Heterorhabditis Bacteriophora Strain Research Articles

Biocontrol potential of six Heterorhabditis bacteriophora strains isolated in the Azores Archipelago.

Entomopathogenic nematodes (EPNs) are closely associated with Popillia japonica and potentially used as their biological control agents, although field results proved inconsistent and evoked a continual pursuit of native EPNs more adapted to the environment. Therefore, we surveyed the Azorean Archipelago to isolate new strains of Heterorhabditis bacteriophora and to evaluate their virulence against the model organism Galleria mellonella under laboratory conditions. Six strains were obtained from pasture and coastal environments and both nematode and symbiont bacteria were molecularly identified. The bioassays revealed that Az172, Az186, and Az171 presented high virulence across the determination of a lethal dose (LD50) and short exposure time experiments with a comparable performance to Az29. After 72 hours, these virulent strains presented a mean determination of a lethal dose of 11 infective juveniles cm-2, a lethal time (LT50) of 34 hours, and achieved 40% mortality after an initial exposure time of only 60 minutes. Az170 exhibited an intermediate performance, whereas Az179 and Az180 were classified as low virulent strains. However, both strains presented the highest reproductive potential with means of 1700 infective juveniles/mg of larvae. The bioassays of the native EPNs obtained revealed that these strains hold the potential to be used in biological control initiatives targeting P. japonica because of their high virulence and locally adapted to environmental conditions.

Genotypic markers associated with cold storage survival of the entomopathogenic nematode Heterorhabditis bacteriophora

Summary Limited shelf life is a major constraint to successful commercialisation of entomopathogenic nematodes (EPN), and to extend shelf life, dauer juveniles (DJ) are formulated and stored at low temperatures (4-8°C). We evaluated the cold storage potential of strains of Heterorhabditis bacteriophora formulated in diatomaceous earth at storage temperatures between 5 and 9°C. When assessing DJ decline to reach 75% survival (MT75) in the formulation for the respective temperatures, H. bacteriophora strain HB4 had the highest survival of 25 days at 9°C, while strain D2D6 survived longest at 8°C for 28 days. A set of 22 H. bacteriophora wild type inbred lines was then phenotyped for cold tolerance in water under oxidative stress in 70 mM H2O2 at 2°C. The MT50 (time to 50% survival) ranged from 11 to 23 days. The phenotypic data were correlated with the respective genotypic data, identifying four single nucleotide polymorphic (SNP) markers associated with cold tolerance. The survival of two lines (PT11 and IR11) with opposite extreme cold tolerance pheno- and genotypes was evaluated in diatomaceous earth formulation at 2°C with the cold tolerant IR11 surviving 3 days longer than PT11. Our study yields a set of valuable SNP markers employable in rapid genotyping of cold tolerance and tracking this trait during the breeding process.

Entomopathogenic nematodes for biological control of Psylliodes chrysocephala (Coleoptera: Chrysomelidae) in oilseed rape

Winter oilseed rape (Brassica napus) is one of the largest crops in Europe and the cabbage stem flea beetle Psylliodes chrysocephala is one of its major pests. Since the ban of neonicotinoids for seed treatment, farmers apply pyrethroids in autumn to control the cabbage stem flea beetle. Current studies show that the insect develops resistance to this group of chemicals. Biological control with entomopathogenic nematodes (EPNs) represents a possible, environmentally friendly alternative control measure. In the present work, we considered three strategies to control the cabbage stem flea beetle: applying the nematodes against the first larval stage in the soil, against the second and third larval stages inside the plant or against the adult beetles. In laboratory experiments, we found the third larval instar to be the most susceptible stage and the adult beetle the less susceptible one. Steinernema feltiae and the cold active SDT1-IL1 Heterorhabditis bacteriophora strain, with a reduction potential of 89 and 76 %, respectively, proved to be the most virulent EPNs against P. chrysocephala in pot experiments at 15 °C. Moreover, we performed four field trials to test the efficacy of H. bacteriophora and S. feltiae against the larvae. The highest reduction in the field trials was 45% and 39%, obtained with SDT1-IL1 and a mixture of H. bacteriophora and S.feltiae, respectively. The present study provides preliminary information about the potential of EPNs to control P. chrysocephala and represents a start point for the development of a competitive and sustainable alternative to pyrethroids.

Indigenous entomopathogenic nematode as biocontrol agents for insect pest management in hilly regions

The present investigation mainly emphasized on the development and use of entomopathogenic nematodes (EPNs) as a bio-insecticide. The success in controlling insect pests in the soil environments increased the production and use of the adapted indigenous EPNs species for insect management in the fields. EPNs as biocontrol agents were capable for high virulence, easy for application, safe for non-target animals and eco-friendly in nature. These nematodes have ubiquitous nature. These occur in low population in their natural habitat which was mass multiplied in the laboratory. In the present investigation, 5 concentrations (30IJs, 60IJs, 90IJs, 120IJs and 150IJs) of Heterorhabditis bacteriophora strain S15 were applied against the 3rd and 4th instar larvae of four major agricultural insect pests, namely Helicoverpa armigera, Spodoptera litura, Agrotis segetum and Mythimna separata under laboratory conditions at different time exposure (24, 48, 72 and 96 hr). It was observed that the 3rd and 4th larval instars of all 4 insects (H. armigera, S. litura, A. segetum and M. separata) were highly susceptible for the pathogenesis caused by H. bacteriophora Sirmaur isolates. Amongst all insects, both the larval instars of M. separata are highly susceptible for EPNs infection with highest 96% and 98% mortality in highest dose @150IJs. In 3rd instar larvae of other insects such as H. armigera, S. litura and A. segetum larval mortality ranges from 84%, 92% and 94% respectively. Among 4th instar larvae of H. armigera, S. litura and A. segetum the pathogenicity varies from 88%, 94% and 96% respectively. The recorded median lethal concentration (LC50) in 3rd instar larvae of H. armigera, S. litura, A. segetum and M. separata varies from 36.15, 30.05, 30.97 and 23.8. Similarly in 4th instar larvae of H. armigera, S. litura, A. segetum and M. separata, LC50 ranged from 31.41, 28.64, 26.92 and 20.64 respectively. Statistically significant variations were observed in the data recorded on the mortality, in all the treatments. EPNs are the best weapon to overcome insect resistance problems and must be employed to manage insect population.

Virulence, penetration rate and reproductive potential of entomopathogenic nematodes from eastern Australia in Queensland fruit fly, Bactrocera tryoni

Queensland fruit fly (Bactrocera tryoni) is Australia’s most significant horticultural pest, yet limited options are available for its biological control. Here, we assessed the virulence and reproductive potential of 32 newly isolated Australian entomopathogenic nematode (EPN) strains of Heterorhabditis bacteriophora, Heterorhabditis indica, Heterorhabditis marelatus, Heterorhabditis zealandica and Steinernema feltiae against the late third larval instar and pupal stages of B. tryoni under laboratory conditions, and compared their performance with four commercially available strains of H. bacteriophora, H. zealandica, S. feltiae and S. carpocapsae. All EPN strains caused significant larval mortality, and, remarkably, 29 also pupal mortality. Among the EPN strains the mean LD50 value, presented as the number of infective juveniles (IJs) per B. tryoni individual, ranged from 25 to 114 IJs per larva, and from 114 to 360 per pupa; therefore, 3.2 to 4.6× more IJs were required to kill pupae. The penetration rates of IJs in B. tryoni larvae varied from 2 to 6.8%, and the number of IJs recovered per larva varied between 2,100 to 5,683. Our findings highlight the potential of Australian EPN strains for controlling larval and pupal stages of B. tryoni. Overall, based on the three tested traits, one H. zealandica strain (Hz.NAR1), three H. indica strains (Hi.ECCH, Hi.HRN and Hi.HIE2), one H. bacteriophora strain (Hb.HIE), one H. marelatus strain (Hm.ENCBF2) and one S. feltiae strain (Sf.Y13) were found the most promising candidates as potential biological control agents against the soil inhabiting stages of B. tryoni. Further laboratory testing is required to assess their persistence under a broader range of environmental conditions before their biological control efficacy can be trialled under field conditions.

Evaluation of the virulence of entomopathogenic nematodes as a biological control agents against Gryllotalpa gryllotalpa (Gryllotalpidae)

AbstractThe European mole cricket Gryllotalpa gryllotalpa Linnaeus (Orthoptera: Gryllotalpidae) is a key pest of several crops in different regions of the world. The mole crickets usually damage seedlings, feeding aboveground on foliage or stem tissue and belowground on roots and tubers. In biological control, entomopathogenic nematodes (EPNs) are used against many insect pests inhabiting the soil as safe alternatives to chemical pesticides. The present study aimed to evaluate the virulence of the EPNs Heterorhabditis bacteriophora strain (HP88) and Steinernema carpocapsae strain (All) against the adults of European mole cricket G. gryllotalpa under laboratory and field conditions. Three concentrations of each of the EPNs H. bacteriophora and S. carpocapsae were tested against G. gryllotalpa. Infected adults of the pest with nematodes, at the concentration of 2000/cup, attained a mortality rate of (96%), when treated with S. carpocapsae and (72%) when treated with H. bacteriophora 8 days post‐treatments. Mortality percentage of G. gryllotalpa adults increased as the concentration of the nematodes increased. Adults' mortality of G. gryllotalpa reached 100% when treated with the combination of the 2 nematode's strains at all concentrations 4 days post‐treatments. At the 3 tested concentrations, 500, 1,000 and 2,000 IJs/adults, H. bacteriophora showed a higher reproductive rate in vivo than S. carpocapsae. A single adult of G. gryllotalpa treated with H. bacteriophora produced the average of 29,972, 74,768 and 140,474 IJs/adult, at the 3 concentrations, respectively. Under field application, a combination of EPNs successfully reduced the loss of the cabbage seedlings and the population's number of the mole crickets after 3 applications in the 2 seasons of (2019 and 2020). Therefore, EPNs as biological control agents can be recommended against the mole cricket G. gryllotalpa in vegetable crops as s pest management practice in an integrated pest management programme.

Efficacy assessment of entomopathogenic nematodes native to Morocco against the white grubs Rhizotrogus obesus Lucas and Geotrogus olcesii Fairmaire (Coleoptera: Scarabaeidae)

The white grubs Rhizotrogus obesus Lucas and Geotrogus olcesii Fairmaire (Coleoptera: Scarabaeidae) are two of the most problematic soil-dwelling pests of some annual crops in Morocco, where they may become a target for entomopathogenic nematodes (EPN). In this study, fourteen EPN strains reported from different fields in Morocco were investigated for their efficacy against both scarab species, under laboratory and greenhouse conditions. In laboratory experiments, seven strains of Heterorhabditis bacteriophora (HB-MOR2, HB-MOR6, HB-MOR1, HB-MOR5, HB-MOR7, HB-MOR4, and HB-MOR8) and one strain of Steinernema feltiae (SF-MOR10), provided remarkable efficacy against R. obesus (38–73% of mortalities). The EPN strains of Steinernema sp.2 (Sfg-MOR12), H. bacteriophora (HB-MOR4), Steinernema sp.1 (Sfg-MOR11), and H. bacteriophora (HB-MOR1) caused mortality rates ranging from 53 to 68% against G. olcesii. In greenhouse experiments where chlorpyrifos-ethyl was used as a positive control, larval mortality of R. obesus caused by both strains of H. bacteriophora, HB-MOR6 (81%) and HB-MOR2 (75%), was higher than that obtained by chlorpyrifos-ethyl (73%). The mortality rate in G. olcesii caused by Steinernema sp.2 (Sfg-MOR12) (77%) was the same as for chlorpyrifos-ethyl treatment (74%). Therefore, it can be concluded that EPN can constitute a promising biological alternative for the sustainable control of R. obesus and G. olcesii.

Potential of native entomopathogenic nematodes for the control of brown marmorated stink bug Halyomorpha halys in Georgia

ABSTRACT The potential of native entomopathogenic nematodes for the control of the brown marmorated stink bug Halyomorpha halys was evaluated in experimental trials in Georgia. Two Georgian strains of Heterorhabditis bacteriophora (HRB, GEO) and Steinernema borjomiense and two Italian strains of H. bacteriophora (HRB, IT) and S. apuliae were used in laboratory assays (22°C and 80% RH) at concentrations of 1000, 500 and 200 infective juveniles (IJs) per adult H. halys. The mortality of tested insects was estimated from the third day after treatment. Differences in mortality of the tested insect were observed between Georgian and Italian strains. At the highest concentration (1000 IJs/adult), mortality (%) of H. halys induced by Georgian strains HRB and S. borjomiense was 53.3 and 40, respectively, at 500 IJs/adult 40 and 33.3, and at 200 IJs/adult 33.3 and 13.3, respectively. Italian strains HRB (IT) and S. apuliae proved more suppressive against the tested insects, with mortality rates of 95.5 and 60, 93.3 and 40 and 73.3 and 33.2%, at concentrations of 1000, 500 and 200 IJs/adult of H. halys, respectively. The present study provides further insights in selection of promising EPN strains to be used against this harmful insect pest, although further studies to test their efficacy under field conditions are needed.

Virulence of new strain of Heterorhabditis bacteriophora from Croatia against Lasioptera rubi

Soil samples from 100 cultivated and natural sites were assessed for the presence of entomopathogenic nematodes. Heterorhabditid nematodes were recovered from three soil samples during spring months, with the overall positive sample rate of 3%. The isolates of entomopathogenic nematodes were identified as three different strains conspecific with Heterorhabditis bacteriophora (Heterorhabditidae). They were found from natural sites and vineyard, while no recovery occurred from intensively cultivated agricultural fields. The morphometrical characteristics of infective juveniles and males showed differences between all Croatian strains and from the original description. Heterorhabditis bacteriophora ISO9 was bioassayed on Lasioptera rubi (Cecidomyiidae) (the raspberry gall midge) larvae at different nematode concentrations under laboratory conditions. The significantly highest mortality was observed in treatments with 50 and 200 infective juveniles per insect larvae within 8 days after inoculation. This is the first report of entomopathogenic nematodes of the family Heterorhabditidae from Croatia, and susceptibility of L. rubi larvae to entomopathogenic nematodes. The Croatian strain H. bacteriophora ISO9 was proved to possess strong insecticidal properties against L. rubi larvae.

Inoculative release of Heterorhabditis bacteriophora Poinar (Oswego) and Steinernema feltiae Filipjev (NY04) mixture can enhance biological control of soil-dwelling pests in turfgrass production systems

ABSTRACTIn this study, the utility of inoculative releases of local strains of Heterorhabditis bacteriophora and Steinernema feltiae isolated from northern New York (NY) State for managing soil-dwelling pests in sod (turfgrass production systems) was evaluated. The objectives of the project were to (a) quantify changes in biocontrol potential during the sod production process and (b) evaluate the potential for the nematodes to suppress populations of soil-dwelling turf pests following harvest and installation at a new site. A field study was conducted on four sod fields in central NY State on soils with varying texture and organic matter content, and a post-harvest sod installation study was performed in the greenhouse on a sandy loam soil. Nematode-induced mortality of Galleria mellonella was increased significantly one month following inoculation but differed between fields with different soil types, likely due to variation in soil organic matter content. In the following year, nematode persistence in sod fields was low, likely driven by moderate to severe drought conditions. In contrast, upon harvest and installation, significant nematode-induced mortality was observed in soil receiving an installation of muck-produced sod. Enhanced nematode-induced mortality was detected in both the greater wax moth and third instar Japanese beetle larvae. This study indicates that inoculation of sod with entomopathogenic nematodes can improve biocontrol potential during sod production and after harvest, however, the utility of this practice is sensitive to soil conditions, and likely most efficacious on coarse-textured soils with high organic matter.

Heterorhabditis bacteriophora pampean-strain VEli (Nematoda): identification and pathogenicity against the strawberry pest Lobiopa insularis (Coleoptera: Nitidulidae)

The pampean region in the Buenos Aires province of Argentina is a major horticultural area. Biological pesticides to control pest insects available in the market are scarce in this country and nonexistent based on entomonematodes. In this paper, we characterized a native entomopathogenic nematode isolated from soil in the pampas, and evaluated its infectivity and reproductive capacity in the strawberry sap beetle Lobiopa insularis. Morphological and molecular-genetic analysis determined this isolate to be a nematode of Heterorhabditis bacteriophora, thereafter designated as the VEli strain. A 901-bp genomic-DNA fragment was isolated (GenBank Accesion No KJ575524). The new strain was pathogenic against L. insularis. In 200 ml containers with autoclaved clay soil (100 ml), at concentrations of 1,000; 5,000 and 10, 000 IJs, mortality in larvae ranged from 25 to 77 %. At a concentration of 10,000 IJs, infectivity was higher in larvae and pupae (77 ± 20% and 64 ± 25 %, respectively) than in adults (2.2 ± 1.1 %). The lethal concentration required to kill 50 and 90 % of the larvae (LC50 and LC90) over 10 days was 2,840 and 23,743 IJs. Differences were observed in the parasitism levels and emergence time of IJs between concentrations and stages, although not for production. Heterorhabditis bacteriophora strain VEli is the first entomopathogenic nematode found to infect and kill L. insularis, capable of completing its life cycle in all strawberry sap beetle stages. This availability would enable the establishment and subsequent dispersal of the strain in strawberry fields. Finally, this report constitutes the first presentation of data on the morphological, morphometric, and molecular-genetic characterization of a native entomopathogenic nematode strain from the Buenos Aires province, Argentina.

The combined approach of strain discovery and the inbred line technique for improving control of Delia radicum with Heterorhabditis bacteriophora

Entomopathogenic nematodes are potent biocontrol agents but their efficacy can be compromised under unfavorable environmental conditions. For example, cold tolerance can be an essential requirement when utilizing entomopathogenic nematodes in cooler regions. Discovery of new nematode species or strains that are adapted to local conditions is one approach that can be used to enhance efficacy. Once a strain is isolated however, serial culturing can lead to loss of beneficial traits (such as temperature tolerance). Trait deterioration can be eliminated or reduced by creating homozygous inbred lines. In a novel approach to strain improvement, we combined strain discovery (to obtain superior traits) with the inbred line technique (to stabilize traits). The efficacy of ten homozygous inbred lines from a Heterorhabditis bacteriophora strain isolated in Ontario, Canada was determined against insect larvae, under low temperature conditions. To assess the impact of serial culturing on nematode performance, two wild-type parent populations (the original ‘non-cultured’ parent population, and a repeatedly ‘sub-cultured’ population) were compared with the ten inbred lines. The first experiment evaluated efficacy against Galleria mellonella under five constant temperature regimes from 8 to 24 °C. At 16 °C, eight inbred lines and the ‘non-cultured’ parents demonstrated efficacy against G. mellonella. At the warmest temperatures, all nematode treatments were effective. In a subsequent experiment, the efficacy of the nematode lines and two commercial H. bacteriophora strains was evaluated at 16 °C against the cabbage maggot, Delia radicum. All treatments except one of the inbred lines caused higher levels of infection than the control. The majority of the inbred lines and the ‘non-cultured’ parental line exhibited superior efficacy compared with the ‘sub-cultured’ population and both commercial strains. This study demonstrates the utility of combining strain discovery with the inbred line approach for improved biological control.

Entomopathogenic fungi and nematodes against larvae of the chestnut weevil, Curculio elephas (Coleoptera: Curculionidae): a laboratory evaluation

ABSTRACTPest control in chestnut orchards is mainly achieved by chemicals, but there is growing focus on the potential use of biological control agents. The larval susceptibility of the chestnut weevil Curculio elephas L. (Coleoptera: Curculionidae) to different strains/species of entomopathogenic fungi and nematodes was evaluated in laboratory by soil cup bioassays. In the experiments with fungi, a wild strain of Metarhizium anisopliae caused the highest larval mortality (about 90%) compared with a commercial and a wild strain of Beauveria bassiana (80% and 77% respectively). Regarding nematodes, the commercial strain of Heterorhabditis bacteriophora was more effective in the control of chestnut larvae (77% insect mortality) than Steinernema carpocapsae (43%).

DETERMINATION OF THE EFFICACY SOME ENTOMOPATHOGENIC NEMATODES AGAINST THE TOMATO LEAFMINER TUTA ABSOLUTA (MEYRICK) AND THE POTATO TUBER MOTH PHTHORIMAEA OPERCULELLA (ZELL.) (LEPIDOPTERA: GELECHIIDAE) UNDER LABORATORY CONDITIONS IN EGYPT

The present study was carried out to determine the efficacy of the entomopathogenic nematodes (EPNs) Steinernema carpocapsae strain All and Heterorhabditis bacteriophora strain HP88 upon the tomato leafminer Tuta absoluta and the potato tuber moth Phthorimaea operculella. Using different concentrations of the entomopathogenic nematodes against third and fourth larval instars and pupae at both T. absoluta and P. operculella revealed a highest susceptibility (highest mortality rate and lowest LC50) in the third instar larvae. Meanwhile pupal stage showed the highest resistance. On comparing the susceptibility of the tested insects towards the two entomopathogenic nematodes, it was clear that T. absoluta was more susceptibility than P. operculella in the three tested stages.

The activity of detoxifying enzymes in the infective juveniles of Heterorhabditis bacteriophora strains: Purification and characterization of two acetylcholinesterases

The infectivity and detoxifying enzyme activities including glutathione-S-transferase (GST), acetylcholinesterase (AChE) and carboxylesterase (CaE) are investigated in the infective juveniles (IJs) of six different strains of Heterorhabditis bacteriophora as a biocontrol agent against insect pests. The specific activities ranged from 10.8–29.8 and 50–220units/mg protein for GST and AChE, respectively; and from 24.7–129 and 22.6–77.3units/mg protein for CaE as estimated by P-nitrophenyl and α-naphthyl acetates, respectively. H. bacteriophora EM2 strain has the highest infectivity and the highest enzymatic activities as well. AChE is the predominant detoxifying enzyme that might imply its major role in the detoxification of insecticide(s). The isoenzyme pattern demonstrated two major slow-moving isoforms in all EPN strains examined. Purification of two AChE isoforms, AChEAII and AChEBI, from H. bacteriophora EM2 strain is performed by ammonium sulfate precipitation, gel filtration on Sephacryl S-200 and chromatography on DEAE-Sepharose. AChEAII and AChEBII have specific activities of 1207 and 1560unit/mg protein, native molecular weights of 180 and 68kDa, and are found in dimeric and monomeric forms, respectively. Both isoforms showed optimum activity at pH8.5 and 35°C. AChEBI exhibited higher thermal stability and higher activation energy than AChEAII. The enzymatic activities of purified AChEs are completely inhibited by Hg+2 and Ni+2 and greatly enhanced by Mn+2. The substrate specificity, the relative efficiency of substrates hydrolysis, substrate inhibition and inhibition by BW284C51, but not by iso-OMPA, clearly indicated that they are true AChEs; their properties are compared with those recorded for insects as target hosts for H. bacteriophora EM2.

Field efficacy against the hazelnut weevil, Curculio nucum and short-term persistence of entomopathogenic nematodes

The hazelnut weevil, Curculio nucum L. (Coleoptera: Curculionidae) is a pest affecting hazelnut orchards in Europe, with an important economical repercussion. Its potential control, short-term field persistence and the vertical distribution of native entomopathogenic nematode strains were tested in Muntanyes de Prades, Tarragona (NE Iberian Peninsula) over two consecutive years. Steinernema feltiae strain D114, Steinernema sp. strain D122 and Heterorhabditis bacteriophora strain DG46 were used in summer and spring applications at a dosage of 5·105 IJs m-2. The three nematode species reduced the hazelnut weevil population, ranging from 32% to 88% efficacy, without significant differences in efficacy or between the two applications. Persistence evaluation was carried out during 9 weeks for S. feltiae (D114), Steinernema sp. (D122) and H. bacteriophora (DG46) and showed all species capable of lasting for this period. Nematodes and larval vertical distribution was assessed. Most of the hazelnut weevil stayed within the first 25 cm although some were found as deep as 40 cm. Entomopathogenic nematodes were found along all 40 cm depth. This study proves the suitability of entomopathogenic nematodes to control the hazelnut weevil.

Differences in Immune Defense Evasion of Selected Inbred Lines of Heterorhabditis Bacteriophora in Two White Grub Species.

We determined virulence of seven Heterorhabditis bacteriophora strain GPS11 inbred lines possessing superior infective juvenile longevity, and heat and ultra violet radiation tolerance against white grubs Popillia japonica and Cyclocephala borealis. At 1 and 2 weeks after treatment, inbred line A2 was significantly more virulent towards P. japonica compared to the parent strain GPS11 and inbred lines A7, A8, A12 and A21; and line A2 caused significantly higher C. borealis mortality than lines A6 and A12. Penetration, encapsulation and survival of two inbred lines, A2 and A12, that showed the highest and lowest virulence against both grub species were then assessed. There were no differences between the two lines for the total number of nematodes penetrated in either P. japonica or C. borealis within the first 24 h, but a significantly higher percentage of penetrated nematodes were alive in line A2 compared to the line A12 in both grub species. P. japonica immune response over time to hemocoel-injected nematodes of A2, A12 and the parent strain was further investigated. While all injected nematodes were encapsulated at 6 h post injection, non-encapsulated living nematodes were detected at 12 and 24 h post injection, showing the breakage out of encapsulation. A higher percentage of non-encapsulated living nematodes and a lower percentage of dead nematodes were found in line A2 as compared to the line A12 after 12 h post injection. These data suggest that virulence differences in the studied H. bacteriophora inbred lines are not due to differences in nematode penetration or recognition by the grub immune system, but are related to the ability of the infective juveniles to break out of encapsulation.

Spatial patterns of the red palm weevil and applied entomopathogenic nematode Heterorhabditis bacteriophora

The distribution and abundance of red palm weevil (Rhynchophorus ferrugineus) within palm tree-infected stems at Ismaelia governorate, Egypt, were investigated. Taylor's power law was fit (P ≤ 0.01) to its larvae, pupae and/or adults. The slope value of this power law indicated the clumped distribution of these insect stages. Sample size optimisation needed to achieve a predetermined level of sampling error for the insect stages was calculated. Also, soil samples were taken after uniform application of entomopathogenic nematode Heterorhabditis bacteriophora strain EG1 to the soil under citrus tree canopies at the rate of 104 infective juveniles/400 cm2 plots, and assayed for the nematode using the Galleria bait method. At the first and second sampling dates, the nematode displayed contagious distribution and attained mean insect mortality of 15.3 and 4, respectively. At the third date of sampling, the nematode showed random distribution according to chi-squared test and caused 1.8 mean insect mortality. Evaluation of H. bacteriophora EG1 as a biocontrol measure for the weevil was discussed based on their investigated dispersion indices.

Evaluation of the action of Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae) isolate HP88 on the biology of engorged females of Rhipicephalus ( Boophilus) microplus (Acari: Ixodidae)

The objective of this work was to evaluate the effect of different concentrations of the entomopathogenic nematode (EPN) Heterorhabditis bacteriophora strain HP88 on the biological parameters of the non-parasite phase of engorged females of the cattle tick Rhipicephalus ( Boophilus) microplus. Six groups were formed, each containing 20 engorged females, which were exposed to the following concentrations of infective juveniles of this nematode: 0, 75, 150, 300, 600 and 1200 EPNs/female, respectively. The following biological parameters were observed: female weight before oviposition, egg mass weight, pre-oviposition period, oviposition period, survival period, hatching percentage, egg production index (%EPI), nutritional index (%NI) and efficacy of treatment. There were no statistically significant differences in the female weight before the oviposition and pre-oviposition period ( p > 0.05) between the groups. The nematode action caused significant alterations ( p < 0.01) in the egg mass weight, oviposition period, survival period, hatching percentage, %EPI and %NI between the treated groups and the control group. Treatment efficacy was higher than 90% in all groups, reaching 99% at a dosage of 1200 EPNs/female. The present study demonstrates that under laboratory conditions, H. bacteriophora HP88 has a deleterious effect on the majority of the parameters of the non-parasitic phase of engorged R. ( B.) microplus females, making this species a potential biological control agent of cattle ticks.

Photorhabdus temperata subsp. stackebrandtii subsp. nov. (Enterobacteriales: Enterobacteriaceae)

The bacterial symbiont of the entomopathogenic nematode Heterorhabditis bacteriophora strain GPS11 was characterized by 16S rRNA gene sequence and physiological traits. The phylogenetic tree built upon 16S rRNA gene sequences clustered the GPS11 bacterial isolate with Photorhabdus temperata strains which have been previously isolated from Heterorhabditis species. The phylogenetic tree further identified four subgroups in P. temperata, and the relationships among these subgroups were confirmed by gyrase subunit B (gyrB) gene sequence analysis. The subgroup containing the GPS11 bacterial isolate differs from other subgroups in sequences of 16S rRNA and gyrB gene, physiological traits, nematode host species, and geographic origin. Therefore, the subgroup comprising the GPS11 bacterial isolate is proposed here as a new subspecies: Photorhabdus temperata subsp. stackebrandtii subsp. nov. (type strain GPS11). The type strain has been deposited in ATCC and DSMZ collections.