Transmission of Pinewood Nematode Through Feeding Wounds of Monochamus carolinensis (Coleoptera: Cerambycidae)

Transmission of pinewood nematode, Bursaphelenchus xylophilus, to mature, field grown Scots pines through feeding wounds of Monochamus carolinensis was investigated by caging nematode-infested beetles on pine branches for 24 hours. Nematodes were transmitted to 31 of 64 branches. Frequency of successful transmission was independent of the sex of the beetle but dependent upon beetle age. Transmission frequencies were highest for beetles 2, 3, and 4 weeks after emergence as adults. The number of nematodes transmitted per branch was low and did not differ between beetle sexes or among beetle age categories. The number of nematodes extracted per branch was correlated with the number of nematodes carried per beetle but was not correlated with the feeding area on the branch.     

Susceptibility of the Colorado Potato Beetle and the Sugarbeet Wireworm to Steinernema feltiae and S. glaseri

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/cm² of soil, and the LC₅₀ based on 6-day mortality was 47.5 nematodes/cm²; in contrast, 100% mortality of SBW larvae was not achieved with even the highest concentration tested, 393 nematodes/cm². 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/cm² 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.     

Horizontal Transmission of Bursaphelenchus xylophilus between Sexes of Monochamus alternatus

Four experiments were conducted using nematode-infested and nematode-free adults of the cerambycid beetle, Monochamus alternatus, to determine horizontal transmission pathways of Bursaphelenchus xylophilus. When nematode-infested beetles of one sex and nematode-free beetles of the opposite sex were paired in containers for 48 or 72 hours, the number of nematodes carried by nematode-free beetles tended to increase with increased number of nematodes carried by nematode-infested beetles. The nematodes acquired by "nematode-free" beetles could be transmitted to pine. A female beetle that received 13 nematodes from a male transmitted one nematode to a Pinus densiflora bolt via an oviposition wound. When the nematode-infested and nematode-free beetles were observed continuously, it was observed that the number of nematodes carried by nematode-free beetles at the end of the first sexual mounting increased as the number of nematodes carried by nematode-infested beetles just before mounting increased. The number of nematodes transferred to nematode-free beetles was positively related to duration time of mounting. There was no difference in transmission efficacy between male-to-female transmission and female-to-male transmission. The horizontal transmission pathways are discussed relative to the persistence of B. xylophilus in resistant pine forests and the control of pine wilt disease.     

Transmission of the Pinewood Nematode, Bursaphelenchus xylophilus,to Slash Pine Trees and Log Bolts by a Cerambycid Beetle,Monochamus titillator, in Florida

Field-collected adults of the southern pine sawyer, Monochamus titillator (F.) (Coleoptera: Cerambycidae), naturally infested with fourth-stage juveniles (dauerlarvae) of the pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer, 1934) Nickle, 1970, were maturation fed on excised shoots of typical slash pine, Pinus elliottii Engelm. var elliottii, for 21 days. During August 1981, a male and female adult beetle were held in a sleeve cage placed on the terminal of a side branch of each of seven replicate, healthy 10-year-old slash pine trees. All seven branch terminals showed evidence of beetle feeding on the bark after 1 week, and pinewood nematodes were present in wood samples taken near these feeding sites. Four of the seven trees showed wilt symptoms in 4-6 weeks and died about 9 weeks after beetle feeding. Pinewood nematodes were recovered from the roots and trunks of the dead trees. Each of seven replicate slash pine log bolts was enclosed in a jar with a pair of the same beetles used in the sleeve cages. After 1 week, wood underlying beetle oviposition sites in the bark of all replicate log bolts was infested with the pinewood nematode.     

Transmission of Bursaphelenchus xylophilus through Oviposition Wounds of Monochamm carolinensis (Coleoptera: Cerambycidae)

Transmission of pinewood nematode through Monochamus carolinensis oviposition wounds was documented. Nematode transmission was measured as the average number of nematodes isolated per oviposition wound excavated and also as the percentage of oviposition wounds from which nematodes were isolated. The influence of three factors that might affect nematode transmission was investigated: age of the beetle vector, number of nematodes carried per beetle, and egg deposition in the oviposition wound. Only the number of nematodes carried by the beetle was found to have a significant effect on transmission. Nematodes were transmitted more frequently and in slightly greater numbers by beetles carrying more nematodes. The influence of pinewood on nematode exit from beetles were investigated by comparing nematode exit from beetles placed over pine chips with those placed over distilled water. Nematodes exited in greater numbers and at a higher frequency from beetles over pine chips than from beetles over distilled water. Apparently, the nematodes are able to detect a factor from the pine chips that promotes their exit from the beetles.     

Factors influencing parasitism of adult Japanese beetles,Polillia japonica (Col.: Scarabaeidae) by entomopathogenic nematodes

Several factors that influence the activity of steinernematid and heterorhabditid nematodes against adult Japanese beetles were examined in the laboratory. The effect of nematode concentration on mortality of adult beetles was evaluated using a Petri plate bioassay. The adults were exposed to 1,000 to 10,000 infective stage juveniles (J3) ofSteinernema glaseri per 10 beetles with or without food for 24 hr after which they were held with food for an additional 6 days. The LC50s for males with and without food during exposure were 3,435 and 2,854 J3s/10 adults, respectively. The LC50s for mixtures of males and females with and without food were 5,228 and 1,762 J3s/10 adults respectively. Although mortality occurred during and shortly after exposure, significant additional mortality was observed 1–4 days following exposure. Exposure of males and females with food to 10,000 J3s/10 adults for 6, 12, 18 or 24 hr resulted in 47, 58, 72 and 77% mortality, respectively. Comparative activity ofS. glaseri, S. carpocapsae (All strain),S. feltiae (Biosys experimental cold adapted strain=bibionis),S. feltiae (Biosys experimental strain 27),Heterorhabditis bacteriophora, andHeterorhabditis sp. (Terceiran isolate) was evaluated against adult Japanese beetles using a 24 hr exposure to 8,000 J3s/10 adults. The most virulent species wereS. glaseri, S. feltiae (=bibionis), the Terceiran isolate ofHeterorhabditis andS. carpocapsae producing 55, 44, 36 and 34% mortality respectively. Our results indicate that adult Japanese beetles infected with entomopathogenic nematodes could serve as a mechanism for nematode dispersal.     

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.     

Pathogens of bark beetles (Curculionidae: Scolytinae) and other beetles in Bulgaria

The presence and diversity of beetle pathogens associated with different tree species in Bulgaria was investigated. In total, 818 specimens belonging to 22 beetle species were examined. Pathogens occurred in 9 host species. The infections were found in the gut (virus, nematodes, protozoans, microsporidia) and haemolymph (nematodes) of the infected insects. The following pathogen species: ItEPV, Beauveria bassiana, B. brongniartii, Isaria farinosa, Gregarina typographi, Gregarina spp., Chytridiopsis typographi, Chytridiopsis sp., and nematodes were revealed. Insects with mycoses were mummified and filled up with fungal structures, formed pseudosclerotium. Most pathogens were established in bark beetles (Scolytinae). For the first time, the fungus B. bassiana was reported in Tomicus piniperda, Orthotomicus longicollis, O. erosus, X. spinole, Taphrorychus villifrons and Phylobius sp. in Bulgaria. We also present the first records of gregarines in O. longicollis, Acanthocinus aedilis, Rhagium inquisitor, Pyrochroa coccinea, and of the microsporidium Chytridiopsis sp. in O. longicollis.     

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.     

The potential use of entomopathogenic nematodesagainst Typhaea stercorea

Four entomopathogenic nematode species, Steinernema carpocapsae, S. feltiae, Heterorhabditis bacteriophoraand H. megidis, were tested in a petri dish assay against larvae and adults of the hairy fungus beetle Typhaea stercorea. In general, adults were less susceptible than larvae and the LC₅₀ decreased with the duration of the exposure to nematodes. S. carpocapsae was the most effective species against adult beetles (LC₅₀ after 96 hours exposure =67 nematodes/adult). Against larvae S.carpocapsae and H. megidis were comparablyeffective with an LC₅₀ of 30 and 55nematodes/larvae, respectively. S. carpocapsaewas tested at 70 and 100% RH against adults in baits of either chicken feed or crushed wheat, both supplemented with horticultural capillary matting pieces in order to obtain a wet weight of 50–60%. At70% RH no significant effect of the nematodes was obtained due to desiccation of the bait. In chickenfeed at 100% RH the mortality reached 80% with 500nematodes/adult. In wheat significant mortality was obtained only at 5000 nematodes/adult. Heavy growth of mould probably limited the nematode infection. When the bait was used in tube traps, desiccation and growth of mould was prevented, but nematode efficacy dropped to 4.4% in the traps and 12% in the surrounding litter. This revised version was published online in July 2006 with corrections to the Cover Date.     

Lethal and sublethal effects of Iranian isolates of Steinernema feltiae and Heterorhabditis bacteriophora on the Colorado potato beetle, Leptinotarsa decemlineata

To determine the LC₅₀ values of two entomopathogenic nematodes against Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae) prepupae, different concentrations of the nematodes were tested in soil. Because of the different temperature requirements of the two nematode species, bioassay experiments were conducted at 20 ± 1°C and 27 ± 2°C for Steinernema feltiae Filipjev (Rhabditida: Steinernematidae) and Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae), respectively. Both the isolates were effective against L. decemlineata. LC₅₀ values of H. bacteriophora against progeny of field-collected adults and laboratory-reared adults were estimated as 8.5 and 7.6 IJ per prepupa, respectively. For S. feltiae the value was calculated as 51.2 IJ per prepupa against offspring of laboratory-reared adults of L. decemlineata only. Cellular encapsulation of both nematode species was observed. Sublethal nematode concentrations caused wing deformation and delayed metamorphosis which may affect Colorado potato beetle adult fitness.     

Efficacy of soil treatments of entomopathogenic nematodes against the larvae, pupae and adults of Tuta absoluta and their interaction with the insecticides used against this insect

The efficacy of soil treatments of three native entomopathogenic nematodes (Steinernema carpocapsae, S. feltiae and Heterorhabditis bacteriophora) against Tuta absoluta larvae, pupae and adults was determined under laboratory conditions. The effect of three insecticides commonly used against T. absoluta, in the survival, infectivity and reproduction of these nematode strains was also evaluated. When dropped into soil to pupate, soil application of nematodes resulted in a high mortality of larvae: 100, 52.3 and 96.7 % efficacy for S. carpocapsae, S. feltiae and H. bacteriophora respectively. No mortality of pupae was observed and mortality of adults emerging from soil was 79.1 % for S. carpocapsae and 0.5 % for S. feltiae. The insecticides tested had a negligible effect on nematode survival, infectivity and reproduction. No sublethal effects were observed. Infective juveniles that survived to insecticide exposition were able to infect Galleria larvae with no significant differences from the control. The Galleria larvae affected by the three insecticides tested served as suitable hosts for the infection and reproduction of the nematodes. These results suggest that larvae of T. absoluta, falling from leaves following insecticide application, could be suitable hosts for nematodes, thereby increasing their concentration and persistence in the soil.     

Susceptibility of the Carrot Weevil (Coleoptera: Curculionidae) to Steinernema feltiae,S. bibionis,and Heterorhabditis heliothidis

Larvae, pupae, and adults of the carrot weevil (Listronotus oregonensis) were infected and killed by the three entomophagous nematodes (Steinernema feltiae, S. bibionis, and Heterorhabditis heliothidis) under controlled conditions. Third-stage larvae were more susceptible than pupae or adults. S. feltiae and S. bibionis were the most aggressive nematode species, causing larval mortality after 24-48 hours in both continuous and 2-hour contact with nematode suspension. The nematodes multiplied sufficiently in all insects at all stages of development; however, production of infective-stage larvae per host cadaver was variable.     

Laboratory Evaluation of Isaria fumosorosea CCM 8367 and Steinernema feltiae Ustinov against Immature Stages of the Colorado Potato Beetle

The Colorado potato beetle, Leptinotarsa decemlineata, has developed resistance to most registered pesticides and has become one of the most difficult insect pests to control. Development of new biopesticides targeting this pest might solve the resistance problem and contribute to sustainable crop production. Laboratory experiments were conducted to assess the efficacy of Isaria fumosorosea (syn. Paecilomyces fumosoroseus) strain CCM 8367 against L. decemlineata when applied alone or combined with the entomopathogenic nematode Steinernema feltiae. The last-instar larvae of the Colorado potato beetle showed the highest susceptibility to I. fumosorosea followed by pre-pupae and pupae. The median lethal concentration (LC₅₀) was estimated to be 1.03×10⁶ blastospores/ml. The strain CCM 8367 was more virulent, causing 92.6% mortality of larvae (LT₅₀ = 5.0 days) compared to the reference strain Apopka 97, which caused 54.5% mortality (LT₅₀ = 7.0 days). The combined application of the fungus with the nematodes increased the mortality up to 98.0%. The best results were obtained when S. feltiae was applied simultaneously with I. fumosorosea (LT₅₀ = 2.0 days); later application negatively affected both the penetration rate and the development of the nematodes. We can conclude that the strain CCM 8367 of I. fumosorosea is a prospective biocontrol agent against immature stages of L. decemlineata. For higher efficacy, application together with an entomopathogenic nematode is recommended.     

Potential vector switching in the evolution of Bursaphelenchus xylophilus group nematodes (Nematoda: Aphelenchoididae)

To show the importance of vector switching of nematodes in the evolution of the Bursaphelenchus xylophilus group, we tested a hypothesis that “Bursaphelenchus doui (or its ancestor) was transferred by Acalolepta fraudatrix, Acalolepta sejuncta, and/or Monochamus subfasciatus (or their ancestral species) from broad‐leaved trees to conifers, switched vectors from these cerambycid beetles to Monochamus beetles in conifers, and then evolved into the common ancestor of Bursaphelenchus mucronatus and B. xylophilus.” We used a simple nematode‐loading method to beetles and produced 20 binary combinations of five B. xylophilus group species and four cerambycid beetle species in the tribe Lamiini. The affinity of the nematodes for the beetles was examined based on phoretic stage formation of the nematodes. Phoretic stages of B. doui appeared in all beetle species examined, namely Acalolepta luxuriosa, Psacothea hilaris, A. fraudatrix, and Monochamus alternatus, although the affinity of the nematode for M. alternatus was weak. This finding indicates that B. doui could switch vectors to conifer‐using Monochamus beetles after transfer by A. fraudatrix from broad‐leaved trees to conifers. We conclude that vector switching of nematodes could have potentially happened during the evolutionary history of the B. xylophilus group.     

Variations in Immune Response of Popillia japonica and Acheta domesticus to Heterorhabditis bacteriophora and Steinernema Species

The infectivities of Steinernema carpocapsae, S. glaseri, S. scapterisci, and Heterorhabditis bacteriophora to Japanese beetle larvae, Popillia japonica, and house cricket adults, Acheta domesticus, were compared using external exposure and hemocoelic injection. Only H. bacteriophora and S. glaseri caused high P. japonica mortality after external exposure. When nematodes were injected, P. japonica had a strong encapsulation and melanization response to all species except S. glaseri. Heterorhabditis bacteriophora and S. carpocapsae were able to overcome the immune response, but S. scapterisci was not. All species except S. scapterisci were able to kill and reproduce within the host. Only S. scapterisci and S. carpocapsae caused A. domesticus mortality after external exposure. When nematodes were injected, A. domesticus had a strong immune response to all species except S. scapterisci. Steinernema carpocapsae effectively overcame the strong immune response and caused high host mortality, but S. glaseri and H. bacteriophora did not. Steinernema scapterisci caused high host mortality and reproduced, S. glaseri and H. bacteriophora caused low host mortality but only S. glaseri reproduced, and S. carpocapsae was able to kill the host but reproduced poorly. Most (ca. 90%) of the S. carpocapsae in the hemocoel of P. japonica became encapsulated and melanized within 8 hours postinjection. The symbiotic bacterium, Xenorhabduf nematophilus, was often released before this encapsulation and melanization.     

Effects of Pesta-Pelletized Steinernema carpocapsae (All) on Western Corn Rootworms and Colorado Potato Beetles

Pesta-pelletized Steinernema carpocapsae (All) nematodes were used in soil treatments in the greenhouse against larvae of Western corn rootworm and prepupae of Colorado potato beetle. The pesta-pellets delivered 100,000 living nematodes/g. Infective-stage nematodes and their associated bacteria survived the pesta-pellet process, emerged from the pellets in large numbers in the soil, and reduced adult emergence of both pest insects by more than 90%.     

<Emphasis Type="Italic">Heterorhabditis amazonensis</Emphasis> for biological control of fungus gnats <Emphasis Type="Italic">Bradysia difformis</Emphasis> in Daisy gerberas

Fungus gnats (FG) have been reported in Venezuelan greenhouses recently. Bradysia difformis Frey (Diptera, Mycetophilidae) was found attacking Daisy gerbera plants [Gerbera sp. L. (Asterales: Asteraceae)] and many other crops in the country causing serious damages. Heterorhabditis amazonensis Andaló et al. (Rhabditida: Heterorhabditidae), was used to assess the mortality of larvae and adults of FG, number of invader nematodes per larvae and to compare the mortality of FG using nematodes and/or ciromazine under laboratory conditions. In a commercial flower farm, different doses of these nematodes were applied for eight weeks to control FG. The results showed 95% of mortality of the B. difformis larvae under laboratory conditions and performed better in the field when 50,000 nematodes were applied in every pot as a base dose to initiate a control programme. According to our results, H. amazonensis has the potential to become a regular organism for controlling fungus gnat larvae in tropical greenhouses.     

Fusarium solani, a weak pathogen of the larval stages of the large elm bark beetle Scolytus scolytus (Coleoptera: Scolytidae)

Fusarium solani, a common colonist of beetle-infested elm bark, has been isolated from dead larvae and adults of the large elm bark beetle, Scolytus scolytus. Two infectivity tests indicated that this fungus is probably a weak pathogen of the larval stages of the beetle. Total mortality was not achieved in either test, but the mortality associated with fungal inoculation was occasionally significantly greater than the mortality in the uninoculated controls in both experiments (P<0.05, experiment A; P<0.025, experiment B). Disease symptoms suggest that the larvae attacked by F. solani may have died from bacterial infection.     

Compatibility and efficacy of the lady beetle <Emphasis Type="Italic">Thalassa montezumae</Emphasis> and the entomopathogenic fungus <Emphasis Type="Italic">Isaria fumosorosea</Emphasis> for biological control of the green croton scale: laboratory and greenhouse investigations

The lady beetle Thalassa montezumae and the entomopathogenic fungus Isaria fumosorosea (Ifr) were assessed alone and in combination to suppress green croton scale, Phalacrococcus howertoni, populations on croton plants using laboratory bioassays and greenhouse cage studies. The acquisition of Ifr blastospores by beetle larvae (3rd instar) and adults during contamination in well plates was used to simulate exposure to direct spraying and subsequent possible fungal infection was assessed. Spore dispersal by the insects was determined after the blastospore-contaminated T. montezumae life stages roamed on agar plates for 24 h by counting the number of colony-forming units (CFUs) produced in the plates. There were no significant differences in survival times at 14 days post-treatment between beetle larvae and adults exposed to Ifr and those exposed to water only. Mean survival time of larvae exposed to Ifr was 14 days and water 12 days, whereas for adults it was 13 days compared to 13 days, respectively. Plates with Ifr blastospore-contaminated T. montezumae adults roaming on the agar surface displayed significantly more fungal trails as CFUs compared to plates with larvae. In greenhouse cage studies, the mean mortality rates of the scale exposed to beetle larvae, either alone (80.8%) or in combination with Ifr (89.1%), were not significantly different. Scale mortality rates in the fungus-only (60.5%) and beetle larvae-only treatments were statistically similar. The treatment with both biocontrol agents had a significantly higher scale mortality rate compared to the treatment with Ifr only. Therefore, spraying Ifr prior to releasing T. montezumae is an effective and compatible biological control strategy for management of the green croton scale on croton plants.