Spatio-temporal dynamics of corn rootworm, Diabrotica spp., adults and their spatial association with environment

Multiyear and multilocation studies were conducted to investigate the within‐ and the between‐year spatial dynamics of corn rootworms Diabrotica spp. (Coleoptera: Chrysomelidae), adults and their spatial associations with environmental factors in cornfields. Grid‐based spatial sampling was conducted by trapping adults emerging from the soil (i.e., ‘emergence‐trap counts’) and by counting the number of adults in the ear zone of corn plant (i.e., ‘ear‐zone count’). Spatial analysis with distance indices (SADIE) was used to determine spatial distribution patterns and to investigate spatial associations. Ear‐zone counts and emergence‐trap counts were spatially dynamic within a year and more frequently aggregated in the middle of growing seasons and random early and late in the season. However, during the peak population periods, spatial distribution of ear‐zone counts and emergence‐trap counts were significantly consistent between years, indicating predictability of future spatial distributions. Spatial distribution of emergence‐trap counts and ear‐zone counts were positively associated with soil moisture and presence of corn plants with silks, respectively. This study demonstrated that within‐year spatial distribution patterns were dynamic and that there was between‐year spatial consistency of corn rootworm adult distributions. Such information can be used to improve preventative management of corn rootworms.     

Estimation of efficacy functions for products used to manage corn rootworm larval injury

Maize, Zea mays L., is an economically important crop grown throughout the world. Corn rootworm, Diabrotica spp. (Coleoptera: Chrysomelidae), larvae constitute a significant economic threat to maize production in the United States, where yield losses and management costs associated with corn rootworm species exceed $1 billion annually. Furthermore, the introduction of the western corn rootworm, D. virgifera virgifera LeConte, into maize‐producing regions of Europe has made managing corn rootworm larval injury an international concern. Larvae injure maize plants by feeding on root tissue and are the primary target of management activities. Products commonly used to protect root systems from injury include chemical insecticides (seed or soil applied) and genetically modified maize hybrids expressing toxins derived from Bacillus thuringiensis Berliner (Bt). The confirmation of field‐evolved resistance to various Bt toxins in populations of the western corn rootworm presents a significant management challenge. We performed a meta‐analysis to provide a broad understanding of the relative efficacy of the primary products currently being used to manage corn rootworm larval injury, including insecticidal seed treatments, soil insecticides and Bt hybrids (with and without the addition of soil insecticide). Our analysis is unique in the breadth of locations and years included – we analysed 135 individual trials conducted from 2003 through 2014 at multiple sites in both Illinois and Nebraska. Panel data were produced by pairing the mean node‐injury rating for each treatment of a given trial with the mean node‐injury rating for untreated maize. Linear regression models were developed to estimate the relationship between the potential for corn rootworm larval injury and product performance. For a given level of injury potential, the parameters estimated reveal differences in the degree of root protection offered by the various product categories analysed. Implications for developing long‐term, integrated, and sustainable practices for managing this important pest of maize are discussed.     

Polymorphic microsatellite loci from northern and Mexican corn rootworms (Insecta: Coleoptera: Chrysomelidae) and cross-amplification with other Diabrotica spp

The northern corn rootworm (Diabrotica barberi) and Mexican corn rootworm (Diabrotica virgifera zeae) are significant agricultural pests. For the northern corn rootworm, and to a lesser extent, the Mexican corn rootworm, high resolution molecular markers are needed. Here we present 14 polymorphic microsatellite loci isolated from libraries constructed using pooled northern and Mexican corn rootworm genomic DNA. Polymorphism in other Diabrotica, including the banded cucumber beetle, southern corn rootworm and western corn rootworm, is described.     

Supercooling and cold-hardiness in eggs of western and northern corn rootworms

Oviposition by northern corn rootworms, Diabrotica barberi Smith and Lawrence, and western corn rootworms, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), key pests of corn in the Great Plains of the USA, occurs in the soil during late summer. Overwintering eggs are exposed to variable soil moisture and temperatures below ?5 °C. The winter mortality of eggs in the soil is a primary factor that determines the potential for larval injury to corn the following spring. Our studies aimed to determine the comparative supercooling capacities of northern and western corn rootworm eggs and to assess egg mortality following brief exposure to extreme low temperature, ranging from ?12.0 to ?21.5 °C, under three moisture regimes. Eggs of northern corn rootworm were supercooled to a temperature as low as ?27 °C, and survived supercooling to a greater extent than did western corn rootworm eggs. Moisture treatment prior to supercooling had little effect on northern corn rootworm eggs. Western corn rootworm eggs were more resistant than northern corn rootworm eggs to the effects of desiccation followed by supercooling. The survival of northern corn rootworm eggs was better than western corn rootworms under dry conditions, followed by exposure to temperatures of ?12.0 and ?17.5 °C, but was very low at ?21.5 °C, regardless of the moisture regime. The results suggest that moisture and temperature may interact in the soil environment to determine the overwintering survival of corn rootworms. It is evident from these studies that both rootworm species experience mortality at temperatures well above the supercooling points of the eggs, but that differences exist in the effects of substrate moisture treatments on the cold‐hardiness of eggs from the two species.     

Western corn rootworm (Diabrotica virgifera virgifera LeConte) population dynamics

1 The western corn rootworm Diabrotica virgifera virgifera LeConte is a major insect pest of field maize, Zea mays L. Larvae can cause substantial injury by feeding on maize roots. Larval feeding may destroy individual roots or root nodes, and reduce plant growth, stability, and yield. Costs associated with managing corn rootworms in continuous maize are annually one of the largest expenditures for insect management in the United States Corn Belt.
2 Even though D. virgifera virgifera has been studied intensively for over 50 years, there is renewed interest in the biology, ecology, and genetics of this species because of its ability to rapidly adapt to management tactics, and its aggressive invasive nature.
3 This article provides a comprehensive review of D. virgifera virgifera population dynamics, specifically: diapause, larval and adult development, seasonality, spatial and temporal dynamics at local and landscape scales, invasiveness in North America and Europe, and non-trophic interactions with other arthropods.
4 Gaps in current knowledge are identified and discussed especially within the context of challenges that scientists in North America and Europe are currently facing regarding pest dynamics and the need to develop appropriate management strategies for each geographic area.     

Polymorphic microsatellite loci from the western corn rootworm (Insecta: Coleoptera: Chrysomelidae) and cross‐amplification with other Diabrotica spp

Corn rootworms (Diabrotica spp.) make up the major insect pest complex of corn in the US and Europe, and there is a need for molecular markers for genetics studies. We used an enrichment strategy to develop microsatellite markers from the western corn rootworm (Diabrotica virgifera virgifera). Of 54 loci isolated, 25 were polymorphic, and of these, 17 were surveyed for variability in 59 wild individuals. In addition, the potential for cross‐amplification of these microsatellites was surveyed for Mexican, northern, and southern corn rootworms. Nine microsatellite loci showed Mendelian inheritance and are likely to be useful in population genetics studies.     

Validation of a nested error component model to estimate damage caused by corn rootworm larvae

The corn rootworm complex (Coleoptera: Chrysomelidae) constitutes a significant threat to maize production in the United States, and more recently, in Europe. We conducted an analysis of readily available field trial data to validate an existing damage function for corn rootworm larvae. We used a nested error component model with unbalanced panel data to describe the relationship between yield loss and root injury caused by these insects. These data were collected by personnel with the Insect Management and Insecticide Evaluation Programme (Department of Crop Sciences, University of Illinois) and represent 19 location‐years. To our knowledge, this is the largest data set used to estimate a damage function for corn rootworm larvae. Unlike many experiments examining the relationship between root injury and yield loss caused by corn rootworm larvae, the data set used for our analysis includes many Bt maize hybrids. Our model suggests that for each node of roots injured by corn rootworm larvae, a yield loss of approximately 15% can be expected. Statistically significant variance components included an effect of location and experimental error. We speculate that variation in weather across experimental sites was the principal factor contributing to the significant effect of location. The substantial experimental error observed for our model highlights the limitations of utilizing a multi‐year, geographically diverse damage function for predicting yield loss because of root injury on a small scale. We discuss major factors contributing to the variance components estimated by our model and suggest techniques for improving future analyses of the damage function for corn rootworm larvae.     

Parasitism of Western Corn Rootworm Larvae and Pupae by Steinernema carpocapsae

Virulence and development of the insect-parasitic nematode, Steinernema carpocapsae (Weiser) (Mexican strain), were evaluated for the immature stages of the western corn rootworm, Diabrotica virgifera virgifera LeConte. Third instar rootworm larvae were five times more susceptible to nematode infection than second instar larvae and 75 times more susceptible than first instar larvae and pupae, based on laboratory bioassays. Rootworm eggs were not susceptible. Nematode development was observed in all susceptible rootworm stages, but a complete life cycle was observed only in second and third instar larvae and pupae. Nematode size was affected by rootworm stage; the smallest infective-stage nematodes were recovered from second instar rootworm larvae. Results of this study suggest that S. carpocapsae should be applied when second and third instar rootworm larvae are predominant in the field.     

Characterizing laboratory colonies of western corn rootworm (Coleoptera: Chrysomelidae) selected for survival on maize containing event DAS-59122-7

Event DAS‐59122‐7 is a novel transgenic trait designed to protect the roots and yield potential of maize from the insect pest corn rootworm Diabrotica spp. (Col.: Chrysomelidae). The increased pest status of corn rootworm, exceptional efficacy of this trait, and anticipated increases in farm efficiency and grower and environmental safety will drive adoption of this trait. Strong grower acceptance of this trait highlights the importance of science‐based and practical resistance management strategies. A non‐diapause trait was introgressed into two laboratory colonies of Diabrotica virgifera virgifera collected from geographically distinct locations: Rochelle, IL and York, NE. Both colonies were divided and each reared on maize containing event DAS‐59122‐7 or its near isoline. Selected and unselected colonies were evaluated for phenotypic change in larval development, injury potential and survival to adulthood during 10 and 11 generations. The F1 generation of both selected colonies displayed increased larval development, survivorship and measurable, but economically insignificant increases in injury potential on DAS‐59122‐7 maize. Survival rates of 0.4 and 1.3% in F1 generations of both selected colonies corroborate field estimates of survival on DAS‐59122‐7 maize. Over later generations, total phenotypic variation declined gradually and irregularly. Despite the absence of random mating, the tolerance trait could not be fixed in either population after 10 or 11 generations of selection. An allele conferring major resistance to DAS‐59122‐7 was not identified in either selected colony. The assessment also concluded that major resistance gene(s) are rare in populations of D. v. virgifera in the United States, and that a minor trait(s) conferring a low level of survival on DAS‐59122‐7 maize was present. The tolerance trait identified in this study was considered minor with respect to its impact on DAS‐59122‐7 maize efficacy, and the role this trait may play in total effective refuge for major resistance genes with recessive inheritance is the basis of future work.     

Baseline susceptibility of western corn rootworm (Coleoptera: Chrysomelidae) to clothianidin

Abstract:  Western corn rootworm, Diabrotica virgifera virgifera LeConte, neonate susceptibility to clothianidin, a contact and systemic neonicotinoid insecticide, was determined from both laboratory and field-collected populations. Neonates were exposed to filter paper treated with increasing clothianidin concentrations and mortality was evaluated after 24 h. Additionally, two populations were exposed to an artificial diet which was surface treated with clothianidin. Although larvae were five- to six-fold more sensitive to treated diet, results with treated filter paper were more reliable in terms of control mortality and required much less manipulation of rootworm larvae. Therefore, initial baseline comparisons were conducted using the filter paper assays. The variation among populations exposed to treated filter paper was generally low, 4.4-fold among laboratory populations tested; however, there was a 14.5-fold difference in susceptibility among all populations tested. In general, clothianidin was very toxic to rootworm neonates, with LC₅₀ values ranging from 1.5 to 21.9 ng/cm². These results indicate the practicability and sensitivity of the paper filter disc assay to establish baseline susceptibility levels, which is an essential first step in resistance management. A baseline response provides a reference for tracking shifts in susceptibility following commercialization of a control agent so that early changes in susceptibility can be detected.     

Selection of entomopathogenic fungi for the control of the western corn rootworm Diabrotica virgifera virgifera

Abstract:  The western corn rootworm Diabrotica virgifera virgifera Le Conte (Col., Chrysomelidae), a serious pest of maize, has been recently introduced into Europe. Several approaches for its control are presently under investigation including microbial agents. During a field survey in Hungary in 2005, naturally occurring entomopathogenic fungi were found to attack this pest. These novel isolates together with standard isolates were tested for virulence against D. v. virgifera larvae and adults. Twenty strains of Metarhizium anisopliae , Beauveria bassiana and Beauveria brongniartii were used in bioassays in the laboratory. Larvae and adults were dipped into a spore suspension with a concentration of 1 × 10⁷ conidia (con.)/ml. They were kept for 14 days at 22°C (±2°C) and 70% relative humidity. The number of infected larvae and adults were counted and infection rates were calculated. Adults were significantly more susceptible to entomopathogenic fungi than larvae. The most virulent isolate infected about 47% of larvae ( M. anisopliae Ma2277), whereas the infection rate in adults was up to 97% ( M. anisopliae Ma2275). Isolates of M. anisopliae caused significantly higher mortalities than isolates of B. brongniartii and B. bassiana . Most of the adult beetles were killed within 12 days. Isolates from D. v. virgifera were more virulent than those from other hosts.     

Western corn rootworm adult movement and possible egg laying in fields bordering maize

Abstract:  Western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, larval damage in maize following soybean was observed in Croatia in 2003 along the edges and within soybean fields which bordered continuous maize fields in previous year. The explanation was that WCR adults moved from the continuous maize to the neighbouring soybean fields to lay eggs. This study was designed to measure how far WCR adults will enter into neighbouring fields to lay eggs. The WCR adult population was monitored in continuous maize fields in 2003 and 2005 by using Pherocon^® AM non-baited yellow sticky traps in the middle and on the borders of the maize field and at different distances and directions into neighbouring fields planted by wheat and soybean. Larval presence and root damage ratings (Iowa State University 1–6 ) were recorded at different locations within the maize field in following years. Approximately, the same concentration of WCR adults was recorded along the edges of the maize fields as recorded in the centres of those fields. A significant number of WCR adults was recorded up to a distance of 50 m into neighbouring fields. Regression analysis showed medium negative correlation between distance from previous maize field and root damage in the following year. Findings indicate that WCR egg lying can reach approximately 20 m into fields neighbouring maize fields and that significant root damage caused by WCR larvae in first-year maize following soybean and wheat can happen up to a distance of 20 m into those fields. Most farmers's fields in Croatia are up to approximately 50 m wide. As an edge effect for WCR egg laying can reach approximately 20 m into fields neighbouring maize fields, our research results indicate that it is possible to see WCR larval damage in rotated fields without those WCR's being the variant form.     

Evaluating multiple approaches for managing western corn rootworm larvae with seed blends

Corn rootworm, Diabrotica spp., larvae represent a significant and widespread economic threat to corn, Zea mays (L.), production in the United States, where control costs and yield losses associated with these insect pests exceed $1 billion annually. Preventing root injury and associated yield loss caused by corn rootworm larvae may be accomplished by the independent use of planting time soil insecticides or transgenic Bt hybrids. However, recent reports of both confirmed and suspected Bt resistance in corn rootworm populations throughout the Corn Belt have led to significant interest in the use of these two management tactics simultaneously. Although this approach has been investigated to some extent previously, information is lacking on how the use of a soil insecticide in tandem with a Bt seed blend—Bt and refuge (non‐Bt) seed mixed into a single product—may affect root protection and yield. We describe an experiment including six trial sites conducted over a three‐year period where various seed blends and soil insecticide/seed blend combinations were evaluated. The predominant species contributing to root injury across all sites was the western corn rootworm (Diabrotica virgifera virgifera LeConte). A weighted technique is presented for evaluating root injury for seed blends that offers a reliable estimate of product performance. The addition of a soil insecticide to the seed blend treatments never resulted in significantly improved root protection and failed to provide a consistent yield benefit. Our results suggest that a soil insecticide/seed blend combination approach is not warranted. Additionally, a subanalysis performed on individual refuge and nearby Bt root systems for seed blend treatments provides insight into the spatial characteristics of root injury in seed blend scenarios.     

Field studies of the temporal response patterns of male Diabrotica virgifera virgifera LeConte and D. barberi Smith and Lawrence to 8R-methyl-2R-decyl propanoate

ABSTRACT. Variations in the diel response of male northern and western corn rootworms attracted to a synthetic pheromone source were examined in southwestern Ontario. Activity was measured by the number of males caught at hourly intervals in sticky traps baited with 8 R -methyl-2 R -decyl propanoate. Northern corn rootworm males were attracted to the pheromone between 22.30 and 0330 hours and were unresponsive at all other times at temperatures above 15°C. Western corn rootworm males had a bimodal response with peaks in attraction from 08.30 to 12.30 hours and again from 15.30 to 18.30 hours. While the period of peak attraction for northern corn rootworm males was well defined, the pattern for western corn rootworm males was erratic and many beetles were caught throughout the day. Evening temperatures below 15°C suppressed the activity of both northern and western corn rootworms. While the nightly period of activity for the northern corn rootworms was shifted by low temperatures to the following morning, that of the western corn rootworm was not altered substantially. As a result, peaks in the response of northern and western corn rootworm males occurred simultaneously under these conditions, suggesting strongly that temporal separation of activity periods was not an important factor in maintaining sexual isolation for these species in Ontario.     

Evaluation of soil‐applied insecticides with Bt maize for managing corn rootworm larval injury

The western corn rootworm, Diabrotica virgifera virgifera LeConte, is considered one of the most significant insect pests of maize in North America. Larvae of other secondary subterranean pests such as grape colaspis, Colaspis brunnea (F.), and Japanese beetle, Popillia japonica Newman, can also injure maize seedlings and cause yield loss. In the past decade, maize hybrids containing Bt proteins have been used to manage the western corn rootworm; additionally, seeds are commonly treated with a neonicotinoid and fungicide combination to control secondary pests. Recently, soil‐applied insecticides have been used in conjunction with rootworm Bt hybrids (and seed‐applied insecticides) in areas with perceived risk for increased rootworm larval or secondary pest damage. We conducted a series of trials from 2009 to 2011 that examined multiple rootworm Bt hybrids and their near‐isolines, along with two soil‐applied insecticides, to determine whether the Bt plus insecticide combination resulted in an increased level of efficacy or yield. We also sampled for Japanese beetle and grape colaspis larvae to determine their potential for reducing yield. Densities of secondary pests in our trials were low and likely had no effect on maize yield. The addition of a soil‐applied insecticide to rootworm Bt hybrids improved efficacy only once across 17 location‐years, when overall corn rootworm injury was highest; an improvement in yield was never observed. Our results suggest that the use of a soil‐applied insecticide with a rootworm Bt hybrid should only be considered in scenarios with potentially significant rootworm larval populations. However, potential negative consequences related to trait durability when soil insecticides are used with rootworm Bt maize should be considered.     

Survival and oviposition of a western corn rootworm variant feeding on soybean

The role of soybean, Glycine max (L.) Merril (Fabaceae), in the circumvention of crop rotation was evaluated by observing the effects of soybean herbivory on western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) survival and oviposition. Field collected D. virgifera adults resistant to annual rotation of corn, Zea mays (L.) (Poaceae), and soybean were given the opportunity to feed on a soybean pre‐treatment diet prior to diet combinations of corn, soybean, and/or water. The number of days that the adults survived after removal of the diet combinations was used as a measure of insect vigor and diet quality. Willingness to feed on a soybean foliage pre‐treatment did not indicate greater ability to benefit nutritionally from soybean herbivory. Adult D. virgifera previously feeding on corn, soybean, or water survived starvation for 8.3 ± 0.15 days, 5.1 ± 0.13 days, and 3.9 ± 0.14 days, respectively. Diabrotica virgifera recover from 2 days of starvation or poor diet if subsequently given access to corn tissues for 2 days. Eggs were laid within 1 day of access to poor diet or starvation. Other D. virgifera, captured as they flew from a cornfield into a soybean field, were maintained on soybean foliage or only water until they died. Of the beetles with access to soybean foliage, 24% fed within 24 h after capture and survived 1 day longer than insects given only water. Only 20% of these D. virgifera were able to lay eggs without consuming additional corn prior to death. Few D. virgifera enter soybean fields capable of immediate oviposition. Female reproductive status and diet quality influence the likelihood of oviposition following exposure to stress. The effects of soybean herbivory may contribute to the proximate mechanism of resistance to crop rotation.     

Larval mortality and development for rotation‐resistant and rotation‐susceptible populations of western corn rootworm on Bt corn

The western corn rootworm, Diabrotica virgifera virgifera LeConte, is one of the most economically important insect pests threatening the production of corn, Zea mays (L.), in the United States. Throughout its history, this insect has displayed considerable adaptability by overcoming a variety of pest management tactics, including the cultural practice of annual crop rotation. Since first reported in Illinois in the late 1980s, populations of the rotation‐resistant western corn rootworm have spread over a wide area of the eastern Corn Belt. Currently, little information is available concerning the interaction of rotation resistance with the use of genetically modified corn expressing insecticidal toxins from Bacillus thuringiensis Berliner (Bt), a popular tactic for preventing larval injury and its associated yield loss. The goal of this greenhouse experiment was to determine whether rotation‐resistant and rotation‐susceptible western corn rootworm larvae differ with respect to survival or development when exposed to single‐ or dual‐toxin (pyramided) Bt corn. Individual corn plants were infested with 225 near‐hatch eggs at the V5 (five leaf collar) growth stage. Larvae developed undisturbed on the root systems for 17 days, after which they were recovered using Berlese–Tullgren funnels. Surviving larvae were counted to estimate mortality, and head capsule widths were measured to assess development. Rotation‐resistant and rotation‐susceptible larvae had statistically similar mean levels of mortality and head capsule widths when exposed to both single‐toxin (Cry3Bb1 or Cry34/35Ab1) and pyramided (Cry3Bb1+ Cry34/35Ab1) Bt corn, suggesting that these two populations do not differ with respect to survival or development when exposed to Bt corn. Additionally, the statistically similar mean levels of mortality for larvae exposed to single‐toxin and pyramided Bt corn suggest that pyramided Bt hybrids containing the Cry3Bb1 and Cry34/35Ab1 toxins do not result in additive mortality for western corn rootworm larvae. Implications for management of this economically important pest are discussed.     

<Emphasis Type="Italic">Trichogramma Brassicae</Emphasis> and SLAM<Superscript>®</Superscript>, an Integrated Approach to Managing European Corn Borer and Corn Rootworms

European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae); western corn rootworm (WCRW), Diabrotica virgifera virgifera LeConte; and northern corn rootworm (NCRW), Diabrotica barberi (Smith & Lawrence) (Coleoptera: Chrysomelidae) are important pests of corn, Zea mays L., that occur simultaneously in the US Corn Belt. Areawide management strategies for northern and western corn rootworm in Iowa, Kansas, South Dakota, Illinois, and Indiana, and for the Mexican strain in Texas, use SLAM^®, which contains cucurbitacin, a feeding stimulant, and a small amount of the insecticide carbaryl. This management strategy is environmentally sound because it introduces a substantially lesser amount of chemical insecticide into the environment than prophylactic soil insecticide applications for WCRW management. To develop a management program for both corn pests, the compatibility of SLAM^® to control CRW, along with a biological agent to manage European corn borer, was investigated. Laboratory studies were conducted using the egg parasitoid Trichogramma brassicae (Bezdenko) (Hymenoptera: Trichogrammatidae). In experiments in which SLAM^® and T. brassicae were evaluated as components of a management system, SLAM^® did not affect parasitism or parasitoid emergence rates. Trichogramma brassicae displayed vigorous biotic fitness, emergence rates, sex ratios, and fecundity over the duration of the study. Data collected under these laboratory conditions indicated that SLAM^® had no adverse effect on the quality of T. brassicae, indicated by its ability to reproduce in the filial generation.     

Assessment of establishment and persistence of entomopathogenic nematodes for biological control of western corn rootworm

Abstract:  The use of entomopathogenic nematodes (EPN) is potentially one ecological approach to control the invasive alien western corn rootworm ( Diabrotica virgifera virgifera LeConte, Col., Chrysomelidae) in Europe. This study investigated the establishment and the short- and long-term persistence of Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae), Heterorhabditis megidis Poinar, Jackson and Klein (Rh., Heterorhabditidae) and Steinernema feltiae Filipjev (Rh., Steinernematidae) in three maize fields in southern Hungary, using the insect-baiting technique. All three EPN species equally established and persisted in maize fields. The timing of application (April or June) did not influence the establishment of EPN species. EPNs persisted for 2–5 months, i.e. they survived up to and throughout D. v. virgifera larval occurrence in the soil. Results demonstrate that D. v. virgifera larvae can potentially be controlled by EPNs during the same year of EPN application but no long-term control effect is expected under intensive maize cultivation practices.