Entomopathogenic Nematodes Isolates Research Articles

Potential of entomopathogenic nematode isolates from Rwanda to control the tomato leaf miner, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae)

Tomato leaf miner, Tuta absoluta (Meyrick), is a major threat to tomato production as it can cause up to 100% yield loss under both greenhouse and open-field conditions. Chemical control, which is associated with several undesirable effects, remains the only option readily available for this pest since its invasion of Rwanda in the year 2015. This study assessed the potential of using local isolates of entomopathogenic nematodes (EPNs) in management of T. absoluta in Rwanda. Six EPNs including four locally isolated strains: Steinernema sp. RW14-M-C2a-3, Steinernema sp. RW14-M-C2b-1, S. carpocapsae RW14-G-R3a-2 and Heterorhabditis bacteriophora RW14-N-C4a, and two exotic species: S. carpocapsae All and H. bacteriophora H06 were evaluated. Three bioassays were conducted in the laboratory, using a tomato leaflet with third instar T. absoluta larva in gallery and 9-cm Petri dishes as bioassay arenas in a completely randomized design with three replications. The EPNs were applied at a volume of 1 ml containing 500 infective juveniles per leaflet, while sterile tap water was used as negative control. Larval mortality was checked continuously for 96 h at 24 h interval. The results revealed that all the tested EPNs were able to find and kill T. absoluta larvae inside the leaf galleries; and their efficacy increased with exposure time. The pathogenicity effects were significantly different (p < 0.05) among EPNs. In the first 24 h after inoculation, the efficacy of local EPN isolates (53.3–96.7%) was significantly higher than the one of exotic species (0.0–26.7%). The efficacy of three Rwandan EPN isolates, Steinernema sp. RW14-M-C2a-3, Steinernema sp. RW14-M-C2b-1, and S. carpocapsae RW14-G-R3a-2 was not significantly different from 24 to 96 h after inoculation, except for S. carpocapsae RW14-G-R3a-2 during 24 h after inoculation in bioassay 3. There was insignificant difference among all the EPN isolates after 96 h of exposure. This is the first study carried out in Rwanda that investigated the potential of locally isolated EPNs against T. absoluta. Field experiments should be conducted to fully explore the possibilities of using local EPN isolates in integrated pest management of T. absoluta in Rwanda.

Potential of four Turkish isolates of entomopathogenic nematodes against three major stored products insect pests

The insecticidal effect of native entomopathogenic nematode (EPN) isolates (Steinernema feltiae UTP-5 isolate, Heterorhabditis bacteriophora UMK-7 isolate, S. feltiae DDKY-11 isolate and H. bacteriophora AVB-15 isolate) recovered from the Cappadocia Region of Turkey were investigated on the adults of S. granarius and R. dominica and the larvae of E. kuehniella at different temperatures (15 °C, 20 °C and 25 °C) and concentrations (250, 500 and 1000 IJs/adult for S. granarius and R. dominica; 10, 50 and 100 IJs/larvae for E. kuehniella) under controlled conditions. Insect mortality was recorded after 4th, 6th and 8th day of exposure time for S. granarius and R. dominica, and 2nd and 4th day for E. kuehniella. The results showed that the efficacy of the isolates at 25 °C was generally higher than the ones at 15 °C and 20 °C. Steinernema feltiae DDKB-17 isolate caused the highest mortality (86%) against S. granarius adults at all temperatures tested. The virulence of the tested isolates on R. dominica adults did not exceed 44% at all temperatures and concentrations tested. UMK-7 and AVB-15 isolates were the most virulent isolates against E. kuehniella larvae with a mortality rate of 100%. Based on the results obtained from the laboratory tests, it appeared that the isolates tested have a good potential for the management of S. granarius, R. dominica and E. kuehniella.

VIRULENCE OF ENTOMOPATHOGENIC NEMATODE TO PUPAE AND ADULTS OF Drosophila suzukii IN LABORATORY

Drosophila suzukii is considered one of the most important pests of fruit farming. Due to its rapid expansion, control alternatives of this fly should be investigated. The use of entomopathogenic nematodes (EPNs) represents an important tool in the control. This study aimed to evaluate the virulence of EPNs isolates in pupae and the repercussion in adults of D. suzukii in laboratory. The experiment was conducted in a completely randomized design with five treatments and five replicates. Each plot consisted of a Petri dish lined with two sheets of filter paper. The isolates Heterorhabditis amazonensis IBCBn 24, Heterorhabditis indica IBCBn 05, Steinernema carpocapsae IBCBn 02 and Steinernema feltiae IBCBn 47 were inoculated into 2 mL at the concentration of 1,000 infective juveniles IJs/mL. The control treatment consisted of 2 mL distilled water. After inoculation, five pupae of D. suzukii were placed in the Petri dishes, which were then sealed and stored in a BOD climate chamber at 26 ± 1ºC, 70 ± 10% RH in the dark. Assessments were performed daily until the emergence of adults. Dead pupae and adults were dissected for the observation and quantification of IJs. The isolates, H. indica IBCBn 05, H. amazonensis IBCBn 24, S. carpocapsae IBCBn 02 and S. feltiae IBCBn 47, infected and made unviable 35.0, 16.0, 13.0 and 43.0% in pupae and 47.0, 80.0, 84.0 and 57.0 % in adults of D. suzukii. H. indica IBCBn 05 obtained the highest number of IJs produced in pupae and adults, 35.0 and 125.0.

Isolation, identification, and pathogenicity of entomopathogenic nematodes occurring in Cappadocia Region, Central Turkey

A survey for naturally occurring entomopathogenic nematodes (EPNs) was conducted in various agricultural fields in central Turkey, Nevsehir, between 2015 and 2016 years. EPNs were recovered from 20 of 112 soil samples (17.9%). Seventeen isolates were identified as Steinernema feltiae (Rhabditida: Steinernematidae). One was unknown Steinernema sp. (Rhabditida: Steinernematidae), and two were Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae). The most common species was S. feltiae, which was recovered from 5 out of 8 sites. The pathogenicity of these isolates was evaluated on the last larval instar of Galleria mellonella L. (Lepidoptera: Pyralidae) at different concentrations (25, 50, 100 IJs) in the laboratory at 25 ± 1 °C. Maximum mortality rate (87%) was achieved from the H. bacteriophora AVB-15 isolate at the lowest concentration after 48 h post-inoculation, while the only isolate that caused maximum mortality of (100%) at the concentration of 50 IJs/ml, 24 h post-inoculation was S. feltiae DDKB-17 isolate. All isolates showed high pathogenicity on G. mellonella last instar ranging from 63 to 100% at the concentration of 100 IJs/ml, 48 h post-inoculation. The present survey revealed that these EPNs are commonly present at Nevsehir, and they might have a good potential in biological control of insect pests.

Entomopathogenic nematodes in pharmaceutical formulations for Rhipicephalus microplus (Acari: Ixodidae) control: In vitro evaluation of compatibility, thermotolerance, and efficiency

This work aimed to investigate candidate biological control agents against the cattle tick Rhipicephalus microplus by studying three entomopathogenic nematodes (EPN) isolates from two species, Heterorhabditis bacteriophora (isolates LPP30 and HP88) and Heterorhabditis baujardi (isolate LPP7). The study comprised the pharmacotechnical development of four test formulations, and their biological evaluation, in three stages: 1) selection of the fittest EPN isolate at 16 ± 1 °C after 120 h of incorporation in the formulations; 2) determination of the effects of temperature and incubation time on the survival of the selected isolate in formulations; 3) evaluation of the in vitro efficacy (infectivity) of the selected EPN formulations and their components against R. microplus females after 72 h exposure. H. baujardi LPP7 presented the highest survival rates in 0.1% carboxymethyl cellulose sodium (CMC) formulations (100% at 25 °C, up to 10 days after incorporation). At the highest tested temperature (34.2 °C), 54% of LPP7 survived for up to 72 h in CMC formulations. The infectivity of LPP7-containing formulation against R. microplus was 99%, reducing oviposition, egg production index and larval hatching. Considering the pressing need for safer acaricide products, incorporation of EPNs in pharmaceutical formulations is presented as a promising approach for the biological control of cattle tick infestations, potentially reducing environmental impact and resistance development associated with chemical acaricides.

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.

Parasitic potential of entomopathogenic nematode Heterorhabditis indica against two Lepidopteran insect pests of cotton, Helicoverpa armigera (Hubner) and Spodoptera litura (Fabricious)

Ten isolates of entomopathogenic nematode (EPN) Heterorhabditis indica were evaluated for their differential infectivity against two major cotton insect pests viz., Helicoverpa armigera and Spodoptera litura. The results of bioassay revealed that the third instar larvae of H. armigera were relatively more susceptible to the EPN isolates compared to the larvae of S. litura. All the ten EPN isolates could reproduce successfully on H. armigera larvae however, the isolates viz., CICR-Su and CICR-SUB exhibited relatively higher virulence. On the other hand, on S. litura larvae only one isolate- CICR-BBFNS2 was able to produce infective juveniles and the rest failed to do so. Analysis of haemolymph composition of EPN infected H. armigera and S. litura larvae indicated the phase change of bacterial symbionts as a primary reason for the failure of EPN to reproduce and multiply in S. litura larvae. Higher percentage of primary phase colonies of bacterial symbionts were recorded in H. armigera larvae which resulted in successful progeny production of EPN while secondary phase colonies were predominant in the larvae of S. litura which resulted in failure of EPN isolates to reproduce. Phylogenetic tree made using internal transcribed spacer (ITS) region of ribosomal DNA of nematodes and 16sRNA of bacterial symbionts revealed the distinctness and uniqueness of the EPN isolate CICR-BBFNS2 which was able to multiply on the larvae of both H. armigera and S. litura from rest of the nine isolates which could multiply only in H. armigera. The results indicate that isolate CICR-BBF-NS2 has potential for deployment in IPM programme for management of H. armigera and S. litura.

In vivo production of entomopathogenic nematodes using Galleria mellonella: costs and effect of diets on nematode pathogenicity.

Five separate diets – beeswax (BW), glycerol (Gly), and three types of dog croquettes (DC1, DC2, and DC3) – were used to rear larvae of the greater wax moth, Galleria mellonella. The larvae were later inoculated with five different isolates of entomopathogenic nematodes (EPN) emerging from the insect larvae reared on the five diets. Insect cadavers were then conserved for 1, 2, 3, and 4 wks (T1, T2, T3, and T4, respectively) to evaluate the nematodes’ progeny production and to assess the pathogenicity of emerging nematodes to G. mellonella larvae. Larvae fed on DC1 weighed the least (0.18 ± 0.03 g) and those fed on Gly weighed the most (0.22 ± 0.04 g). Gly was effective for insect development but was the most expensive to produce at 6.30 US dollars/kg. No significant difference (P = 0.851) was observed between mortality rates of larvae for nematode isolates for the three best diets (Gly, BW, and DC1) during 1 wk (T1) and 3 wks (T3) after processing at 3 d post-inoculation. All nematode isolates emerged and had the highest population density per insect larva at T1 for isolate Ze4 (Heterorhabditis sonorensis) on BW (553.63 ± 311.97 infective juveniles (IJs)/50 μl of suspension, 276,815 IJs/larva) and at T2 on DC1 (488.63 ± 321.37 IJs/50 μl, 244,315 IJs/larva) and for isolate Aglali (H. sonorensis) at T1 on Gly (615.18 ± 309.63 IJs/50 μl, 307,590 IJs/larva). This study shows the costs and effectiveness of different diets on development and production of G. mellonella larvae and the EPN produced in vivo.

Pathogenicity of Entomopathogenic Nematode on Sugarcane White Grub Lepidiota stigma (Coleoptera: Scarabaeidae)

&lt;p&gt;Sugarcane (Saccharum officinarum) is one of high-value commodities in Indonesia for producing sugar. Sugarcane production recently reduced due to insect pests attacked, mainly white grub Lepidiota stigma (Coleoptera: Scarabaeidae). Utilization of entomopathogenic nematodes (EPN) is one of the alternative control methods for sugarcane white grub. The aim of the present&lt;br /&gt;study was to select the higher pathogenicity of EPN isolates for controlling the sugarcane insect pest. The study was conducted in Insect Pathology Laboratory of Indonesia Sweeteners and Fiber Crops Research Institute. Nine isolates of EPN, e.g. DKS-1, AGH-1, DKH-1, DKH-5, NH-1, NH-2, PH-1, PH-2, and PH-4 and one untreated control were tested for their pathogenicity against sugarcane white grub, L. stigma. Each treatment was arranged in Completely Randomized Design with three replications. Every treatment consisted of 30 individuals of the third instar of white grub which treated by 2 × 104 infective juvenile or IJ of EPN&lt;br /&gt;isolates. Parameter observed was the mortality of sugarcane white grub, L. stigma. The result showed that all of EPN isolates tested were promising pathogenic against the white grub with about 10 to 80% of the average percentage of mortality. However, DKS-1 and PH-1 showed more pathogenic against L. stigma with about 80–90% and 70–80% of white grub mortality,&lt;br /&gt;respectively. The highest enhancement of white grub mortality occurred at 72 hours after treatment and it was showed by DKS-1 and PH-1 isolates which increased the percentage of white grub mortality about 57.1 and 50%, respectively. Obtaining the promising isolates of NPS with different host seeking strategies will potentially increase the effectivity of control against white&lt;br /&gt;grub with the result to increase the yield of sugarcane.&lt;/p&gt;

Reproduction of entomopathogenic nematodes of the genera Steinernema Travassos, 1927 (Rhabditida: Steienernematidae) within strawberry blossom weevil larvae Anthonomus rubi Herbst, 1795 (Coleoptera: Curculionidae)

Studies have been conducted on the contamination in the laboratory of larvae of raspberry-straw weevil (Anthonomus rubi Herbst, 1795) with two isolates of entomopathogenic nematodes (ETNs) of the genus Steinernema Travassos, 1927, vchecked the possibility of their reproduction under these conditions, and features of quantitative performance indicators. On the basis of the obtained data, it was established that, regardless of the type of isolate of the EPN, all experimental A. rubi larvae died on the 3rd day after infection, and the yield of larvae nematodes from the dead insects began on day 10—12 and lasted 42—44 days. During this period, on average, from one of the affected specimens of raspberry-straw weevil, from 26965 to 29168 larvae of the EPN was allocated. The total number of larvae of EPN obtained in the infection of 14 larvae of weevil of raspberry-strawberry was 392929 individuals. The migration of newly-formed individuals of the EPN was uneven, the bulk of the larvae (61—66.2%) is released during the second decade of the month, the rest — 24.5%—31.2% in the third decade. In the first and last decades the larvae are significantly less (3.5—5.8%).

Sensitivity of caterpillars of the pine tree lappet moth Dendrolimus pini to native isolates of entomopathogenic nematodes

The pine tree lappet moth Dendrolimus pini (Lasiocampidae) is one of the most dangerous pests of pine Pinus sylvestris stands in Central Europe. To develop biological control method of this pest, the bioassay was made to assess the sensitivity of D. pini caterpillars to native EPMs strains representing two species: Steinernema feltiae and Heterorhabditis megidis in laboratory conditions. The results showed higher activity of S. feltiae strains which parasitized 86.7 - 100% of D. pini caterpillars, compared to the 20-100% of insects parasitized by H. megidis strain. Most nematodes were found in caterpillars treated with S. feltiae (in average 40 IJs/caterpillar), the least – in individuals parasitized by H. megidis (in average IJs/caterpillar). No effect of dose and temperature on parasitizm was found with S. feltiae strains while in H. megidis increased doses resulted in higher extensity of parasitism. The results indicate higher biological activity of S. feltiae strains and advisability of their further studies in field experiments.

Isolation and growth inhibition potential of entomopathogenic nematodes against three public health important mosquito vectors

The prevalence of mosquito vector borne diseases and the resistance of mosquitoes to conventional pesticides have been of important public concern to the mosquito endemic countries. Present study was conducted to identify the native bio-larvicidal potential of the entomopathogenic nematodes; Steinernema siamkayai (KPR-4) Heterohabditis indica (KPR-8), Steinernema glaseri and Steinernema abbasi. The isolated nematodes were subsequently cultured and evaluated their larvicidal potential against the larvae of Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus. Among the tested four different nematode species, the S. abassi exerted the highest mortality against A. aegypti (97.33%), the H. indica (KPR-8) against A. stephensi (97.33%) and the S. siamkayai (KPR-4) against C. quinquefasciatus (98.67%). The maximal mosquito-larvicidal property of EPNs was found with the LC50 and LC90 values (IJs/larvae): S. abbasi = 12.47 & 54.35 on A. aegypti; H. indica KPR-8 = 19.88 & 66.81 on A. stephensi and S. siamkayai KPR-4 = 16.69 & 58.97 on C. quinquefasciatus, respectively. The presently generated data on the molecular and larvicidal characteristics of the entomopathogenic nematodes form an important baseline data that upon further research would lead to the development of eco-friendly mosquito-control agent.

Efficacy of entomopathogenic nematode isolates from Turkey and Kyrgyzstan against the larvae of the mosquito species Culex pipiens L. (Diptera: Culicidae) under laboratory conditions

Mosquitoes (Diptera: Culicidae) are among the most important of the pests in urban entomology, and they not only disturb people but they are also an important health hazard as they are diseases’ vectors. The biological control methods against this pest, which is difficult to fight off, have recently gained importance. A total of five different entomopathogenic nematodes (EPNs), Steinernema feltiae (Aydın isolate), S. carpocapsae (Karadeniz isolate), and Heterorhabditis bacteriophora (Aydın isolate) isolated from Turkey and S. feltiae (KG3) and H. bacteriophora (KG81) isolated from Kyrgyzstan, were tested against the mosquito species Culex pipiens L. (Diptera: Culicidae) larvae under laboratory conditions. The experimental nematode suspensions were determined as 500, 1000, and 1500 IJs ml−1. The mortality rates in the trial were recorded after 24, 48, 72, 96, and 120 h. Dead mosquitoes were dissected under a microscope and confirmed to have died by the EPNs. Larval mortality was observed in all EPN species compared to the control group. H. bacteriophora (KG81) and S. carpocapsae isolates were found to be the most effective isolates with 100% larval mortality. The other isolates were H. bacteriophora (Aydın isolate) (70%), S. feltiae (KG3) (66.67%), and the most ineffective isolate S. feltiae (Aydın isolate) (13.3%).

Evaluation of local isolates of entomopathogenic nematodes for the management of black cutworm, Agrotis ipsilon Hufnagel (Lepidoptera: Noctuidae)

The black cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae) is considered to be one of the serious polyphagous pests that spend a large part of its life in the soil environment, where many microorganisms live including entomopathogenic nematodes (EPNs) (Rhabditida: Steinernematidae and Heterorhabditidae). EPNs have been long used for suppressing the soil-dwelling insects like cutworms and are successful biological control agent against A. ipsilon larvae. In the present study, the efficacy of local EPNs isolates against the fourth larval instar of A. ipsilon was evaluated at different concentrations (10, 25, 50, 100 IJs/larva/Petri dish and 25, 50, 100 IJs/cm2 soil) in two different experiment environments including filter papers in Petri dishes and soil in plastic containers under laboratory conditions at 25 ± 1 °C. Larval mortalities of A. ipsilon were recorded first, second, third, and fourth day post inoculation where the mortality rates increased by increasing the concentrations. The maximum mortality rate (100%) was reached within 2 days after inoculation, inoculating the Heterorhabditis bacteriophora FLH-4-H and H. indica 216-H isolates at concentrations of 50 and 100 IJs/cm2, in the plastic container experiment. The highest mortality rate (90%) was obtained by Steinernema carpocapsae E76-S isolate at a concentration of 100 IJs/larva/Petri on the fourth day after inoculation, in the Petri dish experiment. The lethal concentration values (LC50 and LC90) of the A. ipsilon larval population were 52 IJs and 129 IJs, respectively, for S. feltiae E76-S isolate in the Petri dish tests. In the plastic container experiment, the lowest LC50 and LC90 values found to be 17 IJs and 23 IJs, respectively, for H. bacteriophora FLH-4-H isolate. The results showed that all indigenous EPN isolates had good potentials in the management of A. ipsilon.

Effect of the entomopathogenic nematode-bacterial symbiont complex on Meloidogyne hapla and Nacobbus aberrans in short-term greenhouse trials

Meloidogyne hapla and Nacobbus aberrans are plant-parasitic nematodes that form galls in the roots of infected plants and cause important economic losses. Entomopathogenic nematodes (EPNs) of the genera Steinernema and Heterorhabditis infect and kill insects via toxins produced by their symbiotic bacteria. EPNs have shown to have an antagonistic effect on different plant-parasitic nematode species in field and greenhouse trials. The aim of the present work was to evaluate, in tomato plants in greenhouse, the effect of the application of three Argentine EPN isolates, their symbiotic bacteria and cell-free supernatants, on a population of M. hapla and two populations of N. aberrans. Sixty days after inoculation, the number of galls and egg masses, the nematode reproduction factor (RF) and plant biomass were calculated. With a few exceptions, biomass was not affected by the different treatments. None of the plant-parasitic nematode populations was reduced by infective juvenile inoculation of the different EPN isolates. Bacterial action differed among populations; M. hapla was the most susceptible one, with a significant reduction in the number of galls, egg masses and RF caused by the application of the three bacterial strains. The most significant effect was produced by the cell-free supernatants on nematode RF, with reductions of 62–90%, caused by bacterial metabolites. The different inoculation alternatives of the EPN-bacterial symbiont complex tested in the present work (infective juveniles, bacteria and cell-free supernatant) are compared for the first time for plant-parasitic nematode species.

Long-term Persistence of Native New York Entomopathogenic Nematode Isolates Across Crop Rotation.

Entompathogenic nematodes are found worldwide in a wide array of soil habitats with a broad host range and significant variation in foraging strategies. The primary use of entomopathogenic nematodes (EPNs) in managed plant systems has been focused on inundative releases in a biopesticide strategy. Little effort has been placed in investigating the use of natural occurring or adapted EPN strains for long-term suppression of pest outbreaks in managed systems. This study examined the potential of EPN isolates from Northern New York (NNY), inoculated at a low level (250 million IJ/ha), which are climate adapted and their persistent characteristics preserved to maintain population levels in agricultural fields (N = 82) for multiple years and across crop rotation (alfalfa:corn:alfalfa). Persistence levels for Steinernema carpocapsae (Weiser) (Rhabditida:Steinernematidae) ranged between 8 and 12% of the soil cores assayed in continuous alfalfa and 1-14% of the soil cores assayed in continuous corn rotated from EPN treated alfalfa. Steinernema feltiae (Filipjev) (Rhabditida: Steinernematidae) residual persistence level ranged between 17 and 32% in continuous alfalfa and 22-41% in continuous corn rotated from EPN treated alfalfa. Combined EPN level ranged between 27 and 43% of the soil cores in continuous alfalfa and 28-55% in continuous corn rotated from EPN-treated alfalfa. Inspection of individual fields suggested EPN populations established in prior years at the residual soil core level of 18-35% can respond positively to an increase of susceptible hosts in both alfalfa and corn, often increasing their presence to 100%.

Assessment of entomopathogenic nematodes in Agrotis ipsilon (Lepidoptera: Noctuidae) under laboratory and greenhouse conditions

This work aims to evaluate the potential of Brazilian isolates of entomopathogenic nematodes through the selection of isolates for Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae). In order to analyze the susceptibility at different instars of the insect and different concentrations, to investigate the in vivo production of these isolates, and to perform greenhouse tests in the Laboratory of Entomologia e Controle Microbiano of Universidade Estadual do Norte do Paraná, Brazil. Selection tests were carried out with nine isolates, and those that caused greater percentages of insect mortality were used in concentration tests (0, 50, 100, 150, 200 Infective Juveniles (IJs)/cm2), with different instars of the insect (2nd, 3rd, 4th, 5th, 6th instars and pupa), in the in vivo production of A. ipsilon larvae, and in pathogenicity tests in a greenhouse. In the selection test, the isolate CH3 (Steinernema sp.) caused 100 % of mortality in A. ipsilon, and the isolates GL (Heterorhabditis amazonensis), IBCBn-02 (S. carpocapsae) and IBCB-n05 (H. indica) caused 90 % mortality. In the tests of different concentrations, the isolate IBCB-n02 had CL99 estimated at a concentration of 140 IJs/cm2, and the isolate IBCB-n05 had CL99 estimated at a concentration of 190 IJs/cm2. The most susceptible larval stages were the 3rd and 4th instars. In the in vivo test for production of A. ipsilon larvae, no difference was observed between the two isolates. In the greenhouse test, the isolate IBCB-n02 was the only one that differed from the control treatment.

Pathogenicity Evaluation of Eight Egyptian Isolates of Entomopathogenic Nematodes against Two Tick Species; Argas persicus and Boophilus annulatus

Eight Egyptian isolates of entomopathogenic nematodes (EPN), four belong to the genus Heterorhabditis and four belong to Steinernema were evaluated against adult females of the soft tick Argas persicus and engorged females of the hard tick, Boophilus annulatus in the laboratory. Results indicated that two EPN isolates (A4 and 10k) of Heterorhabditis were rapidly fatal and have high pathogenicity (100% mortality after 72 hours) against A. persicus when treated with 1000 nematode Infective Juveniles (IJs/ml). Meanwhile, only one isolate (5B) of Steinernema gave accumulated mortality of 93.33% against A. persicus.Furthermore, Heterorhabditis isolates (10 K and A4) caused 93.33% and 86% mortality to B. annulatus. The most effective EPN isolates of Steinernema were 5B and AT4 against B. annulatus, they both gave ca 86.7% mortality. The LC50 values of Heterorhabditis spp. (A4 and 10K isolates) after 72 hrs post infection and LT50 were (27.03 and 257.03 IJs/ml) and (11.94 and 19.2 hrs.), respectively against A. persicus. While, the LC90 was 360.24 and 765.72 IJs/ml for A4 and 10K isolates, respectively. Meanwhile, A4 and 10K isolates exhibited relatively high values of LC50, LC90 andLT50 against B. annulatus, represented by (358.57±0.71 and 812.83 IJs/ ml ); (1258.9 and 2485.9 IJs/ml.) and (25.64 and 51.28 hrs.), respectively. The present study cleared that Entomopathogenic nematodes (EPN) isolates of the genus Heterorhabditis spp. (A4 and 10K) are potentially useful biological tools for tick control.

Compartive Efficiency of Egyptian Entomopathogenic Nematodes and Imidacloprid on the Red Palm Weevil, Rhynchophorus ferrugineus Oliv

The efficiency of three isolates of entomopathogenic nematodes indigenous in Egypt, (i. e. Heterorhabditis bacteriophora (EKB20), Steinernema sp. (B32)and Heterorhabiditis sp. (Kasassien isolate)) were determined in the laboratory and field compared with imidacloprid insecticide on the larvae and adults of the red Palm weevil, Rhynchophorus ferrugineus.Results showed that isolates of entomopathogenic nematodes, B32 and EKB20 are more effective than Kasassien against R. ferrugineus larvae at inoculum levels of 1000 and 2000 Infective Juveniles (IJs)/mL. Both isolates of B32 and EKB20 were faster killers achieving more than 90% mortality to the 3rd instar larvae of the red palm weevil after 72 hrs. Field evaluation showed that Kasassien and EKB20 achieved higher levels of control against diferent stages of R. ferrugineus reaching100% reduction than the isolate B32 (80.33 % reduction). When the level of palm weevil infestation was moderate (1-3 weevil tunnels/ tree, the reduction percent was 80.33, 100 and 80.66% when the trees were treated with EPN isolates of EKB20, Kasassien and B32 respectively. There was no significant effect when the trees with higher infestation (more than 3 tunnels/palm tree) level were treated with EPN. While as imidacloprid insecticide gave 100% mortality in the three levels of infestation. The current work has indicated that EPN are considered promising biocontrol agents, if correctly applied and released in an integrated control schemes against the red palm weevil.

Entomopathogenic Nematodes (EPN): Diversity in Indian Tropical Sugarcane Ecosystem and Its Biocontrol Potential Against White Grub Holotrichia serrata F. on Sugarcane

The root-feeding larvae of white grub Holotrichia serrata F. (Coleoptera: Scarabaeidae) causes serious damage in sugarcane fields in India. Generally, the damage caused by white grubs is in patches, but during severe outbreaks the entire crop in a field may be dried up. Entomopathogenic nematodes (EPN) have proven to be the safest and most effective way of controlling white grubs as both organisms spends a large portion of their life cycle in the soil. To ascertain the diversity of EPN, a survey was conducted in the white grub endemic areas of sugarcane ecosystem in the tropical India. Isolation, molecular identification of EPN and laboratory bioassays was conducted at Nematology Laboratory, ICAR-Sugarcane Breeding Institute, Coimbatore, India. Twenty-four EPN were isolated out of 243 samples, and frequency of occurrence of EPN was 9.87%. Among the 24 positive samples, eight EPN (three Heterorhabditis and five Steinernema) were isolated from dead larvae of naturally infested third instar white grub H. serrata. Out of 24 positive samples, 14 (58.3%) were heterorhabditis and 10 were (41.6%) sterinernematids. Sequencing and characterisation of the internal transcribed spacer region was used to identify all nematode isolates to species level. Out of 14 Heterorhabditis isolates, eight have grouped with H. indica in the indica-group and remaining six were unknown species. Out of 10 Steinernema isolates, two were grouped with Steinernema siamkayai and two isolates grouped close to S. backanense, while the remaining six unknown isolates clustered closely with S. affine, but these isolates form a separate cluster which is genetically different from other group of Steinernema spp. Bioassay studies were conducted under laboratory conditions against first, second and third instar larvae of white grub H. serrata. Among the five H. indica isolates, H. indica (DSM78) recorded maximum mortality of the grubs and low median lethal dose (LD50) value for all the three instars of white grub. Two field trials were conducted during June 2011 to evaluate the efficacy of H. indica (DSM78) against naturally infested white grub H. serrata sugarcane fields. In both the trials, reduction in number of grubs observed in the EPN treated plots compared to the control. The reduction in grub population due to EPN treatment ranged between 43.5 and 77% at field trial I and 30–78% at field trial II. The results of the present study clearly indicated that EPN, H. indica isolate DSM78 was found to be effective and a potential EPN isolate against all the three instars of white grub larvae under laboratory bioassays as well as in the field conditions.