Assessing immunocompetence in red palm weevil adult and immature stages in response to bacterial challenge and entomopathogenic nematode infection

Parasites and pathogens can follow different patterns of infection depending on the host developmental stage or sex. In fact, immune function is energetically costly for hosts and trade‐offs exist between immune defenses and life history traits as growth, development and reproduction and organisms should thus optimize immune defense through their life cycle according to their developmental stage. Identifying the most susceptible target and the most virulent pathogen is particularly important in the case of insect pests, in order to develop effective control strategies targeting the most vulnerable individuals with the most effective control agent. Here, we carried out laboratory tests to identify the most susceptible target of infection by infecting different stages of the red palm weevil Rhynchophorus ferrugineus (larvae, pupae, male, and female adults) with both a generic pathogen, antibiotic‐resistant Gram‐negative bacteria Escherichia coli XL1‐Blue, and two specific strains of entomopathogenic nematodes (EPNs), Steinernema carpocapsae ItS‐CAO1 and Heterorhabditis bacteriophora ItH‐LU1. By evaluating bacterial clearance, host mortality and parasite progeny release, we demonstrate that larvae are more resistant than adults to bacterial challenge and they release less EPNs progeny after infection despite a higher mortality compared to adults. Considering the two EPN strains, S. carpocapsae was more virulent than H. bacteriophora both in terms of host mortality and more abundant progeny released by hosts after death. The outcomes attained with unspecific and specific pathogens provide useful information for a more efficient and sustainable management of this invasive pest.     

Attraction of entomopathogenic nematodes Steinernema carpocapsae and Heterorhabditis bacteriophora to the red palm weevil (Rhynchophorus ferrugineus)

The red palm weevil (RPW), Rhynchophorus ferrugineus, is a serious pest of date palms. Its larvae bore deep into the trunk disrupt the vascular tissues and kill the infested trees. Behavioral features of entomopathogenic nematodes (EPNs), reflected by attraction and distribution patterns, are fundamental aspect in determining their parasitic ability and potential management of RPW. We studied the attraction behavior of the EPNs Steinernema carpocapsae and Heterorhabditis bacteriophora to the RPW under simulated natural conditions in tubes to evaluate their infective potential. In all experiments, a certain proportion of infective juveniles (IJs) (16–20%) stayed near the inoculated site and a major proportion (38–48%) was attracted to the host end. Both H. bacteriophora and S. carpocapsae were efficient crawlers, climbing up and descending when locating their insect host. They were efficiently attracted to the various larval sizes and stages of the RPW life cycle. Host localization by ascending movement was more prominent in S. carpocapsae than in H. bacteriophora. In general, H. bacteriophora is classified as a cruiser forager and S. carpocapsae as an ambusher. However, in this study, we discovered a higher percentage of cruiser foragers among S. carpocapsae IJs. They dispersed much faster and their cruising behavior was prominent characteristic in controlling the cryptic RPW concealed in organic habitats.     

Entomopathogenic nematodes fail to parasitize the woolly apple aphid Eriosoma lanigerum as their symbiotic bacteria are suppressed

The restriction of effective insecticides has facilitated the woolly apple aphid (WAA) Eriosoma lanigerum to become a major pest in apple orchards in Western Europe. It has also promoted alternative control strategies such as the use of entomopathogenic nematodes (EPN). We evaluated the control capacity of six commercially available EPN, viz. Heterorhabditis bacteriophora, Heterorhabditis megidis, Steinernema carpocapsae, Steinernema feltiae, Steinernema glaseri and Steinernema kraussei. We assessed the potential of these EPN to colonize and parasitize E. lanigerum in an in vitro multiwell test. Only S. carpocapsae caused higher mortality (20–40%) than the control treatment (water). However, the mortality observed with S. carpocapsae was found to be a test artefact and not induced by its specific entomopathogenic activity. A similar mortality range was recorded when applying the non‐entomopathogenic nematode Pratylenchus thornei in the same multiwell test set‐up. This result warrants careful interpretation of parasitism in these artificial test conditions. The failure of EPN activity was supported in further experiments by frequently finding S. carpocapsae inside living WAA. The presence of the EPN had no effect on aphid reproduction as numbers of ‘large’ embryos in EPN‐colonized and non‐colonized females were similar. In addition, the dauer juveniles did not recover in E. lanigerum reflecting that S. carpocapsae could not develop inside the WAA. We further demonstrated that growth of the EPN‐symbiotic bacteria Xenorhabdus nematophila and Photorhabdus luminescens is inhibited by the body fluid of the WAA, and we speculate that this antibacterial activity is the cause of the unsuccessful parasitization of the WAA by the EPN. This antibiosis inside the body of E. lanigerum would prevent production of the endotoxins by the bacterial symbionts that are essential for entomopathogenicity and insect control.     

Infectivity of entomopathogenic nematode Steinernema carpocapsae Pocheon strain (Nematoda: Steinernematidae) on the green peach aphid Myzus persicae (Hemiptera: Aphididae) and its parasitoids

Infectivity of entomopathogenic nematode (EPN) Steinernema carpocapsae Pocheon strain on the green peach aphid Myzus persicae and its parasitic wasps (e.g., Aphidius colemani, Aphidius gifuensis and Diaeretiella rapae) was evaluated under laboratory conditions. Infective juveniles (IJs) of S. carpocapsae Pocheon strain had low infectivity against nymph and adult stages of M. persicae, showing 2% and 6.7% of mortality, respectively. Application of the EPNs had little effect on mummies caused by the three parasitoid species, allowing them to remain intact. No IJ invaded the host, regardless of EPN application rate. The parasitoid emergence from mummies ranged from 80% to 85% in the presence of EPN while 79–86% was recorded in the absence of EPN. However, the presence of the IJs reduced oviposition by the three parasitoid species, decreasing the rate up to 59% when the nematodes were applied before parasitoid release, while little difference in oviposition was observed when nematodes were applied after parasitoid release.     

Compatibility between Forficula auricularia and entomopathogenic nematodes to be used in pome fruit pest management

Use of predators, parasitoids and entomopathogens as biocontrol agents in pome fruit production can lead to more efficient and sustainable pest management programmes. The European earwig (Forficula auricularia Linnaeus [Dermaptera: Forficulidae]) is a major predator of key pests in pome fruit orchards, and entomopathogenic nematodes (EPNs) of the families Steinernematidae and Heterorhabditidae are obligate parasites of a large number of insect species. Therefore, the interaction between earwigs and EPNs can play an important role in pest management programmes. Susceptibility of the European earwig to Steinernema carpocapsae, Steinernema feltiae (Steinernematidae) and Heterorhabditis bacteriophora (Heterorhabditidae) was evaluated. S. carpocapsae was the only tested EPN capable of killing the European earwig. However, the European earwig can detect the presence of S. carpocapsae and therefore avoid nematode‐treated shelters. An earwig deterrent activity in EPN‐killed codling moth larvae that reduces the foraging of European earwig on insect cadavers containing nematodes and allows nematodes to complete their life cycle was also assessed with the three species of nematodes. These findings suggest a positive compatibility between the European earwig and EPNs.     

Pathogenic effect of entomopathogenic nematode-bacterium complexes on terrestrial isopods

In this study, we evaluated the effect of entomopathogenic nematodes (EPNs) Steinernema carpocapsae, Steinernema feltiae and Heterorhabditis bacteriophora, symbiotically associated with bacteria of the genera Xenorhabdus or Photorhabdus, on the survival of eight terrestrial isopod species. The EPN species S. carpocapsae and H. bacteriophora reduced the survival of six isopod species while S. feltiae reduced survival for two species. Two terrestrial isopod species tested (Armadillidium vulgare and Armadillo officinalis) were found not to be affected by treatment with EPNs while the six other isopod species showed survival reduction with at least one EPN species. By using aposymbiotic S. carpocapsae (i.e. without Xenorhabdus symbionts), we showed that nematodes can be isopod pathogens on their own. Nevertheless, symbiotic nematodes were more pathogenic for isopods than aposymbiotic ones showing that bacteria acted synergistically with their nematodes to kill isopods. By direct injection of entomopathogenic bacteria into isopod hemolymph, we showed that bacteria had a pathogenic effect on terrestrial isopods even if they appeared unable to multiply within isopod hemolymphs. A developmental study of EPNs in isopods showed that two of them (S. carpocapsae and H. bacteriophora) were able to develop while S. feltiae could not. No EPN species were able to produce offspring emerging from isopods. We conclude that EPN and their bacteria can be pathogens for terrestrial isopods but that such hosts represent a reproductive dead-end for them. Thus, terrestrial isopods appear not to be alternative hosts for EPN populations maintained in the absence of insects.     

Immune defence components of Spodoptera exigua larvae against entomopathogenic nematodes and symbiotic bacteria

The current work investigated the immune response of Spodoptera exigua Hübner (Lepidoptera: Noctuidae) when challenged with two entomopathogenic nematodes (EPNs), Steinernema carpocapsae (Weiser) and Heterorhabditis bacteriophora (Poinar). The cellular and humoral defences were considered in this study. The haemocytes were observed around H. bacteriophora, but no haemocyte was found around S. carpocapsae. In larvae treated with H. bacteriophora and S. carpocapsae, total haemocyte counts (THCs) reached maximum levels at 4 and 12 hours post-injection (hpi), respectively, but decreased with the proliferation of symbiotic bacteria. In the humoral defence, there was no significant difference between EPNs on phenoloxidase (PO) activity. Phospholipase A₂ (PLA₂) and protease activity levels in the initial time post-injection were higher in the larvae treated with S. carpocapsae than in H. bacteriophora. In the following, the roles of symbiotic bacteria and axenic infective juveniles (IJs) in suppressing the immune system were studied separately. Maximum THC levels were observed in larvae treated with axenic nematodes and minimum THC levels were recorded in the live Xenorhabdus nematophila treatment. In the humoral defence, PLA₂ activity with axenic S. carpocapsae was suppressed at 4 hpi, while in monoxenic S. carpocapsae the PLA₂ level was increased to the maximum amount at 8 hpi. PO activity with monoxenic S. carpocapsae decreased gradually by 4 hpi; in live X. nematophila, it decreased from 0.5 to 16 hpi, while in axenic S. carpocapsae, it increased slowly from 0.5 to 16 hpi. The current work showed the synergistic effect of nematode and its bacterium in the suppression of the immune system and highlighted the role of the symbiont in inhibition of immune responses.     

The biochemical basis of antimicrobial responses in Manduca sexta

Innate immunity is essential for the wellbeing of vertebrates and invertebrates. Key components of this defense system include pattern recognition receptors that bind to infectious agents, extra-and intra-cellular proteins that relay signals, as well as molecules and cells that eliminate pathogens. We have been studying the defense mechanisms in a biochemical model insect, Manduca sexta. In this insect, hemolin, peptidoglycan recognition proteins, β-1,3-glucan recognition proteins and C-type lectins detect microbial surface molecules and induce immune responses such as phagocytosis, nodulation, encapsulation, melanization and production of antimicrobial peptides. Some of these responses are mediated by extracellular serine proteinase pathways. The proteolytic activation of prophenoloxidase （proPO） yields active phenoloxidase （PO） which catalyzes the formation of quinones and melanin for wound healing and microbe killing. M. sexta hemolymph proteinase 14 （HP 14） precursor interacts with peptidoglycan or β-1,3-glucan, autoactivates, and leads to the activation of other HPs including HP21 and proPO-activating proteinases （PAPs）. PAP-1, -2 and -3 cut proPO to generate active PO in the presence of two serine proteinase homologs. Inhibition of the proteinases by serpins and association of the proteinase homologs with bacteria ensure a localized defense reaction. M. sexta HP1, HP6, HP8, HP17 and other proteinases may also participate in proPO activation or processing of spatzle and plasmatocyte spreading peptide.     

The culturable bacterial community of frass produced by larvae of Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) in the Canary island date palm

Aims: Larvae of the red palm weevil (RPW) Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) feed inside palm stem tissues, making galleries and producing a wet fermenting frass. We characterized the culturable bacteria associated with frass produced by tunnelling larvae inside the Canary island date palms and investigated the role of frass and gut bacteria in plant polymers breakdown. Methods and Results: A culture‐dependent method was used to isolate bacteria from frass and noninfested palm tissues. Bacterial isolates were grouped into operational taxonomic units based on polymorphisms in the ITS‐PCR profiles, and representative isolates were identified by partial sequencing of the 16S rRNA gene. Frass bacteria were dominated by 2,3‐butanediol fermenter Enterobacteriaceae. None of the bacterial isolates was able to degrade cellulose; however, cellulolytic and hemicellulolytic bacteria were isolated from the larval gut enrichment cultures. Conclusions: Frass bacteria are specifically associated with the RPW larvae and might play beneficial roles for RPW, other than nutritional, that deserve further investigations. Breakdown of plant polymers probably occurs inside the larvae digestive system. Significance and Impact of the Study: Frass and gut micro‐organisms of R. ferrugineus should be included in studies of the interactions between RPW, its plant hosts, and its enemies.     

Efficacy of two entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema carpocapsae for control of the leopard moth borer Zeuzera pyrina (Lepidoptera: Cossidae) larvae under laboratory conditions

The biological traits of the entomopathogenic nematodes (EPNs), Steinernema carpocapsae and Heterorhabditis bacteriophora, against the larvae of the leopard moth, Zeuzera pyrina were evaluated in the laboratory. The traits included pathogenicity, penetration potential as well as foraging behaviour. Plate assays were performed using a range of EPN concentrations (5, 10, 20, 50 and 100 infective juveniles (IJs) per larva). The LC₅₀ values for S. carpocapsae and H. bacteriophora were 6.4 and 8.4 IJs larva^?1 after 72 h. Both EPN species caused high mortality in branch experiments. Significantly higher mortality rates occurred in the larger larvae after exposure to S. carpocapsae. Both EPN species successfully penetrated the Z. pyrina larvae as well as larvae of Galleria mellonella L. (Lepidoptera: Galleridae).The proportional response of H. bacteriophora to host-associated cues was strongly higher than S. carpocapsae in Petri dishes containing agar 1, 12 and 24 h after EPN application. These results highlight the efficiency of EPNs for the control of Z. pyrina larvae. However, due to the cryptic habitat of Z. pyrina larvae in their galleries in the trees, field trails need to be conducted to further evaluate this potential.     

Immunosuppression induced by expression of a viral RNase enhances susceptibility of Plutella xylostella to microbial pesticides

Abstract Polydnaviruses are a group of insect DNA viruses and are characterized in their segmented genome that is located in the chromosome(s) of host wasps. A polydnavirus, Cotesia plutellae bracovirus (CpBV), encodes a viral ribonuclease (RNase) T2 in a specific segment #3 (CpBV‐S3). This study tested its effect on gene expression associated with host immune responses in the diamondback moth, Plutella xylostella. Micro‐injection of CpBV‐S3 into nonparasitized larvae induced expression of its two encoded genes, CpBV‐ORF301 (=CpBV‐RNase T2) and CpBV‐ORF302. In response to a bacterial challenge, four antimicrobial peptide genes (hemolin, gloverin, cecropin and lysozyme) and six phenoloxidase (PO)–associated genes (proPO‐activating proteinase, PO, serine proteinase homolog and serpins 1–3) were up‐regulated in their expressions. However, the transient expression of CpBV‐S3 suppressed the expressions of cecropin, PO and serpin 1. Double‐stranded RNA specific to the viral RNase T2 could specifically knockdown the viral gene expression and restored the three gene expressions suppressed in the larvae injected with CpBV‐S3. The inhibitory activity of the viral RNase T2 on the target genes was further proven by the suppression of PO activation in response to bacterial challenge in the larvae injected with CpBV‐S3. This immunosuppression by the expression of the viral RNase T2 resulted in significant increase of pathogen susceptibility of P. xylostella against Bacillus thuringiensis or baculovirus infection.     

Evaluation of hydraulic,pneumatic and mechanical agitation for the spray application of Steinernema carpocapsae (Rhabditida: Steinernematidae)

The application of entomopathogenic nematodes (EPN) is generally done using standard spray application techniques. However, in contrast to chemical pesticides, these biological antagonists must remain viable during and after the application process. For the application of EPN, a good agitation system is indispensable as the nematodes tend to sediment fast in a spray tank without agitation. Three agitation systems, viz. mechanical, pneumatic and hydraulic agitation were tested for their ability to keep Steinernema carpocapsae (Rhabditida: Steinernematidae) suspended in an undamaged way. Hydraulic agitation was tested using a centrifugal and a diaphragm pump. Nematode damage was quantified based on viability and infectivity of the EPN. The ability of the agitation system to keep the nematodes in suspension was examined by comparing the nematode concentration observed in the samples taken at different agitation times. Only the hydraulic agitation using the centrifugal pump damaged the nematodes. After 120 min of recirculation, only 19.3% of the nematodes survived. Infectivity was even reduced to 0%. An additional experiment revealed that the temperature rise, from 21.7 to 45.4°C, was responsible for the observed nematode damage. The concentration measurements showed that the pneumatic agitation was unstable. Agitation during 120 min using the other agitation systems resulted in a significant loss of nematodes at 15 cm above the spray tank bottom. In conclusion, mechanical and hydraulic agitation using a diaphragm pump can be recommended when S. carpocapsae is applied, although attention should be paid to possible nematode loss during application.     

Biocontrol potential of the entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema carpocapsae on cucurbit fly,Dacus ciliatus (Diptera: Tephritidae)

Entomopathogenic nematodes (EPNs) from the families Steinernematidae and Hererorhabditidae are considered excellent biological control agents against many insects that damage the roots of crops. In a regional survey, native EPNs were isolated, and laboratory and greenhouse experiments were conducted to determine the infectivity of EPNs against the cucurbit fly, Dacus ciliatus Loew (Diptera: Tephritidae). Preliminary experiments showed high virulence by a native strain of Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae) and a commercial strain of Steinernema carpocapsae Weiser (Rhabditida: Steinernematidae). These two strains were employed for further analysis while another native species, Steinernema feltiae, was excluded due to low virulence. In laboratory experiments, larvae and adult flies were susceptible to nematode infection, but both nematode species induced low mortality on pupae. S. carpocapsae had a significantly lower LC₅₀ value against larvae than H. bacteriophora in filter paper assays. Both species of EPNs were effective against adult flies but S. carpocapsae caused higher adult mortality. When EPN species were applied to naturally infested fruit (150 and 300 IJs/cm²), the mortality rates of D. ciliatus larvae were 28% for S. carpocapsae and 12% for H. bacteriophora. Both EPN strains successfully reproduced and emerged from larvae of D. ciliates. In a greenhouse experiment, H. bacteriophora and S. carpocapsae had similar effects on fly larvae. Higher rates of larval mortality were observed in sandy loam and sand soils than in clay loam. The efficacy of S. carpocapsae and H. bacteriophora was higher at 25 and 30°C than at 19°C. The results indicated that S. carpocapsae had the best potential as a biocontrol agent of D. ciliatus, based on its higher virulence and better ability to locate the fly larvae within infected fruits.     

Biological control of Tipula paludosa (Diptera: Nematocera) using entomopathogenic nematodes (Steinernema spp.) and Bacillus thuringiensis subsp. israelensis

Tipula paludosa (Diptera: Nematocera) is the major insect pest in grassland in Northwest Europe and has been accidentally introduced to North America. Oviposition occurs during late August and first instars hatch from September until mid-October. Laboratory and field trials were conducted to assess the control potential of entomopathogenic nematodes (EPN) (Steinernema carpocapsae and S. feltiae) and Bacillus thuringiensis subsp. israelensis (Bti) against T. paludosa and to investigate whether synergistic effects can be exploited by simultaneous application of nematodes and Bti. Results indicate that the early instars of the insect are most susceptible to nematodes and Bti. In the field the neonates prevail when temperatures tend to drop below 10 °C. S. carpocapsae, reaching >80% control, is more effective against young stages of T. paludosa than S. feltiae (<50%), but the potential of S. carpocapsae might be limited by temperatures below 12 °C. Mortality of T. paludosa caused by Bti was not affected by temperature even at 4 °C but the lethal time increased with decreasing temperatures. Synergistic effects of Bti and EPN against T. paludosa were observed in 3 out of 10 combinations in laboratory assays but not in a field trial. The potential of S. carpocapsae was demonstrated in field trials against early instars in October reaching an efficacy of >80% with 0.5 million nematodes m⁻² at soil temperatures ranging between 3 and 18 °C. Results with Bti were strongly influenced by the larval stage and concentration. Against early instars in autumn between 74 and 83% control was achieved with 13 kg ha⁻¹ Bti of 5,700 International Toxic Units (ITUs) and 20 kg ha⁻¹ of 3,000 ITUs. Applications in spring against third and fourth instars achieved between 0 and 32% reduction. The results indicate that application of Bti and nematodes will only be successful and economically feasible during the early instars and that the success of S. carpocapsae is dependent on temperatures >12 °C. Synergistic effects between S. carpocapsae and Bti require more detailed investigations in the field to determine maximal effect.     

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.     

Susceptibility of three lepidopteran pests to five entomopathogenic nematodes and in vivo mass production of these nematodes

Abstract

An investigation was conducted in pots to access the susceptibility of three lepidopteran pests, namely, gram pod borer, Helicoverpa armigera, greater wax moth, Galleria mellonella, and rice moth, Corcyra cephalonica, to two recently described species, Steinernema masoodi, S. seemae, and three indigenous S. carpocapsae, S. glaseri and S. thermophilum entomopathogenic nematodes (EPN). The suitability of these lepidopterans for the in vivo mass production of the nematodes was also estimated. Among the five species of EPN, S. masoodi, S. seemae and S. carpocapsae were found most pathogenic to C. cephalonica, bringing about mortality within 24 h, followed by H. armigera (36, 38 and 48 h, respectively) and G. mellonella (30, 36 and 48 h, respectively). The other species of EPN, viz., S. glaseri and S. thermophilum was the least pathogenic, which killed the larvae of C. cephalonica in 29 and 36 h, respectively, G. mellonella in 48 h, and H. armigera in 38 and 56 h, respectively. Galleria mellonella was found the most suitable host for the mass production of infective juveniles (IJs) of S. seemae, which yielded higher IJs than S. carpocapsae. Helicoverpa armigera was the next best suitable alternate host, which produced maximum IJs in case of S. seemae followed by S. masoodi, S. carpocapsae, S. glaseri and S. thermophilum. Rice moth, Corcyra cephalonica was the least suitable host. The susceptibility of H. armigera to five tested EPN species and susceptibility of G. mellonella and C. cephalonica to S. masoodi and S. seemae are new records.     

Entomopathogenic nematodes as potential biological control agents of Popillia japonica (Coleoptera,Scarabaeidae) in Piedmont Region (Italy)

In 2014, the Japanese beetle Popillia japonica (Coleoptera: Scarabaeidae) was first spotted in northern Italy in the Nature Park of the Ticino Valley, its first detection in continental Europe. This polyphagous invasive species has the potential to cause serious losses to horticulture and agriculture. Particularly for its management in a Nature Park, environmentally friendly strategies are necessary. To develop baseline data for a biological control approach to the Italian outbreak of P. japonica, we conducted laboratory and field experiments testing several indigenous and commercial strains of the entomopathogenic nematode (EPN) species Heterorhabditis bacteriophora and Steinernema carpocapsae against P. japonica larvae. In the laboratory, strains of H. bacteriophora caused greater mortality (ranging from 57% to 100%) than those of S. carpocapsae (3% to 77%). In micro‐plot field tests carried out at three different times, the most virulent indigenous EPN strain, H. bacteriophora ItH‐LU1, showed again the best results ranging from 44% to 93% against young larvae. Finally, in a large‐plot field trial, the commercial H. bacteriophora product provided 46% larval mortality. This study shows that H. bacteriophora strains have good potential as biological control agents of larvae of the invasive P. japonica in northern Italy.     

Efficacy of entomopathogenic nematodes against potato tuber moth,Phthorimaea operculella (Lepidoptera: Gelechiidae) under laboratory conditions

The susceptibility of potato tuber moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae) to native and commercial strains of entomopathogenic nematodes (EPNs) was studied under laboratory conditions. Native strains of EPNs were collected from northeastern Iran and characterised as Steinernema feltiae and Heterorhabditis bacteriophora (FUM 7) using classic methods as well as analysis of internal transcribed spacer (ITS) and D2/D3 sequences of 28S genes. Plate assays were performed to evaluate the efficiency of five EPN strains belonging to four species including Steinernema carpocapsae (commercial strain), S. feltiae, Steinernem glaseri and H. bacteriophora (FUM 7 and commercial strains). This initial assessment with 0, 75, 150, 250, 375 and 500 IJs/ml concentrations showed that S. carpocapsae and H. bacteriophora caused the highest mortality in both larval and prepupal stages of P. operculella, PTM. Thereafter, these three strains (i.e. S. carpocapsae, H. bacteriophora FUM 7 and the commercial strains) were selected for complementary assays to determine the effects of soil type (loamy, loamy–sandy and sandy) on the virulence of EPNs against the second (L2) and fourth instar (L4) larvae as well as prepupa. A soil column assay was conducted using 500 and 2000 IJs in 2-ml distilled water. Mortality in the L2 larvae was not affected by the EPN strain or soil type, while there was a significant interactive effect of nematode strains and soil type on larval mortality. The results also showed that EPN strains have higher efficiency in lighter soils and caused higher mortality on early larvae than that in loamy soil. In L4 larvae, mortality of PTM was significantly influenced by nematode strain and applied concentrations of infective juveniles. The larval mortality induced by S. carpocapsae was higher than those caused either by a commercial or the FUM 7 strain of H. bacteriophora. Prepupa were the most susceptible stage.