Endophyte infection in perennial ryegrass reduces the susceptibility of black cutworm to an entomopathogenic nematode

Abstract The perennial ryegrass, Lolium perenne, forms a symbiotic relationship with Neotyphodium lolii, a fungus that produces alkaloids. This relationship provides a competitive advantage to the host plant in grassland communities by increasing drought tolerance, and disease and herbivore resistance. Black cutworm, Agrotis ipsilon, is among the few insect species that are able to feed and develop on endophytic perennial ryegrass. Some insects can use plant secondary compounds to defend themselves against predators, therefore we hypothesized that the cutworms fed on endophytic grasses would exhibit greater defense against a lethal endoparasitic nematode, Steinernema carpocapsae. Laboratory experiments involving 4–5th instars support the hypothesis that A. ipsilon feeding on grass clippings from field plots with high (> 90%) incidence of endophyte infected perennial ryegrass are less susceptible to entomopathogenic nematodes than larvae fed grass clippings from plants with little or no incidence of endophyte. Laboratory studies resulted in similar overall mortality after 48 h. Field studies, however, show decreased susceptibility to S. carpocapsae when larvae were confined to areas of endophytic grass (> 75% infected). Early instars (2–3rd) fed on endophyte free grass suffered greater overall mortality at all nematode concentrations than 4–5th instars fed similarly. Early (2–3rd) instars were equally susceptible to nematode attack regardless of food source. Our results indicate that the fungal endosymbionts of grasses can influence the biology of natural enemies of an herbivorous insect.     

Comparative study of the entomopathogenic nematode, Steinernema carpocapsae, reared on mutant and wild-type Xenorhabdus nematophila

The symbiotic interaction between Steinernema carpocapsae and Xenorhabdus nematophila was investigated by comparing the reproduction, morphology, longevity, behavior, and efficacy of the infective juvenile (IJ) from nematodes reared on mutant or wild-type bacterium. Nematodes reared on the mutant X. nematophila HGB151, in which an insertion of the bacterial gene, rpoS, eliminates the retention of the bacterium in the intestinal vesicle of the nematode, produced IJs without their symbiotic bacterium. Nematodes reared on the wild-type bacterium (HGB007) produced IJs with their symbiotic bacterium. One or the other bacterial strain injected into Galleria mellonella larvae followed by exposing the larvae to IJs that were initially symbiotic bacterium free produced progeny IJs with or without their Xenorhabdus-symbiotic bacterium. The two bacterial strains were not significantly different in their effect on IJ production, sex ratio, or IJ morphology. IJ longevity in storage was not influenced by the presence or absence of the bacterial symbiont at 5 and 15 °C, but IJs without their bacterium had greater longevity than IJs with their bacterium at 25 and 30 °C, suggesting that there was a negative cost to the nematode for maintaining the bacterial symbiont at these temperatures. IJs with or without their symbiotic bacterium were equally infectious to Spodoptera exigua larvae in laboratory and greenhouse and across a range of soil moistures, but the absence of the bacterial symbiont inhibited nematodes from producing IJ progeny within the host cadavers. In some situations, such as where no establishment of an alien entomopathogenic nematode is desired in the environment, the use of S. carpocapsae IJs without their symbiotic bacterium may be used to control some soil insect pests.     

Effect of temperature on the development of <Emphasis Type="Italic">Steinernema carpocapsae</Emphasis> and <Emphasis Type="Italic">Steinernema feltiae</Emphasis> (Nematoda: Rhabditida) in liquid culture

For commercial use of the entomopathogenic nematodes Steinernema carpocapsae and Steinernema feltiae in biological control of insect pests, they are produced in liquid culture on artificial media pre-incubated with their symbiotic bacteria Xenorhabdus nematophila and Xenorhabdus bovienii, respectively. After 1 day of the bacterial culture, nematode dauer juveniles (DJs) are inoculated, which recover development. The adult nematodes produce DJ offspring, which are harvested and can be sprayed. This study determined optimal temperatures to obtain high DJ progeny within a short process time. Temperatures assessed were 23°C, 25°C, 27°C, and 29°C for S. carpocapsae and 20°C, 23°C, 25°C, and 27°C for S. feltiae. The recovery of inoculated DJs was hardly affected and was reduced only in S. carpocapsae at 29°C. The fecundity (eggs in uterus) in S. carpocapsae reached a maximum at 27°C; whereas, maximum yields were recorded at 25°C. For both Steinernema spp., highest DJ densities were obtained after 15 days incubation at 25°C. Optimal culture temperature for both nematode species is 25°C. S. carpocapsae was more sensible to suboptimal temperature than S. feltiae. Results on total DJ density and DJ proportion of the total nematode population were more variable at non-optimal temperature condition for S. carpocapsae than for S. feltiae. Suboptimal culture temperature also reduced DJ infectivity.     

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.     

In-silico analysis of genomic distribution and functional association of hipBA toxin-antitoxin (TA) homologs in entomopathogen Xenorhabdus nematophila

Bacteria have a particular strategy to invade the host immune system by forming an undetectable dormant state that may resuscitate and cause disease even after inhabiting for years in a host body. Several mechanisms are known to be responsible for bacterial dormancy, among them the hipBA toxin-antitoxin (TA) system which was initially identified in Escherichia coli. Here we explore the genomic distribution and functional association of hipBA TA homologs from an entomopathogenic bacterium Xenorhabdus nematophila. This bacterium is a symbiotic model with the nematode Steinernema carpocapsae. We found that HipA toxin homologs are more closely related than HipB antitoxins and have satisfactory adenine (for HipA homologs) and nucleic acid (for HipB homologs) ligand partners with a typical TA interaction network that may promote the X. nematophila towards a stringent response to form the dormant state. Such homologs distribution is an inclusion in the current TA repertoire of X. nematophila.     

Comparison of Assays for the Determination of Entomogenous Nematode Infectivity

Injection, contact, and soil assays were used to compare infectivity of Heterorhabditis bacteriophora strain HP88 and Steinernema carpocapsae strain All to final instar Galleria mellonella larvae. Under comparable assay conditions, H. bacteriophora produced less Galleria mortality and showed greater within-assay variability in infectivity than S. carpocapsae. Injection of individual S. carpocapsae or H. bacteriophora infective juveniles into Galleria indicated that a comparatively greater percentage of S. carpocapsae was capable of initiating infection. In addition to nematode species, other major components of variability in assay estimations of nematode infectivity were number of nematodes used in the assay, assay type, date of the assay, and possibly, Galleria age.     

Contribution of ergot alkaloids to suppression of a grass-feeding caterpillar assessed with gene knockout endophytes in perennial ryegrass

Neotyphodium and Epichloë species (Ascomycota: Clavicipitaceae) are fungal symbionts (endophytes) of grasses. Many of these endophytes produce alkaloids that enhance their hosts’ resistance to insects or are toxic to grazing mammals. The goals of eliminating from forage grasses factors such as ergot alkaloids that are responsible for livestock disorders, while retaining pasture sustainability, and of developing resistant turf grasses, require better understanding of how particular alkaloids affect insect herbivores. We used perennial ryegrass Lolium perenne L. (Poaceae) symbiotic with Neotyphodium lolii × Epichloë typhina isolate Lp1 (a natural interspecific hybrid), as well as with genetically modified strains of Lp1 with altered ergot alkaloid profiles, to test effects of ergot alkaloids on feeding, growth, and survival of the black cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae), a generalist grass‐feeding caterpillar. Neonates or late instars were provided clippings from glasshouse‐grown plants in choice and rearing trials. Wild‐type endophytic grass showed strong antixenosis and antibiosis, especially to neonates. Plant‐endophyte symbiota from which complex ergot alkaloids (ergovaline and lysergic acid amides such as ergine) or all ergot alkaloids were eliminated by endophyte gene knockout retained significant resistance against neonates. However, this activity was reduced compared to that of wild‐type Lp1, providing the first direct genetic evidence that ergot alkaloids contribute to insect resistance of endophytic grasses. Similarity of larval response to the two mutants suggested that ergovaline and/or ergine account for the somewhat greater potency of wild‐type Lp1 compared to the knockouts, whereas simpler ergot alkaloids contribute little to that added resistance. All of the endophyte strains also produced peramine, which was probably their primary resistance component. This study suggests that ergot alkaloids can be eliminated from an endophyte of perennial ryegrass while retaining significant insect resistance.     

Pepsin-like aspartic protease (Sc-ASP155) cloning,molecular characterization and gene expression analysis in developmental stages of nematode Steinernema carpocapsae

Steinernema carpocapsae is an insect parasitic nematode associated with the bacterium Xenorhabdus nematophila. These symbiotic complexes are virulent against the insect host. Many protease genes were shown previously to be induced during parasitism, including one predicted to encode an aspartic protease, which was cloned and analyzed in this study. A cDNA encoding Sc-ASP155 was cloned based on the EST fragment. The full-length cDNA of Sc-ASP155 consists of 955 nucleotides with multiple domains, including a signal peptide (aa1–15), a pro-peptide region (aa16–45), and a typical catalytic aspartic domain (aa71–230). The putative 230 amino acid residues have a calculated molecular mass of 23,812 Da and a theoretical pI of 5.01. Sc-ASP155 blastp analysis showed 40–62% amino acid sequence identity to aspartic proteases from parasitic and free-living nematodes. Expression analysis showed that the sc-asp155 gene was up-regulated during the initial parasitic stage, especially in L3 gut and 6 h induced nematodes. Sequence comparison revealed that Sc-ASP155 was a member of an aspartic protease family and phylogenetic analysis indicated that Sc-ASP155 was clustered with Sc-ASP113. In situ hybridization showed that sc-asp155 was expressed in subventral cells. Additionally, we determined that sc-asp155 is a single-copy gene in S. carpocapsae. Homology modeling showed that Sc-ASP155 adopts a typical aspartic protease structure. The up-regulated Sc-ASP155 expression revealed that this protease could play a role in the parasitic process. In this study, we have cloned the gene and determined the expression of the pepsin-like aspartic protease Sc-ASP155 in S. carpocapsae.     

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

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

NilD CRISPR RNA contributes to Xenorhabdus nematophila colonization of symbiotic host nematodes

The bacterium Xenorhabdus nematophila is a mutualist of entomopathogenic Steinernema carpocapsae nematodes and facilitates infection of insect hosts. X. nematophila colonizes the intestine of S. carpocapsae which carries it between insects. In the X. nematophila colonization‐defective mutant nilD6::Tn5, the transposon is inserted in a region lacking obvious coding potential. We demonstrate that the transposon disrupts expression of a single CRISPR RNA, NilD RNA. A variant NilD RNA also is expressed by X. nematophila strains from S. anatoliense and S. websteri nematodes. Only nilD from the S. carpocapsae strain of X. nematophila rescued the colonization defect of the nilD6::Tn5 mutant, and this mutant was defective in colonizing all three nematode host species. NilD expression depends on the presence of the associated Cas6e but not Cas3, components of the Type I‐E CRISPR‐associated machinery. While cas6e deletion in the complemented strain abolished nematode colonization, its disruption in the wild‐type parent did not. Likewise, nilD deletion in the parental strain did not impact colonization of the nematode, revealing that the requirement for NilD is evident only in certain genetic backgrounds. Our data demonstrate that NilD RNA is conditionally necessary for mutualistic host colonization and suggest that it functions to regulate endogenous gene expression.     

Virulence of entomopathogenic nematodes and their symbiotic bacteria against the hazelnut weevil Curculio nucum

The virulence of different entomopathogenic nematode strains of the families Steinernematidae and Heterorhabditidae, isolates from Catalonia (NE Iberian Peninsula), and their symbiotic bacteria was assessed with regard to the larvae and adults of the hazelnut weevil, Curculio nucum L. (Coleoptera: Curculionidae). The nematode strains screened included one Steinernema affine, five Steinernema feltiae, one Steinernema carpocapsae, one Steinernema sp. (a new species not yet described) and one Heterorhabditis bacteriophora. The pathogenicity of all the strains of nematodes was tested on larvae and only four of them on adults of the hazelnut weevil. Larval mortality ranged from 10% with S. affine to 79% with Steinernema sp. Adult mortality was higher in S. carpocapsae, achieving 100% adult weevil mortality. The pathogenicity of the symbiotic bacteria Xenorhabdus bovienii, X. kozodoii, X. nematophila and Photorhabdus luminescens was studied in larvae and adults of C. nucum. In the larvae, X. kozodoii showed a LT50 of 22.7 h, and in the adults, it was 20.5 h. All nematodes species except S. affine tested against larvae showed great potential to control the insect, whereas S. carpocapsae was the most effective for controlling adults.     

Entomopathogenic nematodes induce components of systemic resistance in plants: Biochemical and molecular evidence

Antagonism between entomopathogenic nematodes (EPNs) and plant-parasitic nematodes (PPNs) has been documented over the past two decades but its mechanism and ecological significance remain elusive. We investigated the effects of Steinernema carpocapsae and its symbiotic bacterium, Xenorhabdus nematophila applied to the potting medium on pyrogallol peroxidase (P-peroxidase), guaiacol peroxidase (G-peroxidase) and catalase activities in Hosta sp. and Arabidopsis thaliana leaves as components of induced systemic resistance. We found that P-peroxidase activity was significantly higher in the leaves from hosta plants treated with S. carpocapsae infective juveniles (IJs) and S. carpocapsae infected insect cadavers than in the leaves from the control plants 2 weeks after treatment. The G-peroxidase activity was significantly higher in S. carpocapsae infected cadaver and X. nematophila treatments 10 and 15 days after treatment (DAT) and in S. carpocapsae IJs treatment 5 and 15 DAT. The catalase activity in hosta leaves was significantly higher in S. carpocapsae infected cadaver and X. nematophilus treatments compared with the control 5 and 15 DAT and in S. carpocapsae IJs treatment 5 and 10 DAT. Further, the catalase activity in A. thaliana leaves was significantly higher in S. carpocapsae IJs treatment than in the control 7 DAT. We also determined the effects of S. carpocapsae infected cadavers and S. carpocapsae IJs on PR1-gene expression in transgenic A. thaliana leaves through GUS (β-glucuronidase) activity assay and found that the PR1-gene was expressed in leaves from all treatments except the control. Thus, we conclude that the EPNs and their symbiotic bacteria can induce systemic resistance in plants which may explain the elusive antagonistic effect of EPNs on PPNs.     

Identification and Functional Characterization of a Xenorhabdus nematophila Oligopeptide Permease

The bacterium Xenorhabdus nematophila is a mutualist of Steinernema carpocapsae nematodes and a pathogen of insects. Presently, it is not known what nutrients the bacterium uses to thrive in these host environments. In other symbiotic bacteria, oligopeptide permeases have been shown to be important in host interactions, and we therefore sought to determine if oligopeptide uptake is essential for growth or symbiotic functions of X. nematophila in laboratory or host environments. We identified an X. nematophila oligopeptide permease (opp) operon of two sequential oppA genes, predicted to encode oligopeptide-binding proteins, and putative permease-encoding genes oppB, oppC, oppD, and oppF. Peptide-feeding studies indicated that this opp operon encodes a functional oligopeptide permease. We constructed strains with mutations in oppA₁, oppA₂, or oppB and examined the ability of each mutant strain to grow in a peptide-rich laboratory medium and to interact with the two hosts. We found that the opp mutant strains had altered growth phenotypes in the laboratory medium and in hemolymph isolated from larval insects. However, the opp mutant strains were capable of initiating and maintaining both mutualistic and pathogenic host interactions. These data demonstrate that the opp genes allow X. nematophila to utilize peptides as a nutrient source but that this function is not essential for the existence of X. nematophila in either of its host niches. To our knowledge, this study represents the first experimental analysis of the role of oligopeptide transport in mediating a mutualistic invertebrate-bacterium interaction.     

Characterization of the pleiotropic phenotype of an ompR strain of Xenorhabdus nematophila

Xenorhabdus nematophila is an insect pathogen that forms a symbiotic association with the nematode, Steinernema carpocapsae. Xenorhabdus is carried into the insect host by the nematode, is released into the hemolymph and participates in killing the insect. The bacteria grow to high concentrations supporting the development of the nematode in the hemolymph. OmpR is a global regulatory protein involved in the regulation of porin genes, motility, acid tolerance and virulence in several enteric bacteria. To study the role of ompR in the lifecyle of Xenorhabdus, an ompR -minus strain was constructed. The ompR strain produced markedly reduced levels of the porin protein, OpnP and was both hypermotile and exhibited a hyperhemolysis phenotype. Inactivation of flhDC, the master regulator for flagella synthesis, eliminated hemolysin production in the ompR strain, suggesting that ompR regulates hemolysin production via flhDC. The ompR mutant strain was virulent towards insect hosts. However, when nematodes were grown on a mixture of the wild-type and the ompR strain, only the wild-type strain was recovered indicating that ompR is required for competitive symbiotic interaction with the nematode. The role of ompR in the symbiosis between the bacterium and the nematode is under investigation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of insect host age and diet on the fitness of the entomopathogenic nematode-bacteria mutualism

Insect host age and diet were evaluated as potential factors that could affect the fitness of the entomopathogenic nematode-bacterium mutualistic partnership. Two nematode species were considered: Steinernema carpocapsae and Heterorhabditis sonorensis, together with their symbionts Xenorhabdus nematophila and Photorhabdus luminescens, respectively. The tobacco hornworm, Manduca sexta, was used as the insect host. Insect developmental stage was a factor that impacted nematode virulence. Non-wandering 5th instar M. sexta were found to be more susceptible to nematode infection compared to wandering 5th instars. This was more noticeable for S. carpocapsae than for H. sonorensis. The nutritional status of the host also had an effect on the fitness of the two nematode species tested. In general, insects fed with the reduced diet content were less susceptible to nematode parasitism. The least observed mortality (0.5 %) was in those M. sexta larvae exposed to the low H. sonorensis dose. Host diet also had an effect on the production of IJ progeny in the insect cadavers. For both nematode species tested, the highest yield of emerging IJs was observed from those insect hosts fed with the low nutrient diet and exposed to the highest nematode inoculum. However, for both nematode species tested, the nutritional status of the host did not significantly affect time of emergence of IJ progeny or the reassociation with their bacterial symbionts (expressed as cfu/IJ). This is the first study on the effect of insect host physiology on both EPN and their symbiotic bacteria fitness.     

Quantification of Invasion of Two Strains of Steinernema carpocapsae (Weiser) into Three Lepidopteran Larvae

Studies with last instar larvae of the fall armyworm, Spodoptera frugiperda (J. E. Smith), the black cutworm, Agrotis ipsilon (Hufnagel), and the greater wax moth, Galleria mellonella (L.) were used to quantify the invasive ability of two strains (All and Mexican) of Steinernema carpocapsae and to determine how factors in the bioassay procedure affect both nematode invasion and host mortality. Nematode invasive ability was variable, with 10-50% of nematodes successfully infecting the host. The percentage of infectives invading the host (invasion efficiency) was positively related to increases in length of host exposure time and number of hosts per arena, negatively related to increases in substrate surface area per host, and not affected by nematode concentration. There was a direct relationship between concentration applied and the number of nematodes invading the host. Mortality was less affected than invasion efficiency by bioassay conditions and appears to be a much less sensitive index of nematode activity than invasive ability.     

Entomopathogenic Nematodes for Biological Control of Pryeria sinica Moore (Lepidoptera: Zygaenidae) and Persistence on Euonymus japonica Thunberg Foliage

Korean entomopathogenic nematodes (EPN), Steinernema carpocapsae Pocheon strain, Steinernema sp. GSNUS-4 strain, Steinernema sp. GSNUS-14 strain, Steinernema sp. GSNUS-16 strain, Heterorhabditis sp. GSNUH-2 strain, and Heterorhabditis sp. GSNUH-3 strain were evaluated for the biological control of pellucid zygaenid, Pryeria sinica Moore (Lepidoptera: Zygaenidae), a leaf-feeding insect pest of Euonymus japonica Thunberg. In addition, persistence of S. carpocapsae Pocheon strain on E. japonica foliage was checked.Nematode species, strain, and concentration influenced infectivity of Korean EPNs against 3rd instar of P. sinica larvae. S. carpocapsae Pocheon strain was the most effective nematode.Larval mortality by S. carpocapsae Pocheon strain in the exposure experiments was low in both light and dark conditions representing 36.7% at 100 infective juveniles/larva in each dark and light condition. Mortality by fenitrothion, however, was 100% 3 days later. Infectivity of S. carpocapsae Pocheon strain was not different between non-exposed foliage and exposed foliage to sunlight for 30 min.Mortality of 4th instar of P. sinica larvae was very low for both S. carpocapsae Pocheon alone and S carpocapsae Pocheon with spreading agent. Application time of nematodes, shading, and leaf position on the tree influenced persistence of S. carpocapsae Pocheon strain on E. japonica foliage. Nematode survival on foliage was positively correlated with shading level and higher at the lowest level than the middle or upper level of the tree.     

The integrated use of chemical insecticides and the entomopathogenic nematode,Steinernema carpocapsae (Nematoda: Steinernematidae), for the control of sweetpotato whitefly,Bemisia tabaci (Hemiptera: Aleyrodidae)

The integration of chemical insecticides and infective juveniles of the entomopathogenic nematode Steinernema carpocapsae (Wesier) (Nematoda: Steinernematidae), to control second instars of the sweetpotato whitefly, Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) was investigated. Using a sand bioassay, the effects of direct exposure of S. carpocapsae for 24 h to field rate dilutions of four insecticides (spiromesifen, thiacloprid, imidacloprid and pymetrozine) on infectivity to Galleria mellonella larvae were tested. Although all chemicals tested, except spiromesifen, produced acceptable nematode infectivity rates, they were all significantly less than the water control. The effect of insecticide treatment (dry residues of spiromesifen, thiacloprid and pymetrozine and soil drench of imidacloprid) on the efficacy of the nematode against B. tabaci was also investigated. Nematodes in combination with thiacloprid and spiromesifen gave higher B. tabaci mortality (86.5% and 94.3% respectively) compared to using nematodes alone (75.2%) on tomato plants. There was no significant difference in B. tabaci mortality when using the chemicals imidacloprid, pymetrozine and spiromesifen in conjunction with nematodes compared to using the chemicals alone. However, using thiacloprid in combination with the nematodes produced significantly higher B. tabaci mortality than using the chemical alone. The integration of S. carpocapsae and these chemical agents into current integrated pest management programmes for the control of B. tabaci is discussed.