Synergism of imidacloprid and entomopathogenic nematodes against white grubs: the mechanism

Entomopathogenic nematodes and the chloronicotinyl insecticide, imidacloprid, interact synergistically on the mortality of third-instar white grubs (Coleoptera: Scarabaeidae). The degree of interaction, however, varies with nematode species, being synergistic for Steinernema glaseri (Steiner) and Heterorhabditis bacteriophora Poinar, but only additive for Steinernema kushidai Mamiya. The mechanism of the interaction between imidacloprid and these three entomopathogenic nematodes was studied in the laboratory. In vials with soil and grass, mortality, speed of kill, and nematode establishment were negatively affected by imidacloprid with S. kushidai but positively affected with S. glaseri and H. bacteriophora. In all other experiments, imidacloprid had a similar effect for all three nematode species on various factors important for the successful nematode infection in white grubs. Nematode attraction to grubs was not affected by imidacloprid treatment of the grubs. Establishment of intra-hemocoelically injected nematodes was always higher in imidacloprid-treated grubs but the differences were small and in most cases not significant. The major factor responsible for synergistic interactions between imidacloprid and entomopathogenic nematodes appears to be the general disruption of normal nerve function due to imidacloprid resulting in drastically reduced activity of the grubs. This sluggishness facilitates host attachment of infective juvenile nematodes. Grooming and evasive behavior in response to nematode attack was also reduced in imidacloprid-treated grubs. The degree to which different white grub species responded to entomopathogenic nematode attack varied considerably. Untreated Popillia japonica Newman (Coleoptera: Scarabaeidae) grubs were the most responsive to nematode attack among the species tested. Untreated Cyclocephala borealis Arrow (Coleoptera: Scarabaeidae) grubs showed a weaker grooming and no evasion response, and untreated C. hirta LeConte (Coleoptera: Scarabaeidae) grubs showed no significant response. Chewing/biting behavior was significantly increased in the presence of nematodes in untreated P. japonica and C. borealis but not in C. hirta and imidacloprid-treated P. japonica and C. borealis. Our observations, however, did not provide an explanation for the lack of synergism between imidacloprid and S. kushidai.     

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.     

Influences of host size and host species on theinfectivity and development of Heterorhabditismegidis (strain NLH-E87.3)

The infectivity, time to first emergence of infective juveniles (IJs), total number of IJs per insect and IJs body length of the entomopathogenic nematode Heterorhabditis megidis (strain NLH-E87.3) after development in larvae of two insect hosts, Galleria mellonella (greater wax moth) and Otiorhynchus sulcatus (vine weevil) was studied. At a dose of 30 IJs, larvae of G. mellonella show to be significantly more susceptible than O. sulcatus larvae. At a dose of one IJ, vine weevil larvae were more susceptible. The number of invading infective juveniles (IJs) increased with host size while the host mortality at a dose of one IJ decreased with the increase of host size. Time to first emergence was longer at a dose of one IJ per larva and increased with the increase of host size in both insect species. Reproduction of IJs differed between host species, host sizes and doses of nematodes. Generally, the IJs body size increased with an increasing host size. The longest infective juveniles were produced at the lowest IJ doses. Results are discussed in relation to the influence of different host species and their different sizes on the performance of H. megidis (strain NLH-E87.3) as a biological control agent.     

Effectiveness of Steinernema spp. and Heterorhabditis bacteriophora against Popillia japonica in the Azores

Steinernema carpocapsae (Breton strain), S. glaseri, and Heterorhabditis bacteriophora were evaluated for their potential to control immature stages of the Japanese beetle, Popillia japonica, on Terceira Island (the Azores). In bioassays carried out at temperatures higher than 15 C, S. glaseri and H. bacteriophora caused 100% mortality of larvae, whereas S. carpocapsae caused 56% larval mortality. At temperatures slightly below 15 C, only S. glaseri remained effective. In field plots, in September, S. glaseri and S. carpocapsae reduced larval populations by 91% and 44%, respectively, when applied at the rate of 10⁶ nematodes/m². In April, S. glaseri caused 31% reduction in numbers of larvae, but S. carpocapsae was ineffective. In colder months (November-February) neither steinernematids nor H. bacteriophora reduced larval populations. Increasing the application rate from 10⁶ to 5 x 10⁶ infective stage S. glaseri per m² increased efficacy from 63% to 79% mortality.     

Entomopathogenic nematodes (Steinernema spp. and Heterorhabditis bacteriophora) and a fungus Beauveria brongniartii for biological control of the white grubs, Ectinohoplia rufipes and Exomala orientalis, in Korean golf courses

Three species of entomopathogenicnematodes, a combination of two nematodespecies, an entomopathogenic fungal species,and a combination of a nematode and fungalspecies were evaluated against the white grubsEctinohoplia rufipes and Exomalaorientalis (Coleoptera: Scarabaeidae) in the field. The nematodes were acommercial formulation of Steinernemacarpocapsae (BioSafe) and S. glaseri from Dongrae and from Hanrim, and Heterorhabditis bacteriophora from Hamyang,Republic of Korea. The entomopathogenic funguswas Beauveria brongniartii, produced onSabouraud maltose agar plus 1% yeast (SMAY),rice bran, or compost. The combinationtreatment was S. carpocapsae with H.bacteriophora or B. brongniartii. Fieldapplications were made in August or Septemberagainst third instars at a golf course infestedwith E. rufipes in Gyeongnam Province in1991, and one in Pusan with E. orientalisin 1992 and 1993. In 1991, a significantreduction of 70.2 to 79.4% of E. rufipeslarvae was observed in the nematode, fungal andchemical (fenitrothion) treatments comparedwith a 15.7% reduction in the control. In1992, the E. orientalis larval populationwas reduced between 62.7 and 82.8% in thetreatments compared to 10.7% in the control.In 1993, larval reductions in plots treatedwith nematodes (78.3 to 97%) and B.brongniartii propagated on rice bran (84.5%)were significantly better than in plots treatedwith B. brongniartii propagated on SMAY(63.6%) or compost (59.6%). Combining twonematode species did not enhance the efficacycompared to treatments with one nematodespecies alone, but combining S.carpocapsae with B. brongniartiiproduced on SMAY resulted in a significantincrease in grub mortality over the applicationof the fungus alone produced on SMAY orcompost. The high efficacy of the nematode andmost fungal treatments was attributed to theclose proximity of the white grubs to the soilsurface which allowed for excellentpathogen-host contact and to favorable soiltemperatures, sandy soil, post irrigationapplication and/or rain and a minimal thatchlayer in the turfgrass.     

Orientation of Heterorhabditis megidis to insect hosts and plant roots in a Y-tube sand olfactometer

The host-searching behaviour of Heterorhabditis megidis strain NLH-E 87.3 in the presence of insect hosts and plant roots, offered individually and in combination, was studied using a newly developed Y-tube olfactometer filled with sand. Within a period of 24 hours infective juveniles (IJs) were significantly attracted to living G. mellonella larvae and caused 100% larval mortality. Otiorhynchus sulcatus larvae, however, did not elicit host-oriented movement of IJs and no larval mortality was observed. Roots of strawberry plants induced a negative response in IJs. The combination of strawberry roots and O. sulcatus larvae, however, strongly attracted IJs leading to 37% host mortality. It was shown that this type of Y-tube choice arena is a useful tool in studying the searching behaviour of entomopathogenic nematodes in a semi-natural habitat.     

Isolation and identification of entomopathogenic nematodes from citrus orchards in South Africa and their biocontrol potential against false codling moth

A survey was conducted to determine the diversity and frequency of endemic entomopathogenic nematodes (EPN) in citrus orchards in the Western Cape, Eastern Cape and Mpumalanga provinces of South Africa. The main aim of the survey was to obtain nematodes as biological control agents against false codling moth (FCM), Thaumatotibia leucotreta, a key pest of citrus in South Africa. From a total of 202 samples, 35 (17%) tested positive for the presence of EPN. Of these, four isolates (11%) were found to be steinernematids, while 31 (89%) were heterorhabditids. Sequencing and characterisation of the internal transcribed spacer (ITS) region was used to identify all nematode isolates to species level. Morphometrics, morphology and biology of the infective juvenile (IJ) and the first-generation male were used to support molecular identification and characterisation. The Steinernema spp. identified were Steinernema khoisanae, Steinernema yirgalemense and Steinernema citrae. This is the first report of S. yirgalemense in South Africa, while for S. citrae it is the second new steinernematid to be identified from South Africa. Heterorhabditis species identified include Heterorhabditis bacteriophora, Heterorhabditis zealandica and an unknown species of Heterorhabditis. Laboratory bioassays, using 24-well bioassay disks, have shown isolates of all six species found during the survey, to be highly virulent against the last instar of FCM larvae. S. yirgalemense, at a concentration of 50 IJs/FCM larva caused 100% mortality and 74% at a concentration of 200 IJs/pupa. Using a sand bioassay, S. yirgalemense gave 93% control of cocooned pupae and emerging moths at a concentration of 20 IJs/cm². This is the first report on the potential use of EPN to control the soil-borne life stages of FCM, which includes larvae, pupae and emerging moths. It was shown that emerging moths were infected with nematodes, which may aid in control and dispersal.     

Susceptibility of Hoplia philanthus (Coleoptera: Scarabaeidae) larvae and pupae to entomopathogenic nematodes (Rhabditida: Steinernematidae, Heterorhabditidae)

Biological control potential of nine entomopathogenic nematodes, Heterorhabditis bacteriophora CLO51 strain (HbCLO51), H. megidis VBM30 strain (HmVBM30), H. indica, Steinernema scarabaei, S. feltiae, S. arenarium, S. carpocapsae Belgian strain (ScBE), S. glaseri Belgian strain (SgBE) and S. glaseri NC strain (SgNC), was tested against second-, and third-instar larvae and pupae of Hoplia philanthus in laboratory and greenhouse experiments. The susceptibility of the developmental stages of H. philanthus differed greatly among tested nematode species/strains. In the laboratory experiments, SgBE, SgNC, HbCLO51 and HmVBM30 were highly virulent to third-instar larvae and pupae while SgBE was only virulent to second-instar larvae. Pupae were highly susceptible to HbCLO51, HmVBM30, SgBE and SgNC (57–100%) followed by H. indica and S. scarabaei (57–76%). In pot experiments, HbCLO51, SgBE and S. scarabaei were highly virulent to the third-instar larvae compared to the second-instar larvae. Our observations, combined with those of previous studies on other nematode and white grub species, show that nematode virulence against white grub developmental stages varies with white grub and nematode species.     

Biological control of tobacco cutworm,Spodoptera litura Fabricius with entomopathogenic nematodes

The efficacies of several entomopathogenic nematodes ofSteinernema andHeterorhabditis spp. were examined against tobacco cutworm,Spodoptera litura Fabricius.H. bacteriophora HY showed 100% mortality after 20 h against 2nd instar of tobacco cutworm. In the case of 3–4th instar,S. carpocapsae PC.,H. bacteriophora HY andS. monticola CR showed 100% mortality after 47 h. In the case of 5–6th instar,S. carpocapsae PC proved more effective than the others. Generally, the number of nematodes harvested increased as their size decreased. Also, the highest number of nematodes was obtained in the 5–6th instar ofS. litura byH. bacteriophora HY, showing about 1.3×10⁶ nematodes per larva.In vitro culturedS. carpocapsae PG showed 100% mortality after 73 h against 5–6th instar tobacco cutworm, indicating that nematodes producedin vitro can be potentially used for the biological control ofS. litura instead of nematodesin vivo.     

Susceptibility of Delia radicum to steinernematid nematodes

Isolates of different Steinernema species (S. affine, S. bicornutum, S. feltiae and Steinernema C1) were used in mortality assays with third instar larvae of Delia radicum (L.) (Diptera: Anthomyiidae). The nematode isolates had been obtained by baiting soil regularly grown with cabbage. One isolate (S. feltiae) was the result of a natural infection of a D. radicum puparium. The highest mortality (77%) was obtained with an isolate of S. feltiae (DK1). The isolate DK1 was also used in tests with all larval stages of D. radicum. Mortality around 60% was observed for second and third instar larvae, while first instar larvae showed very low or no susceptibility. Maximum mortality of second and third instar larvae was reached applying only 25 nematodes per larva. Observations of larvae that pupated revealed that some of these puparia contained nematodes. Experiments with hatching puparia showed that a high proportion was infected by nematodes if the flies were prevented from leaving nematode-containing soil. In addition to mortality, the ability of the nematodes to successfully reproduce in the insects was studied. It was found that the species S. feltiae and S. bicornutum reproduced in D. radicum larvae and adults with S. feltiae being the most successful.     

Virulence of entomopathogenic nematodes to larvae of the guava weevil, Conotrachelus psidii (Coleoptera: Curculionidae), in laboratory and greenhouse experiments

The guava weevil, Conotrachelus psidii, is a major pest of guava in Brazil and causes severe reduction in fruit quality. This weevil is difficult to control with insecticides because adults emerge over a long period, and larvae develop to the fourth-instar inside the fruit and move to the soil for pupation. We assessed the virulence of entomopathogenic nematodes to fourth-instar larvae in soil by comparing their susceptibility to nine species or strains: Heterorhabditis bacteriophora HP88, H. baujardi LPP7, and LPP1, H. indica Hom1, Steinernema carpocapsae All and Mexican, S. feltiae SN, S. glaseri NC, and S. riobrave 355. In petri dish assays with sterile sand at a concentration of 100 infective juveniles (IJs) of a given nematode species/strain, larval mortality ranged from 33.5 to 84.5%, with the heterorhabditids being the most virulent. In sand column assays with H. baujardi LPP7, H. indica Hom1, or S. riobrave 355 at concentrations of 100, 200, and 500 IJs, mortality was greater than the control only for H. baujardi (62.7%) and H. indica (68.3%) at the highest concentration. For H. baujardi LPP7 in a petri dish assay, the time required to kill 50 and 90% of the larvae (LT₅₀ and LT₉₀) for 100 IJs was 6.3 and 9.9 days, whereas the lethal concentration required to kill 50 and 90% of the larvae (LC₅₀ and LC₉₀) over 7 days was 52 and 122.2 IJs. In a greenhouse study with guava trees in 20-L pots, 10 weevil larvae per pot, and concentrations of 500, 1000 or 2000 IJs, H. baujardi LPP7 caused 30 and 58% mortality at the two highest concentrations. These results show that H. baujardi is virulent to fourth-instar larvae and has potential as a biological control agent in IPM programs.     

Characterization of biocontrol traits in the entomopathogenic nematode Heterorhabditis georgiana (Kesha strain), and phylogenetic analysis of the nematode’s symbiotic bacteria

Our objective was to estimate the biocontrol potential of the recently discovered entomopathogenic nematode species Heterorhabditis georgiana (Kesha strain). Additionally, we conducted a phylogenetic characterization of the nematode’s symbiotic bacterium. In laboratory experiments, we compared H. georgiana to other entomopathogenic nematodes for virulence, environmental tolerance (to heat, desiccation, and cold), and host seeking ability. Virulence assays targeted Acheta domesticus, Agrotis ipsilon, Diaprepes abbreviatus, Musca domestica, Plodia interpunctella, Solenopsis invicta, and Tenebrio molitor. Each assay included H. georgiana and five or six of the following species: Heterorhabditis floridensis, Heterorhabditis indica, Heterorhabditis mexicana, Steinernema carpocapsae, Steinernema feltiae, Steinernema rarum, and Steinernema riobrave. Environmental tolerance assays included Heterorhabditis bacteriophora, H. georgiana, H. indica, S. carpocapsae, S. feltiae, and S. riobrave (except cold tolerance did not include S. carpocapsae or S. riobrave). Host seeking ability was assessed in H. bacteriophora, H. georgiana, S. carpocapsae, and Steinernema glaseri, all of which showed positive orientation to the host with S. glaseri having greater movement toward the host than S. carpocapsae (and the heterorhabditids being intermediate). Temperature range data (tested at 10, 13, 17, 25, 30 and 35 °C) indicated that H. georgiana can infect Galleria mellonella between 13 and 35 °C (with higher infection at 17–30 °C), and could reproduce between 17 and 30 °C (with higher nematode yields at 25 °C). Compared with other nematode species, H. georgiana expressed low or intermediate capabilities in all virulence and environmental tolerance assays indicating a relatively low biocontrol potential. Some novel observations resulted from comparisons among other species tested. In virulence assays, H. indica caused the highest mortality in P. interpunctella followed by S. riobrave; S. carpocapsae caused the highest mortality in A. domesticus followed by H. indica; and S. riobrave was the most virulent nematode to S. invicta. In cold tolerance, S. feltiae exhibited superior ability to cause mortality in G. mellonella (100%) at 10 °C, yet H. bacteriophora and H. georgiana exhibited the ability to produce attenuated infections at 10 °C, i.e., the infections resumed and produced mortality at 25 °C. In contrast, H. indica did not show an ability to cause attenuated infections. Based on the phylogenetic analysis, the bacterium associated with H. georgiana was identified as Photorhabdus luminescens akhurstii.     

Investigations on the parasitism ofOtiorrhynchus salicicola andO. Sulcatus [Col.: Curculionidae] byHeterorhabditis sp. [Nematoda]

Experiments were conducted to study the efficacy of the insect parasitic nematodeHeterorhabditis sp. (HW79) as a biological control agent ofOtiorrhynchus salicicola. This weevil species is reported as a pest of ornamental plants in Switzerland and Italy. Dipping plastic boxes containing heavily infested cuttings of laurel (Prunus laurocerasus) in a nematode suspension resulted in approximately 100% parasitisation of full-grown larvae, pupae and non-emerged young adults. The average dose resulting from dipping varied between 56,000 and 62,000 nematodes per liter soil. This experiment was run under natural outdoor conditions. In a further outdoor experiment, pottedLigustrum plants were inoculated with eggs ofO. salicicola and later 20,000 infective juvenile nematodes per liter soil were added to the soil surface. The resulting weevil mortality in the treated pots was 78%. In seven greenhouse tests using the same nematode dose in pots with horticultural soil to which weevil larvae had been added, weevil mortality varied between 76% and 100%, the arithmetic average being 90%. These results indicate that Heterorhabditid nematodes may provide an effective means of controllingO. salicicola. In an other experiment usingO. sulcatus larvae, the influence of application time on nematode efficacy was investigated. When nematodes were added a few days before weevil larvae had hatched from the eggs, no parasitic effect was obtained. Nematode applications done shortly after larval hatching however, resulted in complete weevil control. These results are of significance in timing nematode applications in practice.      

Evaluation of certain entomopathogenic nematodes against the melon ladybird,Epilachna chrysomelina F. (Coleoptera: Coccinellidae)

Three native Egyptian nematode isolates; Heterorhabditis taysearae and Heterorhabditis sp. S1 (Heterorhabditidae) and Steinernema carpocapsae S2 (Steinernematidae) as well as H. bacteriophora Hp88 as an imported species, were used in the present work to evaluate their activities against larvae and adults of the melon ladybird, Epilachna chrysomelina. The target pest was found to be susceptible to all tested entomopathogenic nematodes under laboratory conditions of 30±5°C.

In the greenhouse, a single spray of nematode suspension (1000 infective juveniles per ml) of each of H. taysearae, H. bacteriophora Hp88 and Steinernema carpocapsae S2 on squash seedlings was enough to give a reasonable mortality of 4^th larval instar E. chrysomelina, reaching 65.2, 44.0 and 84.0%, respectively, one week after application. This gives evidence that the Egyptian nematode isolates could tolerate high temperature and could be recommended for application in the control programmes of E. chrysomelina larvae in cucurbit fields.     

Diversity and distribution of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) in Turkey

The diversity and distribution of entomopathogenic nematodes in thefamilies Steinernematidae and Heterorhabditidae were assessed throughout anextensive soil survey in Turkey during 1999 and 2000. Entomopathogenic nematodeswere recovered from six out of seven regions sampled, with 22 positive sites(2%) out of 1080 sites sampled. A single nematode isolate was recovered at eachof the positive sites, of which 15 were steinernematid isolates and seven wereheterorhabditid isolates representing a total of four species. Based onmorphometric and molecular data, the nematode species were identified asHeterorhabditis bacteriophora, Steinernemafeltiae, S. affine, andSteinernema n. sp. The most common species was S.feltiae, which was isolated from 10 sites in six regions, followed byH. bacteriophora from seven sites in five regions,S. affine from four sites in two regions, andSteinernema n. sp. from one site. Heterorhabditisbacteriophora and S. feltiae have been found inmany parts of the world, whereas S. affine, so far, hasonly been recovered in Europe until our survey. Steinernemaaffine was isolated from the European (Marmara) as well as theAsiatic region (Middle Anatolia) of Turkey. A new undescribedSteinernema sp. was isolated from the most eastern region(East Anatolia) of Turkey. Soils of the positive sites were classified as sandy,sandy loam, or loam (68.2%) and sandy–clay–loam or clay loam (31.8%) and the pHranged from 5.6 to 7.9. The habitats from which the entomopathogenic nematodeswere isolated were broadly classified as disturbed (59.1%), which includedagricultural fields and poplar planted for lumber and wind breaks, andundisturbed (40.9%), which included pine forest, grassland, marsh and reed sites.Steinernema feltiae, S. affine, andH. bacteriophora were recovered from both disturbed andundisturbed habitats. The new Steinernema sp. was recoveredfrom grassland. Our survey showed that these nematodes occur widely throughoutTurkey, but at a frequency below that reported for other parts of the world.     

Morphological and physiological comparisons ofHelicobasidium mompa andH. purpureum

Morphological and physiological comparisons were made of sevenHelicobasidium mompa isolates and fourH. purpureum isolates. Colonies of theH. mompa isolates were thin, dense, or hard and dense, and most were pale brown to brown or dark brown, while that of isolate 344c was pinkish. Colonies ofH. purpureum isolates were hard and dense, and their colonies were dark brown. Diameters of hyphae were similar forH. mompa andH. purpureum. Dimensions of conidia and morphology of conidiophores ofH. mompa isolate 344c were close to those ofH. purpureum reported previously.H. mompa isolates grew well at 23°C, 25°C or 27°C, while all isolates ofH. purpureum grew well at 23°C. Growth rates ofH. purpureum isolates was almost the same as those ofH. mompa isolates with slow growth. Polygaracturonase activity at pH 3 was variable among the isolates for bothH. mompa andH. purpureum. Itaconic acid was produced abundantly by three isolates ofH. mompa but not produced by isolate AH130, whereas all isoaltes ofH. purpureum produced a small amount of itaconic acid.     

Steinernema scarabaei, an entomopathogenic nematode for control of the European chafer

A new entomopathogenic nematode species, Steinernema scarabaei, was evaluated for efficacy against two white grub species, the European chafer, Rhizotrogus majalis, and the Japanese beetle, Popillia japonica, in laboratory, greenhouse, and field trials. In laboratory assays, S. scarabaei caused greater mortality than Heterorhabditis bacteriophora. S. scarabaei was highly virulent with an LC₅₀ of 5.5–6.0 and 5.7 infective juveniles (IJs) per third-instar larva in R. majalis and P. japonica, respectively. In a greenhouse trial, S. scarabaei provided greater mortality of R. majalis at all application rates (0.156–1.25 × 10⁹ IJs/ha) than Steinernema glaseri and H. bacteriophora (both at 1.25 × 10⁹ IJs/ha). Combination of imidacloprid and S. scarabaei resulted in an antagonistic interaction. In a fall field trial, S. scarabaei provided 88 and 75% control of R. majalis at 2.5 × 10⁹ and 10⁹ IJs/ha, respectively, and 54% control of P. japonica at 10⁹ IJs/ha; H. bacteriophora had no effect on mortality of either white grub species. In a spring field trial, unusually cool temperatures impeded nematode activity. Against R. majalis, S. scarabaei provided moderate control (56–59%), whereas Heterorhabditis marelatus provided no control. Mortality of P. japonica was moderate (49–66%) in both S. scarabaei and H. marelatus treatments. Overwinter persistence of S. scarabaei activity was demonstrated in a spring assay of soil from fall treated plots in which nematode infection was absent from control plots and present in treated plots.     

Effect of potting media on the efficacy and dispersal of entomopathogenic nematodes for the control of black vine weevil, Otiorhynchus sulcatus (Coleoptera: Curculionidae)

The effect of five commercial potting media, peat, bark, coir, and peat blended with 10% and 20% compost green waste (CGW) on the virulence of six commercially available entomopathogenic nematodes (EPN), Heterorhabditis bacteriophora UWS1, Heterorhabditis megidis, Heterorhabditis downesi, Steinernema feltiae, Steinernema carpocapsae, and Steinernema kraussei was tested against third-instar black vine weevil (BVW), Otiorhynchus sulcatus. Media type was shown to significantly affect EPN virulence. Heterorhabditis species caused 100% larval mortality in all media whereas Steinernema species caused 100% larval mortality only in the peat blended with 20% CGW. A later experiment investigated the effect of potting media on the virulence of EPN species against BVW by comparing the vertical dispersal of EPN in the presence and absence of BVW larva. Media type significantly influenced EPN dispersal. Dispersal of H. bacteriophora was higher than H. megidis, H. downesi, or S. kraussei in all media, whereas, S. feltiae and S. carpocapsae dispersal was much reduced and restricted to peat blended with 20% CGW and coir, respectively. In the absence of larvae, most of the EPN species remained in the same segment they were applied in, suggesting that the larvae responded to host volatile cues. Greenhouse trials were conducted to evaluate the efficacy of most virulent strain, H. bacteriophora in conditions more representative of those in the field, using 2.5 × 10⁹ infective juveniles/ha. The efficacy of H. bacteriophora UWS1 against third-instar BVW was 100% in peat, and peat blended with 10% and 20% CGW but only 70% in bark and coir, 2 weeks after application. These studies suggest that potting media significantly affects the efficacy and dispersal of EPN for BVW control.     

Synergism of Entomopathogenic Nematodes and Imidacloprid against White Grubs: Greenhouse and Field Evaluation

In previous greenhouse studies, the insecticide imidacloprid and the entomopathogenic nematode Heterorhabditis bacteriophora Poinar interacted synergistically against third instars of the masked chafers Cyclocephala hirta LeConte and C. pasadenae Casey (Coleoptera: Scarabaeidae). We tested this interaction for two additional nematode species and three additional scarab species under field conditions. In greenhouse tests, H. bacteriophora and Steinernema glaseri (Steiner) interacted synergistically against third instars of the Japanese beetle, Popillia japonica Newman, the oriental beetle, Exomala orientalis Waterhouse, and the masked chafers Cyclocephala borealis Arrow, C. pasadenae, and C. hirta. The degree of interaction varied with nematode species. The strongest synergism occurred between imidacloprid and S. glaseri. Synergism between imidacloprid and H. bacteriophora was weaker and the interaction was not always significant. Combinations of imidacloprid and S. kushidai Mamiya only resulted in additive mortality. The synergistic interaction was also observed in field trials but the results were more variable than those under greenhouse conditions. The combination of nematodes and imidacloprid could be used for curative treatments of white grub infestations, especially against scarab species that are less susceptible to nematodes and/or imidacloprid. This combination has a low environmental impact and high compatibility with natural biological control of turfgrass insects. The possible roles of these combinations in augmentative control approaches are discussed.     

Heterorhabditis sonorensis n. sp. (Nematoda: Heterorhabditidae), a natural pathogen of the seasonal cicada Diceroprocta ornea (Walker) (Homoptera: Cicadidae) in the Sonoran desert

A new Heterorhabditis species was isolated from nymphal stages of the seasonal cicada Diceroprocta ornea (Walker) in an asparagus field in the state of Sonora, Mexico. Concomitantly, another isolate of the same nematode species was also collected from an oak woodland habitat in the Chiricahua mountain range in southeastern Arizona. Morphological and molecular studies together with cross-hybridization tests indicate these two isolates are conspecific and represent a new undescribed Heterorhabditis sp. This new species is distinguished from other species in this genus by a combination of several qualitative and quantitative morphological traits. Key diagnostic features include: presence of a pronounced post-anal swelling in the hermaphrodite; male with nine pairs of bursal rays, with pairs 4 and 7 bent outwards and one pair of papillae placed on the cloacal opening, value of D% (average: 79); infective juveniles with a well developed cuticular tooth, long tail (average: 105 μm) and values of D% (average: 90) and E% (average: 99). In addition to these diagnostic characters, cross-hybridization tests between the new species with H. bacteriophora and H. mexicana yielded no fertile progeny. Comparison of ITS rDNA sequences with other available sequences of described species depicted the two isolates as a new species. Phylogenetic analysis of these sequence data placed H. sonorensis n. sp. as a member of the indica-group.