Insect cellular and chemical limitations to pathogen development: the Colorado potato beetle, the nematode Heterorhabditis marelatus, and its symbiotic bacteria

This research examines possible factors limiting pathogen development and reproduction in a novel host insect. The nematode Heterorhabditis marelatus and its symbiotic bacterium, Photorhabdus luminescens, kill 98% of nematode-treated Colorado potato beetle (CPB) prepupae, but the nematode reproduces in only 1-6% of beetles. We examined nematode/bacterial inhibition at each step of the normal developmental pathway to determine host feature(s) limiting nematode reproduction. We found that in vivo encapsulation of nematodes occurred in only 1.6% of CPB, and in 5% of in vitro hanging drops of hemolymph. Thus, the cellular defense system did not strongly limit nematode reproduction in the CPB. The symbiotic bacterium was negatively affected by a heat-labile factor found in the CPB's hemolymph which often caused the bacterium to switch from the primary form that produces antibiotics and nutrients necessary for the nematodes' development, to a secondary form that provides only limited nutrients. A 58 kDa protein was isolated and bioassayed for activity against P. luminescens, but caused a delay in bacterial growth rather than the primary-secondary form switch. Thus, the identity of the heat-labile factor could not be confirmed as being the 58 kDa protein. The heat-labile factor did not directly affect the nematode. The addition of lipids in the form of olive oil to heated CPB hemolymph allowed nematodes to reproduce in 17% of hanging drops, in contrast to zero reproduction in hemolymph without oil. Reproductive nematodes were smaller when grown in CPB hemolymph than in hemolymph of the highly susceptible Galleria mellonella. These data suggest that both the toxic heat-labile factor and a lack of appropriate nutrients alter the CPB-bacterium-nematode interaction. These factors preclude the use of this otherwise highly effective nematode-bacterial complex in the longterm control of the CPB.     

Lethal and sublethal effects of Iranian isolates of Steinernema feltiae and Heterorhabditis bacteriophora on the Colorado potato beetle, Leptinotarsa decemlineata

To determine the LC₅₀ values of two entomopathogenic nematodes against Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae) prepupae, different concentrations of the nematodes were tested in soil. Because of the different temperature requirements of the two nematode species, bioassay experiments were conducted at 20 ± 1°C and 27 ± 2°C for Steinernema feltiae Filipjev (Rhabditida: Steinernematidae) and Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae), respectively. Both the isolates were effective against L. decemlineata. LC₅₀ values of H. bacteriophora against progeny of field-collected adults and laboratory-reared adults were estimated as 8.5 and 7.6 IJ per prepupa, respectively. For S. feltiae the value was calculated as 51.2 IJ per prepupa against offspring of laboratory-reared adults of L. decemlineata only. Cellular encapsulation of both nematode species was observed. Sublethal nematode concentrations caused wing deformation and delayed metamorphosis which may affect Colorado potato beetle adult fitness.     

Effectiveness of entomopathogenic nematodes Steinernema feltiae and Heterorhabditis bacteriophora on the Colorado potato beetle Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae) under laboratory and greenhouse conditions

In laboratory and greenhouse studies, the invading ability, virulence, and mortality caused by Stinernema feltiae and Heterorhabditis bacteriophora were compared. After one and two days of exposure to either nematode species, the mortality of Colordo potato beetle (CPB) Leptinotarsa decemlineata larvae at different instars, third and fourth, was recorded and the number of nematodes invading cadavers was more than the number of nematodes inside the larvae at the late last instar (one day before pre-pupa). Two concentrations, 250 and 500 IJs/dish, infective juvenile nematodes/0.5 ml were tested on different CPB larval instar. S. feltiae was more effective, with fourth instar rather than third and late last instar. On the other hand, H. bacteriophora showed a very weak effect with L. decemlineata. Also it was clear that S. feltiae was more effective and faster than H. bacteriophora: more than 70% of larvae were killed within 24 hours compared with H. bacteriophora which killed 40% of larvae within 48–72 hours. A significant difference in invading efficiency was observed with concentration 2500 IJs/pot in the greenhouse test. The number of adult females found in the cadavers of L. decemlineata larvae was always higher than the number of males. Foliage application of S. feltiae and H. bacteriophora resulted in a significant reduction of the number of damaged leaves and a lower index of damage compared with that in the control. We conclude that S. feltiae has significant potential and can help in the management of the Colorado potato beetle.     

Compatibility of Soil Amendments with Entomopathogenic Nematodes

The impact of inorganic and organic fertilizers on the infectivity, reproduction, and population dynamics of entomopathogenic nematodes was investigated. Prolonged (10- to 20-day) laboratory exposure to high inorganic fertilizer concentrations inhibited nematode infectivity and reproduction, whereas short (1-day) exposures increased infectivity. Heterorhabditis bacteriophora was more sensitive to adverse effects than were two species of Steinernema. In field studies, organic manure resulted in increased densities of a native population of Steinernema feltiae, whereas NPK fertilizer suppressed nematode densities regardless of manure applications. Inorganic fertilizers are likely to be compatible with nematodes in tank mixes and should not reduce the effectiveness of nematodes used for short-term control as biological insecticides, but may interfere with attempts to use nematodes as inoculative agents for long-term control. Organic manure used as fertilizer may encourage nematode establishment and recycling.     

Orientation of Colorado potato beetle to natural and synthetic blends of volatiles emitted by potato plants

1 Behavioural responses of the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), to volatiles emitted from solanaceous host plants (potato and tomato), a non‐host legume (soybean), and 13 synthetic blends or three individual chemicals emitted by potato plants were investigated in laboratory bioassays. 2 Both male and female CPB were attracted to volatiles emitted by mechanically damaged potato foliage, but not to mechanically damaged tomato foliage; CPB offered a choice between the two damaged solanaceous plants did not show a preference. 3 Among 16 odourous blends or individual chemical components of potato plant emissions tested, six blends were attractive, two were repellent, and eight elicited no preference in laboratory bioassays. Volatile blends containing relatively high amounts of the green leaf volatiles (E)‐2‐hexen‐1‐ol and (Z)‐3‐hexen‐1‐ol, or the sesquiterpene β‐ caryophyllene, were unattractive or repellent. Minimal blends attractive to CPB were comprised of (Z)‐3‐hexenyl acetate (±)‐linalool and methyl salicylate: the combination of all three chemicals elicited sexually dimorphic attraction of males; two component blends comprised of (Z)‐3‐hexenyl acetate and either (±)‐linalool and methyl salicylate attracted both sexes. Individual compounds were inactive. No significant difference was noted between two attractive blends, or an attractive synthetic blend vs. mechanically damaged potato foliage. 4 These results show that CPB are attracted to blends of specific chemicals emitted by their host plants and provide a basis for the use of plant attractants as a component of integrated management of pestiferous populations.     

Effects of Zygorrhynchus moelleri on the growth of potato and reproduction of potato cyst nematodes

Laboratory, pot and field experiments investigated the effects of the fungus Zygorrhynchus moelleri on the growth of potato and on the reproduction of the potato cyst nematodes (PCN), Globodera pallida and G rostochiensis. Preliminary laboratory tests showed that Z. moelleri growth was favoured by temperatures and pH ranges commonly present in field soils. The fungus colonised potato roots in vitro and in compost or field soil. It also stimulated in vitro root growth of three potato cultivars. In pot experiments Z. moelleri stimulated potato growth, particularly in the presence of PCN attack. In field plots infested with a mixture of G pallida and G. rostochiensis, tuber yields were not increased after application of the fungus but, in G pallida‐infested plots, yields were significantly increased after drills were inoculated with Z. moelleri. The application of Z. moelleri had no apparent effects on nematode reproduction. Factors influencing the interactions between Z. moelleri, potato and potato cyst nematodes are discussed and the potential role of the fungus as a plant growth promoter in organic potato production considered.     

Horizontal transmission of Beauveria bassiana (Bals.) Vuill

1 Factors influencing horizontal transmission of the entomopathogen Beauveria bassiana in the Colorado potato beetle (CPB) were examined through a series of laboratory studies. 2 Cadaver density, cadaver life stage, ambient temperature and conidial density were the factors manipulated. 3 Mortality and sporulation of burrowing CPB prepupae both increased significantly with increased sporulating second‐instar cadaver density on the soil surface. 4 Mortality rates were significantly higher when prepupae were released into laboratory arenas containing third‐instar cadavers compared to second‐instar cadavers. 5 Mortality and sporulation decreased significantly as temperature increased from 15 °C to 30 °C, however, no temperature‐dependent behavioural response by prepupae could be identified as a potential cause. 6 An 86.1% decrease in conidial density per cadaver had no significant effect on mortality or sporulation of prepupae, indicating that this level of environmental degradation of cadavers may not significantly reduce the probability of horizontal transmission.     

Transgenic Bt potato and conventional insecticides for Colorado potato beetle management: comparative efficacy and non-target impacts

Field studies were conducted in 1992 and 1993 in Hermiston, Oregon, to evaluate the efficacy of transgenic Bt potato (Newleaf^®, which expresses the insecticidal protein Cry3Aa) and conventional insecticide spray programs against the important potato pest, Leptinotarsa decemlineata (Say), Colorado potato beetle (CPB), and their relative impact on non-target arthropods in potato ecosystems. Results from the two years of field trials demonstrated that Newleaf potato plants were highly effective in suppressing populations of CPB, and provided better CPB control than weekly sprays of a microbial Bt-based formulation containing Cry3Aa, bi-weekly applications of permethrin, or early- and mid-season applications of systemic insecticides (phorate and disulfoton). When compared with conventional potato plants not treated with any insecticides, the effective control of CPB by Newleaf potato plants or weekly sprays of a Bt-based formulation did not significantly impact the abundance of beneficial predators or secondary potato pests. In contrast to Newleaf potato plants or microbial Bt formulations, however, bi-weekly applications of permethrin significantly reduced the abundance of several major generalist predators such as spiders (Araneae), big-eyed bugs (Geocorus sp.), damsel bugs (Nabid sp.), and minute pirate bugs (Orius sp.), and resulted in significant increases in the abundance of green peach aphid (GPA), Myzus persicae (Sulzer) – vector of viral diseases, on the treated potato plots. While systemic insecticides appeared to have reduced the abundance of some plant sap-feeding insects such as GPA, lygus bugs, and leafhoppers, early and mid-season applications of these insecticides had no significant impact on populations of the major beneficial predators. Thus, transgenic Bt potato, Bt-based microbial formulations and systemic insecticides appeared to be compatible with the development of integrated pest management (IPM) against other potato pests such as GPA because these CPB control measures have little impact on major natural enemies. In contrast, the broad-spectrum pyrethroid insecticide (permethrin) is less compatible with IPM programs against GPA and the potato leafroll viral disease.     

Synthesis of active oryzacystatin I in transgenic potato plants

Summary Transformation of potato (Solanum tuberosum L.) with cysteine proteinase inhibitor (PI) genes represents a potential way of controlling the major insect pest Colorado potato beetle (CPB; Leptinotarsa decemlineata Say). The present study describes the Agrobacterium-mediated transformation of potato (cv. Kennebec) with an oryzacystatin I (OCI) cDNA clone linked to a CaMV 35S promoter. The transgenic plants accumulated active OCI in potato leaves, as demonstrated by the papain-inhibitory activity of transgenic plant leaf extracts. In addition to their anti-papain activity, the extracts also caused a partial but significant inhibition of CPB digestive proteinases, similar to that observed with pure inhibitors. Recombinant OCI did not alter the activity of the major potato leaf endogenous proteinases, which seemed to be of the serine-type. Therefore we suggest that the OCI cDNA can be used for the production of CPB-resistant transgenic potato plants without interfering with endogenous proteinases of these plants.Abbreviations CPB Colorado potato beetle - E-64 trans-epoxy-succinyl-L-leucylamido (4-guanidino) butane - OCI oryzacystatin I - PI proteinase inhibitor - PMSF phenylmethylsulfonyl fluoride     

Modelling Beauveria bassiana horizontal transmission

1 We modelled horizontal transmission of Beauveria bassiana in Colorado potato beetle (CPB) between larval cadavers and soil inhabiting prepupae. 2 The rate of disease transmission, based on the probability of a prepupa contacting sporulating cadavers on the soil surface, is a non‐linear function of cadaver density and also dependent upon temperature. 3 The spatial pattern of cadavers is needed to estimate prepupal contacts with cadavers. Observational field studies determined Johnson distributions to model the spatial pattern of cadavers and prepupae in the field. The model also implicitly incorporates within‐field larval spatial pattern into estimates of field‐level horizontal transmission. 4 Potential for horizontal transmission is higher in simulations using weather data from the warmer year of 1995, than in simulations of the cooler growing season of 1993. 5 Simulations of CPB populations under northern Maine climatic conditions recorded in 1993 and 1995 suggest that horizontal transmission can range from 3 to 24% depending upon the timing of primary infection of larvae in the field. 6 Two simulated sequential applications of B. bassiana targeted at peak first instars resulted in maximum horizontal infection in both years. 7 Sensitivity analysis suggests that horizontal transmission is most sensitive to changes in the proportion of cadavers that sporulate and least sensitive to changes in the time between larval death and the onset of cadaver sporulation. 8 Field validation of the model indicates good prediction of one measure of horizontal infection, the proportion of prepupae which eventually sporulated after being released in controlled field experiments.     

Endophytes of fescue grasses enhance susceptibility ofPopillia japonica larvae to an entomopathogenic nematode

We evaluated tritrophic level interactions among fungal endophytes (Acremonium spp.) of fescue grasses (Festuca spp.), the root-feeding Japanese beetlePopillia japonica Newman larvae, and the entomopathogenic nematodeHeterorhabditis bacteriophora Poinar. Third-instarP. japonica larvae were introduced into pots containing endophyteinfected or endophyte-free plants of tall fescueFestuca arundinacea Schreber (cultivars Kentucky 31 and Georgia Jesup Improved) and the Chewings fescueFestuca rubra commutata Guad. (cultivars F-93 and Jamestown II). After two weeks, the surviving larvae were recovered, and their susceptibility to nematodes was evaluated in sand columns. Endophytes enhanced the rate of nematode-induced mortality in all cultivars except Georgia Jesup Improved, and increased the proportion of dead larvae with nematodes in all cultivars except Jamestown II. Endophytes in the cultivar Kentucky 31 were associated with improved nematode establishment in the larvae. No effect on nematode reproduction was found. Since endophytes produce biologically active alkaloids, we tested the effects of an ergot alkaloid, ergotamine tartrate, on the feeding behavior and weight ofP. japonica larvae in agar medium. The alkaloid caused feeding deterrence, and reduced the consumption of medium by the larvae, resulting in weight loss. These larvae were more susceptible toH. bacteriophora than the untreated larvae. Unfed ‘starved’ larvae were more susceptible to nematodes than those fed on untreated agar. Our results support the hypothesis that endophyte-induced starvation ofP. japonica would reduce larval vigor, and render them more susceptible to entomopathogenic nematodes.     

Colorado potato beetle resistant somatic hybrid potato plants produced via protoplast electrofusion

Leptines are natural glycoalkaloids found only in certain selections of the wild potato speciesSolanum chacoense. These rare glycoalkaloids have been identified to be phytochemical defensive agents against insect herbivores such as the Colorado potato beetle (CPB). In an attempt to introduce this CPB resistance into the cultivated potatoS. tuberosum, interspecific somatic hybrid plants were developed between a dihaploid ofS. tuberosum and a high leptine-producing germplasm selection ofS. chacoense. The somatic hybrid was fused using protoplast electrofusion and regeneration techniques. Selection of interspecies fusion cell lines was based on hybrid vigor in protoplast-callus (p-callus) growth, on shoot regeneration from p-calli, and on characteristic appearance of anthocyanin pigment. This selection system was highly efficient and 12 of 13 fully regenerated plants were identified as somatic interspecies hybrids, as determined by the analyses of morphologic biochemical, and isozyme markers. In vitro insect bioassays demonstrated that the hybrids averaged a threefold reduction in leaf consumption by the CPB when compared to cultivated potatoes.     

Field evidence for indirect interactions between foliar‐feeding insect and root‐feeding nematode communities on Nicotiana tabacum

Abstract 1. As herbivory often elicits systemic changes in plant traits, indirect interactions via induced plant responses may be a pervasive feature structuring herbivore communities. Although the importance of this phenomenon has been emphasised for herbivorous insects, it is unknown if and how induced responses contribute to the organisation of other major phytoparasitic taxa. 2. Survey and experimental field studies were used to investigate the role of plants in linking the dynamics of foliar‐feeding insects and root‐feeding nematodes on tobacco, Nicotiana tabacum. 3. Plant‐mediated interactions between insects and nematodes could largely be differentiated by insect feeding guild, with positive insect–nematode interactions predominating with leaf‐chewing insects (caterpillars) and negative interactions occurring with sap‐feeding insects (aphids). For example, insect defoliation was positively correlated with the abundance of root‐feeding nematodes, but aphids and nematodes were negatively correlated. Experimental field manipulations of foliar insect and nematode root herbivory also tended to support this outcome. 4. Overall, these results suggest that plants indirectly link the dynamics of divergent consumer taxa in spatially distinct ecosystems. This lends support to the growing perception that plants play a critical role in propagating indirect effects among a diverse assemblage of consumers.     

Survival and fecundity of Bt-susceptible Colorado potato beetle adults after consumption of transgenic potato containing Bacillus thuringiensis subsp. tenebrionis Cry3A toxin

Survival and fecundity of Colorado potato beetle adults, Leptinotarsa decemlineata (Say), that had or had not fed previously on non-transgenic potato before exposure to transgenic potato containing the Bacillus thuringiensis subsp. tenebrionis Cry3A toxin (Bt) was investigated. In the laboratory, < 5% of first-generation adults survived after two weeks when restricted to Bt foliage since eclosion, but over 85% of adults that had fed initially on non-Bt potato survived exposure to Bt potato for two weeks. In field experiments, less than 0.5% of adults that were exclusively provided Bt potato plants survived overwinter, whereas 44% to 57% survived overwinter when fed non-Bt potato plants for two weeks before being provided Bt potato as a final pre-overwintering host. Survival through the winter increased as the duration of initial feeding on non-Bt potato increased and was similar for beetles provided either tubers or Bt potato plants as a final pre-overwintering host. Only overwintered beetles that fed initially on non-Bt potato before encountering either tubers or Bt potato as a final pre-overwintering host laid eggs the following spring. Survival and reproduction of potato beetle adults after colonizing Bt potato fields should not be adversely affected as long as they have had sufficient time to feed initially on non-Bt potato. Implications for how potato production practices in the Mid-Atlantic US may affect the utility of general resistance management plans for Bt potato are discussed.     

The bacterial community of entomophilic nematodes and host beetles

Insects form the most species‐rich lineage of Eukaryotes and each is a potential host for organisms from multiple phyla, including fungi, protozoa, mites, bacteria and nematodes. In particular, beetles are known to be associated with distinct bacterial communities and entomophilic nematodes. While entomopathogenic nematodes require symbiotic bacteria to kill and reproduce inside their insect hosts, the microbial ecology that facilitates other types of nematode–insect associations is largely unknown. To illuminate detailed patterns of the tritrophic beetle–nematode–bacteria relationship, we surveyed the nematode infestation profiles of scarab beetles in the greater Los Angeles area over a five‐year period and found distinct nematode infestation patterns for certain beetle hosts. Over a single season, we characterized the bacterial communities of beetles and their associated nematodes using high‐throughput sequencing of the 16S rRNA gene. We found significant differences in bacterial community composition among the five prevalent beetle host species, independent of geographical origin. Anaerobes Synergistaceae and sulphate‐reducing Desulfovibrionaceae were most abundant in Amblonoxia beetles, while Enterobacteriaceae and Lachnospiraceae were common in Cyclocephala beetles. Unlike entomopathogenic nematodes that carry bacterial symbionts, insect‐associated nematodes do not alter the beetles' native bacterial communities, nor do their microbiomes differ according to nematode or beetle host species. The conservation of Diplogastrid nematodes associations with Melolonthinae beetles and sulphate‐reducing bacteria suggests a possible link between beetle–bacterial communities and their associated nematodes. Our results establish a starting point towards understanding the dynamic interactions between soil macroinvertebrates and their microbiota in a highly accessible urban environment.     

Predation of Colorado potato beetle eggs by a polyphagous ladybeetle in the presence of alternate prey: potential impact on resistance evolution

The influence of prey choice on the predation of a target prey item by a polyphagous insect predator was investigated in field plot studies. The target prey consisted of eggs of the Colorado potato beetle (CPB), Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), and the predator was the 12‐spotted ladybeetle, Coleomegilla maculata Lengi (Coleoptera: Coccinellidae). Eggs of the European corn borer (ECB), Ostrinia nubilalis Hübner (Lepidoptera: Pyralidae), and nymphs and adults of the green peach aphid, Myzus persicae Sulzer (Homoptera: Aphididae), comprised the alternative prey choices. The objectives of these studies were to: (1) examine predation in a multiprey scenario likely to occur in an agroecosystem, and (2) use the data to simulate the impact of predator‐induced mortality on the evolution of resistance to Bt‐transgenic plants in the target herbivore. Simulations of the rate of resistance evolution were carried out using a deterministic genetic model. Experiments were performed using potato field plots planted in a manner reflecting a 25% or 50% non‐transgenic refuge. CPB eggs were infested so as to mimic the densities of resistant and susceptible populations that might occur in commercial Bt‐transgenic plantings. Densities of predators and alternate prey species were chosen to represent those that might typically occur in potato crops in the eastern USA. Simulation results indicated that when ECB eggs were present, predation on CPB eggs either became inversely spatially density‐dependent, or increased significantly in a density‐dependent manner. When aphids were present, predation became positively density‐dependent. Model simulations predicted that ECB egg presence is beneficial, in that resistance was delayed by up to 40 pest generations (as compared to the scenario with CPB as the only prey), while aphid presence accelerated resistance evolution by 18 generations. Results suggest that resistance management strategies should take into account the composition of prey species available to generalist predators typically present, so as to best delay pest adaptation to Bt‐toxins.     

Behavioural responses of larvae of Colorado potato beetle,Leptinotarsa decemlineata (Coleoptera: Chrysomelidae), to host plant volatile blends attractive to adults

Abstract

• 1 Orientation of second‐ and fourth‐instar Colorado potato beetle (CPB) Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), to volatiles emitted from a solanaceous host, potato, and seven synthetic blends or three individual chemicals emitted by potato plants were investigated in laboratory bioassays.
• 2 Both second‐ and fourth‐instar CPB were attracted to intact and mechanically damaged (MD) potato foliage. When offered a choice between intact and MD foliage, no preference was observed.
• 3 Among seven synthetic blends tested (of which six are attractive to adult CPB), second‐ and fourth‐instar CPB were attracted only to a single three‐component blend comprising (±)‐linalool, methyl salicylate, and (Z)‐3‐hexenyl acetate. Individual compounds and two‐component blends were inactive. No significant difference was noted between larval responses to the attractive synthetic blend vs. MD potato foliage.
• 4 Second‐and fourth‐instar larvae had similar thresholds for behaviour for the three‐component blend (50 µg source load). Female CPB were attracted to source loads 10× below the larval threshold (5 µg). Male CPB were the most sensitive life form tested with a behavioural threshold at 0.5 µg source load which was 10× and 100× below female and larval thresholds, respectively
• 5 This is the first report of a synthetic chemical attractant for CPB larvae. As both larval and adult CPB are attracted to a single chemical blend, the usefulness of the attractant as a component of an attracticide or ‘push‐pull’ strategies for management of pestiferous populations is enhanced.

     

Field efficacy of entomopathogenic nematodes against the beetle Maladera matrida (Coleoptera: Scarabaeidae)

Single, double and triple releases of the entomopathogenic nematode Heterorhabditis bacteriophora Poinar, reduced the population of the beetle Maladera matrida Argaman, infesting peanuts (’Shulamit’ cv.) by 70, 75 and 93% respectively in microplot tests. Simultaneous and late (2 weeks after infestation) applications reduced beetle numbers by 63 and 79% respectively, in the microplots, while early application (2 weeks prior to infestation) did not reduce the beetle population. In a field trial, reductions in insect population and damage to the crop were achieved by early treatment with the nematode as well as by Heptachlor, leading to reductions in the insect population of 60 and 90% respectively, when recorded 4 weeks after nematode application. However, the nematode treatment did not maintain its effectiveness for a longer period and pest damage increased to the same level as the untreated control after 7 weeks. When the nematodes were applied at different concentrations (0.25–1.0 x 10⁶ infective juveniles (IJs) m^‐2) their effectiveness was not related to the concentration level. The only significant (P < 0.05) reduction in insect levels was recorded in the treatment with 0.5 X 10⁶ IJs m^‐2. In a second field trial, both H. bacteriophora and Steinernema glaseri reduced insect populations significantly (P < 0.05) by approximately 50% in comparison to the control. In the third trial, treatment with H. bacteriophora resulted in a decrease in insect population of 90% while treatment with S. carpocapsae reduced the grub numbers by 40% in comparison to the control. A differential susceptibility of various grub developmental stages was recorded in the field. The small grubs (I‐4 mm long, lst‐2nd larval stage) were not affected by the nematode treatments while the numbers of medium and large size grubs were reduced by 2‐ and 3‐fold respectively in the various tests. Nematodes were recovered by ‘nematode traps’ containing Galleria mellonella larvae from treated field plots 78 days after application. The implications of the results from the present studies on the use of entomopathogenic nematodes are discussed in relation to the development of an integrated pest management programme.