have mechanisms to escape encapsulation and melanization. We investigated the immune responses of larvae for the greater wax moth (Galleria mellonella), tobacco hornworm (Manduca sexta), Japanese beetle (Popillia japonica), northern masked chafer (Cyclocephala borealis), oriental beetle (Exomala orientalis) and adult house crickets (Acheta domesticus), challenged with infective juveniles from different species and strains of entomopathogenic nematodes. The in vivo immune responses of hosts were correlated with nematode specificity and survival found by infection assays. In P. japonica, 45% of injected infective juveniles from Steinernema glaseri NC strain survived; whereas the hemocytes from the beetle strongly encapsulated and melanized the Heterorhabditis bacteriophora HP88 strain, S. glaseri FL strain, Steinernema scarabaei and Steinernema feltiae. Overall, H. bacteriophora was intensively melanized in resistant insect species (E. orientalis, P. japonica and C. borealis) and had the least ability to escape the host immune response. Steinernema glaseri NC strain suppressed the immune responses in susceptible hosts (M. sexta, E. orientalis and P. japonica), whereas S. glaseri FL strain was less successful. Using an in vitro assay, we found that hemocytes from G. mellonella, P. japonica, M. sexta and A. domestica recognized both nematode species quickly. However, many S. glaseri in M. sexta and H. bacteriophora in G. mellonella escaped from hemocyte encapsulation by 24h. These data indicate that, while host recognition underlies some of the differences between resistant and susceptible host species, escape from encapsulation following recognition can also allow successful infection. Co-injected surface-coat proteins from S. glaseri did not protect H. bacteriophora in M. sexta but did protect H. bacteriophora in E. orientalis larva; therefore, surface coat proteins do not universally convey host susceptibility. Comparisons of surface coat proteins by native and SDS-PAGE demonstrated different protein compositions between H. bacteriophora and S. glaseri and between the two strains of S. glaseri.     

Distribution and adult activity of Popillia quadriguttata (Coleoptera: Scarabaeidae) on golf courses in Korea

Japanese beetle traps baited with the Popillia japonica Newman (Coleoptera: Scarabaeidae) pheromone lure and a eugenol feeding attractant were placed at five golf courses in Korea to determine how well they work for detecting activity of a closely related species, Popillia quadriguttata (F.) (Coleoptera: Scarabaeidae), a turf pest in Korea. The traps also were used to determine the time of day and time of year that P. quadriguttata is most active. Nineteen scarab species of 13 genera were attracted to the Japanese beetle traps with P. quadriguttata clearly being the most abundant (383 beetles per trap), followed by Adoretus tenuimaculatus Waterhouse (10 per trap), Popilliaflavosellata Fairmaire (seven per trap), Exomala orientalis Waterhouse (four per trap), and Maladera japonica (two per trap). Other scarab species were trapped at a rate of <1.0 per trap. Popillia quadriguttata adults were active over a 5-wk period in late June and early July. At Yongwon Golf Club in 2002, peak adult activity was during the last week of June in visual counts and approximately 1 wk later in the Japanese beetle traps. In Korea, P. quadriguttata adults are most active between 12:00 p.m. and 4:00 p.m. This information should be helpful to golf course superintendents in Korea and to entomologists interested in finding natural enemies of P. quadriguttata to evaluate as potential biocontrol organisms for the very closely related species, the Japanese beetle.     

Host density effects on efficacy of entomopathogenic nematodes against white grub (Coleoptera: Scarabaeidae) species

We tested the effect of host density on entomopathogenic nematode efficacy in 1-L pots with grass and soil. In four experiments, combinations ranged from somewhat resistant hosts (oriental beetle, Anomala orientalis, or northern masked chafer, Cyclocephala borealis, with Heterorhabditis bacteriophora) over more susceptible hosts (Japanese beetle, Popillia japonica, with Steinernema glaseri) to a highly susceptible host (P. japonica and S. scarabaei). In each experiment, four larval densities were exposed to two nematode rates over a 14-day period. A significant effect of host density on nematode efficacy occurred only in the A. orientalis–H. bacteriophora combination, but there was no clear trend in the data. This suggests that an exhaustion of available nematode populations to less lethal levels by high host numbers was counteracted by other factors such as increased chances for nematode-host contact and increased host susceptibility due to stress via reduced food resources and increased aggression between larvae.     

Effect of insecticides on Tiphia vernalis (Hymenoptera: Tiphiidae) oviposition and survival of progeny to cocoon stage when parasitizing Popillia japonica (Coleoptera: Scarabaeidae) larvae

The effect of insecticides on oviposition of Tiphia vernalis Rohwer and subsequent survival of parasitoid progeny to the cocoon stage was determined in the laboratory by using larval Japanese beetle, Popillia japonica Newman, as the host. Insecticides tested were imidacloprid, thiamethoxam, halofenozide, chlorpyrifos, and carbaryl at labeled rates. Female T. vernalis were allowed 2 d to parasitize P. japonica larvae after the parasitoids had received a 4-d exposure to insecticide-treated soil. Another group of female T. vernalis were allowed 2 d to parasitize P. japonica larvae that had been exposed to insecticide-treated soil for 3-4 d. Percentage of parasitism of P. japonica larvae in these trials after exposure of adult parasitoids to carbaryl, chlorpyrifos, halofenozide, or imidacloprid-treated soil (23.3-50.0%) or adult parasitoids to chlorpyrifos, halofenozide, or imidacloprid-treated grubs (33.0-56.7%) was not negatively affected relative to the control treatment (21.7-54.2%). A third group of adult T. vernalis and P. japonica larvae were simultaneously exposed to chlorpyrifos or carbaryl treatments. Percentage parasitism in these trials was lower for T. vernalis adults exposed to the chlorpyrifos and carbaryl (15.0-25.0%) relative to the control (57.5-62.5%) with the exception of one trial with carbaryl (40.0%). However, exposure of the parasitoid and P. japonica to chlorpyrifos 0.5X, carbaryl 0.5X, imidacloprid, halofenozide, or thiamethoxam in several trials resulted in parasitism that was equivalent or greater than (45.0-80.0%) the untreated control (57.5-62.5%). Japanese beetle larval mortality in these trials was greater in the insecticide and parasitoid combination (97.5-100.0%) than with insecticides alone (45.0-100.0%). Percentage of survival of T. vernalis progeny to the cocoon stage was not negatively affected by a 4-d adult parasitoid exposure to carbaryl and chlorpyrifos treated soil (11.7-16.7% versus 18.3% control) or a 2-d exposure to P. japonica-treated larvae (16.7-18.3% versus 28.3% control). However, simultaneous exposure of T. vernalis progeny and P. japonica larvae to chlorpyrifos- and carbaryl-treated soil resulted in no parasitoids surviving to the cocoon stage. Between neonicotinoids, thiamethoxam had more adverse impact on percentage parasitism (52.5%) and survival to the cocoon stage (10.0%) than imidacloprid (80.0 and 32.5%, respectively). Results of this study indicate soil incorporation of imidacloprid and halofenozide had minimal effect on the number of P. japonica larvae parasitized by T. vernalis or survival of T. vernalis progeny to the cocoon stage; therefore, they are more suitable for use with T. vernalis. In contrast, chlorpyrifos, carbaryl, and thiamethoxam lowered the number of T. vernalis progeny surviving to the cocoon stage, and carbaryl and chlorpyrifos reduced the number of P. japonica larvae parasitized. The soil incorporation of insecticides is discussed as one explanation for the minimal effects of some insecticides on T. vernalis.     

Life history and spatial distribution of Oriental beetle (Coleoptera: Scarabaeidae) in golf courses in Korea

Larval and adult activity of the oriental beetle Exomala orientalis (Waterhouse), a pest of turfgrass in Korea, was investigated at four golf clubs in Pusan, Korea, from 1995 to 1999. Adult emergence was first observed on the greens in late May with peak activity occurring 2 wk later. During the day, E. orientalis adults were most active between 1800 and 2200 hours. First instars were found mostly in early July, second instars mostly in late July, and third instars from August to April. The density of larvae in fixed plots decreased steadily from the time of egg laying to pupation: 667/m3 on 26 July, 267/m3 on 29 August, and 122/m3 on 2 October 1997. All the observed E. orientalis completed one generation per year. Adult females were observed feeding on flowers of a late-blooming variety of Japanese chestnut (Castanea crenata Sieb & Zucc). E. orientalis larval densities were higher in greens with Japanese chestnut nearby, and where magpie, Pica pica sericea (Gould), feeding was observed. More E. orientalis adults emerged from the right, left, and back of greens than from the front or middle. The intensity of emergence was inversely proportional to the amount of golfer traffic on various parts of the green. Counting emergence holes may be a way that golf course superintendents can predict which greens and tees are most likely to be damaged from E. orientalis larvae without destructive sampling.     

Management of early-instar Japanese beetle (Coleoptera: Searabaeidae) in field-grown nursery crops.

Numerous field studies were conducted in commercial nurseries in Tennessee from 1996 through 1999 to evaluate chemical and biological treatments, application timing and rates, and method of application for control of early instars of Japanese beetle, Popillia japonica Newman. Insecticide treatments included bifenthrin, bendiocarb, chlorpyrifos, carbaryl, fipronil, halofenozide, imidacloprid, permethrin, tefluthrin, thiamethoxam, and trichlorfon. Biological treatments included entomopathogenic nematodes (Heterorhabditis bacteriophora HP88 or H. marelatus, Bacillus thuringiensis Berliner subspecies japonensis Buibui strain, and Beauveria bassiana (Balsamo) Vuillemin. All treatments were applied on the soil surface or injected into the soil around the base of each tree. Tree type and size varied among and within tests, however, the sampling unit (61-cm-diameter root ball) remained the same throughout all tests. The biological treatments provided poor-to-moderate control (0-75%) of Japanese beetle larvae. Imidacloprid was the most frequently evaluated insecticide and achieved 91-100, 87-100, 83-100, and 41-100% control with applications in May, June, July, and August, respectively. Halofenozide treatments were not significantly different from imidacloprid treatments with one exception. Halofenozide provided 60-87, 85-100, and 82-92 control with applications made in June, July, and August, respectively. Fipronil and thiamethoxam were evaluated to a lesser extent but both performed similarly to imidacloprid. Most other insecticide treatments were less successful in reducing numbers of Japanese beetle larvae and with few exceptions achieved <50% control.     

Differences in penetration routes and establishment rates of four entomopathogenic nematode species into four white grub species

We compared the penetration of the entomopathogenic nematodes Steinernema scarabaei (AMK001 strain), S. glaseri (NC1 strain), Heterorhabditis zealandica (X1 strain), and H. bacteriophora (GPS11 strain) into third-instars of the scarabs Popillia japonica, Anomala orientalis, Cyclocephala borealis, and Rhizotrogus majalis. When larvae were exposed to nematodes for 6-72 h larval mortality and nematode establishment rate and occasionally speed of kill often showed the same pattern within nematode-white grub combinations. But no two nematodes or white grub species had the same pattern for these observations for all white grub or nematode species, respectively. Mortality, establishment, and speed of kill followed a similar pattern for H. zealandica, S. glaseri, and S. scarabaei, but there was no clear relationship for H. bacteriophora. Significant nematode establishment was only observed after at least 48 h exposure in most nematode-white grub combinations. Faster establishment was observed only for H. zealandica in A. orientalis and R. majalis (after 24 h) and for S. scarabaei in P. japonica and R. majalis (after 12 h). Nematode establishment after 72 h in the different scarab species was generally low for S. glaseri (<1.5%) and H. bacteriophora (<3%), higher for H. zealandica (2-5%), and the highest for S. scarabaei (1-14%). However, in another experiment establishment was generally higher after 96h exposure. Nematode penetration sites were determined by comparing nematode establishment in larvae with mouth, anus, mouth+anus, or none sealed with glue. The trends for each nematode species were very similar in the different white grub species. H. zealandica and H. bacteriophora showed excellent cuticular penetration ability but may also penetrate through mouth and/or anus. S. glaseri also penetrated through the cuticle but lower establishment in larvae with mouth or mouth+anus sealed suggested that the mouth is an important penetration site. S. scarabaei showed a preference for the mouth as a penetration site, but it showed some cuticular penetration ability and may also use the anus as a penetration site. The methodology used cannot exclude that cuticular penetration also included penetration through the spiracles. To fully understand the effect of nematode and white grub species on nematode virulence, future studies will have to compare host immune response to the penetrating IJs and the role of the symbiotic bacteria in these interactions.     

取食不同品种棉花的棉蚜对龟纹瓢虫功能反应的影响

本文以GK12、33B和SGK3213种转基因棉和对应的3种常规棉泗棉3号、33及石远321为材料,通过室内测定,研究了取食转基因棉的棉蚜却h／sgossypiiGlover对龟纹瓢虫Propylaea japonica Thunberg1—4龄幼虫功能反应的影响。结果指出,龟纹瓢虫对来自转基因棉田蚜虫的捕食量大多高于常规棉田,且捕食量随猎物密度的增加而增大。在1、2龄幼虫期,龟纹瓢虫取食转双价基因棉田蚜虫的数量多高于取食转单价基因棉田的蚜虫数量,但是在3、4龄幼虫时,龟纹瓢虫取食转双价棉田蚜虫的数量多低于取食转单价棉田的蚜虫数量。龟纹瓢虫对来自转基因棉田棉蚜的捕食功能反应符合HollingII型。龟纹瓢虫1龄幼虫取食GK12和SGK3212个转基因棉田蚜虫的最大日捕食量大于对照,处理时间短于对照;龟纹瓢虫P．japonica2、3龄幼虫取食33B和SGK3212个转基因棉田蚜虫A．gossypii的日最大捕食量大于对照,处理时间短于对照;4龄时取食转基因棉田蚜虫的数量都大于对照,处理时间均短于对照,其中取食转双价基因棉SGK321棉田蚜虫的日最大捕食量小于取食单伊棉田蚜虫的日最大捕食量。     

Damage loss assessment and control of the cereal leaf beetle (Coleoptera: Chrysomelidae) in winter wheat

Cereal leaf beetle, Oulema melanopus (L.), invaded northern Alabama and Georgia more than a decade ago and since has become an economic pest of winter wheat and other cereal crops in the southeastern United States. A series of trials was conducted beginning in 1995 to determine optimal rate and timing of applications of selected foliar insecticides for managing cereal leaf beetle in soft red winter wheat. These trials, cage studies with larvae, and a manual defoliation experiment were used to provide information on cereal leafbeetle yield loss relationships and to develop economic decision rules for cereal leaf beetle in soft red winter wheat. Malathion, methomyl, carbaryl, and spinosad effectively controlled larval infestations when treatments were applied after most eggs had hatched. Encapsulated endotoxin of Bacillus thuringiensis, methyl parathion, and disulfoton applied at the lowest labeled rates were not effective treatments. Organophosphate insecticides generally were not effective when applied before most eggs had hatched. The most effective treatments were the low rates of lambda cyhalothrin when applied early while adults were still laying eggs and before or near 50% egg hatch. These early applications applied at or before spike emergence virtually eliminated cereal leaf beetle injury. The manual defoliation study demonstrated that defoliation before spike emergence has greater impact on grain yield and yield components than defoliation after spike emergence. Furthermore, flag leaf defoliation causes more damage than injury to lower leaves. Grain test weight and kernel weight were not affected by larval injury in most trials. Regression of larval numbers and yield losses calculated a yield loss of 12.65% or 459 kg/ha per larva per stem, which at current application costs suggested an economic threshold of 0.4 larvae per stem during the spike emergence to anthesis stages.     

Does host range influence susceptibility of herbivorous insects to non-host plant proteinase inhibitors?

We examined the influence of proteinase inhibitors on digestive enzymes and development of oriental beetle,Exomala orientalis Waterhouse, European chafer,Rhizotrogus majalis (Razoumowsky),Phyllophaga white grub,Phyllophaga anxia (LeConte), cranberry root grub,Lichnanthe vulpina (Hentz), Japanese beetle,Popillia japonica Newman, Asiatic garden beetle, Maladera castanea (Arrow) (Coleoptera: Scarabaeidae), and the black cutworm,Agrotis ipsilon (Rottemburg) (Lepidoptera: Noctuidae). We demonstrated that all species within our test group had alkaline midguts that contained proteinase activity that could be inhibited,in vitro with serine proteinase inhibitors. Our data suggests that host range may influence the susceptibility to non-host inhibitors. Chronic ingestion of the serine proteinase inhibitor, Kunitz-soybean trypsin inhibitor (STI), significantly reduced proteolytic activityin vivo in those species with relatively specialized feeding habits (i.e., cranberry root grub, Japanese beetle, Asiatic garden beetle, and black cutworm). Chronic ingestion of STI also resulted in reduced larval growth and delayed pupation for black cutworm, and elevated larval mortality for Japanese beetle. However, chronic ingestion of STI did not influence larval survival for those species with relatively generalized feeding habits (i.e., oriental beetle, European chafer). Based on these results, we propose mechanistically-based criteria for selecting proteinase inhibitors for phytochemical defense against herbivorous insects.     

Drench treatments for management of larval Japanese beetle (Coleoptera: Scarabaeidae) in field-grown balled and burlapped nursery plants

Insecticide drenches were applied to postharvest field-grown nursery plants harvested as 60-cm-diameter balled and burlapped (B&B) root balls for controlling third instars of Japanese beetle, Popillia japonica Newman (Coleoptera: Scarabaeidae). Bifenthrin, chlorpyrifos, lambda-cyhalothrin, and thiamethoxam were drench-applied in fall and spring tests at volumes of runoff (1X; approximately equal 2.57 liters per drench per root ball) or twice runoff (2X). Tests also examined consecutive drenches (two, four, or six) and B&B rotation between drenches. Fall-applied drenches did not meet the Domestic Japanese Beetle Harmonization Plan (DJHP) standards of < or =1 grub and ranged from 0 to 90% control. However, most fall-applied drenches significantly reduced grub numbers relative to the untreated root balls. Spring-applied drenches were more effective than fall drenches: chlorpyrifos treatments gave 94-100% control, whereas other spring-applied treatments were less consistent, including thiamethoxam (83-100% control) and bifenthrin (61-100% control). Lambda-cyhalothrin was not effective. A higher drench volume (2X) did not significantly improve treatment efficacy; however, grub numbers decreased as the number of drenches increased for fall-applied chlorpyrifos and thiamethoxam and spring-applied bifenthrin. Rotation of root balls significantly reduced grub numbers compared with nonrotated treatments for fall-applied chlorpyrifos (six drenches) and bifenthrin (two or six drenches), but these treatments did not meet DJHP standards. The study indicates chlorpyrifos, bifenthrin, and thiamethoxam drenches can control Japanese beetle in the spring and may provide a new postharvest option to certify B&B plants for Japanese beetle.     

SPLAT-OrB reveals competitive attraction as a mechanism of mating disruption in oriental beetle (Coleoptera: Scarabaeidae)

This study compared the efficacy of SPLAT-OrB, a new pheromone formulation for oriental beetle mating disruption that can be mechanically applied, with hand-applied plastic dispensers in commercial blueberry fields. Both formulations were tested at 2.5 and 5 g of the major sex pheromone component (Z)-7-tetradecen-2-one per hectare, and evaluated by measuring trap shut-down, mating success of caged females, and the number of grubs in sentinel blueberry pots baited with tethered females. All pheromone-treated plots had fewer male captures in traps and lower mating success of caged females compared with untreated plots. SPLAT-OrB, and plastic dispensers at the higher rate, also reduced the number of grubs in sentinel pots. To understand the mechanism of mating disruption in oriental beetle, males were observed approaching the pheromone sources in disrupted plots. In addition, male oriental beetle captures were quantified in plots treated with varying SPLAT-OrB dollop densities per ha. Consistent with predictions for competitive attraction, field observations revealed males approaching the pheromone source and male captures decreasing concavely with increasing dollop density. In a mark-release-recapture study, male oriental beetles responded to SPLAT-OrB dollops and plastic dispensers at least 60 m from the source. Additionally, SPLAT-OrB emitted pheromone that was attractive to male oriental beetles for >5 wk; however, emission rates and attraction dropped rapidly during the first 3-4 wk. This study demonstrates the feasibility of using SPLAT-OrB for oriental beetle mating disruption as an alternative to hand-applied plastic dispensers, and conclusively reveals that a principal mechanism is the competitive attraction of males.     

Interaction between halofenozide and the entomopathogenic nematode Heterorhabditis marelatus for control of Japanese beetle (Coleoptera: Scarabaeidae) larvae

Japanese beetle, Popillia japonica Newman is a major pest of turf and ornamentals. Laboratory bioassays were conducted to evaluate the potential interactions between a biological control agent, Heterorhabditis marelatus (Nematoda: Heterorhabditidae) IN strain and the insecticide halofenozide against both overwintered and nonoverwintered 3rd instars of Japanese beetle. Treatments consisted of all combinations of 2 rates of halofenozid with H. marelatus nematodes Imidacloprid was used as a standard. Percentage larval mortality was evaluated at 7, 14, and 21 d after treatment. No deleterious effects were observed. The nematode treatments generally produced significantly greater larval mortality relative to both chemical treatments. Twenty-one days after treatment, both rates of nematodes resulted in 100% mortality, whereas insecticide treatments did not surpass 60% mortality. No synergism was detected in any of the combination treatments. There were no significant differences in nematode reproduction in larvae exposed to halofenozide and nematodes versus larvae exposed to only nematodes.     

Dispersal of Steinernema glaseri (Nematoda: Steinernematidae) in adult Japanese beetles, Popillia japonica (Coleoptera: Scarabaeidae)

Japanese beetle adults, Popillia japonica, can become infected with and disperse the entomopathogenic nematode, Steinernema glaseri, under laboratory and field conditions. After a 24-h exposure to 10 000 infective juveniles/20 adult beetles, 45% of the beetles died within 4 days post-treatment, but only 59% of these were infected with the nematode. Corresponding control mortality was 6.5%. An average of 238 infective juveniles were produced/beetle. Beetles exposed to 4000 and 10 000 infectives/10 adults carried with them an average of 17 and 59 infectives/adult on external body surfaces respectively. When beetles that had been exposed to 4000 infectives/20 adults were transferred to, and held in, cages containing soil for 2 weeks, up to 89% of the adults died, as did 74% of the P. japonica larvae that were subsequently placed in the cages. When adults that had been exposed to 50 000 infectives/250 beetles in moist sand for 16 h were released into screened cages in the field at soil temperatures of over 25 C, the soil beneath 83% of the cages tested positive for the nematode, using Galleria mellonella larvae as bait, 2 weeks after releasing the beetles. No nematodes were detected in control plots. The potential of infected adult P. japonica for dispersing S. glaseri by flight was investigated by exposing adults to 50 000 infectives/250 beetles, marking and releasing them in the field and recapturing them in lure-baited Japanese beetle traps. Less than 1% of the treated beetles were recaptured, but 33% of these had one or more nematodes in their hemocoels. Accordingly, this approach does not appear to be feasible for large-scale augmentation and dispersal of the nematode using currently developed methods of infection. If improvements in mass-inoculation methods can be made that enable a rapid high percentage of infection while still permitting flight, this concept could be employed to establish new foci of infection or for the introduction of other species of nematodes.     

Identification of two entomopathogenic bacteria from a nematode pathogenic to the Oriental beetle, Blitopertha orientalis

A pathogenic nematode, Butlerius sp., was isolated from Oriental beetle, Blitopertha orientalis. The infective juveniles exhibited dose- as well as time-dependent entomopathogenicity on the larvae of B. orientalis. Two bacterial species, Providencia vermicola (KACC 91278) and Flavobacterium sp. (KACC 91279), were isolated from the infective juveniles and identified. P. vermicola outnumbered Flavobacterium sp. in the nematode host, in which the colony density of P. vermicola was found to be 21 times higher than that of Flavobacterium sp. However, when the two bacterial species were cocultured in culture media without the nematode host, they showed similar growth rates. Both bacteria induced significant entomopathogenicity against Spodoptera exigua larvae infesting economically important vegetable crops, where P. vermicola was more potent than Flavobacterium sp.     

The nematode Pristionchus pacificus (Nematoda: Diplogastridae) is associated with the oriental beetle Exomala orientalis (Coleoptera: Scarabaeidae) in Japan

Pristionchus pacificus has been developed as a nematode satellite organism in evolutionary developmental biology. Detailed studies of vulva development revealed multiple differences in genetic and molecular control in P. pacificus compared to the model organism Caenorhabditis elegans. To place evolutionary developmental biology in a comprehensive evolutionary context, such studies have to be complemented with ecology. In recent field studies in western Europe and eastern North America we found 11 Pristionchus species that are closely associated with scarab beetles and the Colorado potato beetle. However, P. pacificus was not commonly found in association with scarab beetles in these studies. Here, we describe the results of a similar survey of scarab beetles in Japan. Pristionchus pacificus was the most common Pristionchus species on scarab beetles in Japan, with 40 out of 43 (93%) isolates. The other Pristionchus isolates represent three novel species, which we refer to as Pristionchus sp. 11, Pristionchus sp. 14, and Pristionchus sp. 15. Thirty-seven of the established P. pacificus strains were found on the oriental beetle Exomala orientalis. Laboratory studies with the sex pheromone (Z)-7-tetradecen-2-one of the oriental beetle revealed that P. pacificus shows strong olfactory attraction to the beetle's sex pheromone, which provides a potential mechanism for the recognition and interaction of P. pacificus and E. orientalis. Together, this study identifies P. pacificus as the most common Pristionchus nematode in field studies in Japan, identifies E. orientalis as an important host species, and provides the basis for the ecology of P. pacificus.     

Two new bacterial pathogens of Colorado potato beetle Coleoptera: Chrysomelidae)

Other than Bacillus thuringiensis Berliner, few bacteria are lethal to the Colorado potato beetle (Leptinotarsa decemlineata [Say]), a major pest of potatoes and eggplant. Expanded use of biologicals for the control of Colorado potato beetle will improve resistance management, reduce pesticide use, and produce novel compounds for potential use in transgenic plants. Using freeze-dried, rehydrated artificial diet in pellet form to screen bacteria lethal to other insects, we determined that strains of Photorhabdus luminescens killed Colorado potato beetle larvae. The LC50 for second instar larvae of strain HM5-1 was 6.4 +/- 1.87 x 10(7) cells per diet pellet. In an attempt to find additional naturally occurring P. luminescens strains toxic to Colorado potato beetle larvae, we recovered, from soil, bacteria that produced a purple pigment. This bacterial strain, identified as Chromobacterium sp. by 16S ribosomal DNA sequencing, was also toxic to Colorado potato beetle larvae within 3 d. The LC50 for second instar larvae for these bacteria was 2.0 +/- 0.79 x 10(8) cells per diet pellet, while the LC50 was approximately 1 log lower for third instar larvae. P. luminescens appeared to kill by means of a protein toxin that may be similar to the described lepidopteran protein toxins. Based on the heat and acid stability, the toxin or toxins that Chromobacterium sp. produces, while not fully characterized, do not appear to be typical proteins. In both bacteria, the toxins are made after exponential growth ceases.     

Solid state fermentation of broiler litter for production of biocontrol agents

Several varieties of heat-sterilized broiler litter with 60% (wet basis, wb) moisture content were substrate in solid-state fermentations to produce biocontrol agents. Litter varieties included litter produced by one flock of broilers from medicated and non-medicated controlled rations, and litter produced by two flocks and four flocks on a single application of bedding material from medicated commercial sources. Litter preparations were inoculated with monocultures of Bacillus thuringiensis serovar japonensis strain Buibui, a pathogen of Japanese beetle larvae (Popillia japonica), or Pseudomonas fluorescens 2-79. B. thuringiensis did not grow in unextracted 1-flock litter nor in water extracted litter, but grew in methanol extracted litter to 5 x 10(10) cell forming units (CFU)/g litter (dry weight, dw) and a spore count of 1 x 10(10) CFU/g litter (dw). B. thuringiensis also grew in unprocessed 2-flock and 4-flock litter, achieving cell counts of 3 x 10(9) and 1 x 10(9) CFU/g litter (dw), respectively, and spore counts of 1 x 10(9) CFU/g litter (dw). P. fluorescens grew in medicated 1-flock litter with no extraction to a cell density greater than 4 x 10(11) CFU/g litter (dw). Bioassays in soil containing over 0.5% (db) litter fermented with B. thuringiensis resulted in over 90% mortality in 21 days for first instars of Japanese beetle when compared to a control treatment using compost without fermented litter. The investigations demonstrate that bacterial biocontrol agents produced via solid substrate fermentations using broiler poultry litter have potential in biocontrol applications in the soil environment.