All three larval instars of Diabrotica virgifera virgifera LeConte (western corn rootworm, Coleoptera: Chrysomelidae) feed on the roots of maize, Zea mays (L.). We assessed the efficacies of the following four agents in controlling these larvae: (1) the entomopathogenic fungus Metarhizium anisopliae (Metsch.) Sorokin (Hypocreales: Clavicipitaceae), (2) the nematode Heterorhabditis bacteriophora Poinar (Nematoda: Rhabditida), (3) a tefluthrin-based soil insecticide and (4) clothianidin-coated seeds. The agents were applied in field plot experiments in southern Hungary in 2006 and 2007. Efficacy was assessed by comparing the number of emerging D. v. virgifera adults and corresponding root damage among treatments and untreated controls. All agents significantly reduced D. v. virgifera numbers and root damage, but the relative success of each treatment was variable. On average across fields and years, the nematode and the two insecticides reduced D. v. virgifera by 65 ± 34% SD, while the fungus reduced D. v. virgifera by 31 ± 7%. According to the node injury scale, the agents prevented 23-95% of potential root damage. Large-scale commercialisation of these biological agents could offer viable and practical control options against D. v. virgifera.

Genetically engineered maize producing the insecticidal protein Cry3Bb1 from Bacillus thuringiensis (Bt maize) is protected against corn rootworms (Diabrotica spp.), which are serious maize pests in North America and Europe. The aim of the present study was to investigate the interaction of Bt maize (event MON88017) and the entomopathogenic fungus Metarhizium anisopliae for controlling the western corn rootworm, Diabrotica virgifera virgifera. Exposure to Cry3Bb1 expressed in Bt maize seedlings resulted in decreased weight gain in D. v. virgifera larvae but did not influence susceptibility to M. anisopliae. Adult beetles were not affected by Cry3Bb1 in their food, but mortality when feeding on maize leaves was higher than when feeding on silk. Adults were more susceptible to the fungus than larvae. The results indicate that the effects of Bt maize and M. anisopliae on D. v. virgifera are additive and that Bt maize does not interfere with the biological control provided by entomopathogenic fungi.

The entomopathogenic fungus Metarhizium anisopliae (Metsch.) Sorokin (Hypocreales: Clavicipitaceae) was applied in maize fields to control the Western Corn Rootworm Diabrotica virgifera virgifera Le Conte (Coleoptera: Chrysomelidae). Establishment and persistence of two strains of M. anisopliae were investigated after application as ‘fungal colonized barley kernels’ (FCBK) into the soil and as a spore suspension on maize leaves and on the soil surface in 2006 and 2007 at two locations in Hungary. The applied fungal strains were able to establish at both locations and a long‐term persistence of at least 15 months could be recorded in the soil. A positive correlation between density of colony forming units (CFU) in the soil and the soil inhabiting stages of the host insect D. v. virgifera could be found. M. anisopliae spores applied on maize leaves were able to survive for no longer than 3 days after application, whereas on the soil surface a noticeably increase of fungus densities were found after treatments. Molecular markers were used to identify the applied M. anisopliae strains before and after application of FCBK into the soil of the maize field.

No abstract

The Western Corn Rootworm D. virgifera virgifera Le Conte (Coleoptera: Chrysomelidae), a serious pest of maize, has been recently introduced into Europe. Several approaches for its control are presently under investigation including microbial agents. In order to get information on the role of naturally occurring pathogens in the regulation of Diabrotica populations, we started an investigation in established populations in Hungary, Romania, Serbia, Austria, and Italy in 2005 and 2006. In infested maize fields in Hungary, plants and their root systems were grubbed out and larvae and pupae were collected. Adult D. v. virgifera were collected in Hungary, Austria, Romania, Serbia and Italy. Additionally, the occurrence of entomopathogenic fungi in soils of maize fields was determined using Galleria mellonella and Tenebrio molitor larvae as bait insects. The density of entomopathogenic fungi was obtained by plating soil suspension on selective medium. Metarhizium anisopliae and Beauveria spp. infections were found in 1.4% of field collected larvae, 0.2% of field collected pupae and 0.05% of field collected adults. Whereas natural infections of D. v. virgifera were rarely found, a high density of insect pathogenic fungi was recorded in Hungarian soils. M. anisopliae could be detected in every maize field either using the ''bait method'' or a ''selective medium'' method. This is the first report of a natural occurrence of entomoparasitic nematodes (Heterorhabditis sp., Steinernema sp.) in Diabrotica v. virgifera in Europe.

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 · 10 7 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.

The entomoparasitic nematode (EPN) Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae) is a promising candidate for the biological control of larvae of the maize pest Western Corn Rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). An easily applicable and economically feasible method is the inundative release of EPNs together with sowing of maize in spring. At that time, however, D. v. virgifera eggs are still diapausing and larval hatch will only start about four to 6 weeks after EPN application, depending on climatic conditions. Efficacy of the nematode application against the targeted pest species therefore depends on the ability of the biocontrol agent to persist in the pest's environment, at least until emergence of the first larvae. To address this key issue, 18 field trials were carried out at six locations in Germany, Austria and Hungary between 2010 and 2011. Fields differed in their soil type and in the presence or absence of a host. Nematodes were either applied as fluid stream (suspended in water) or as microgranules (without water) into the soil during the sowing process. Persistence of the nematodes was determined by examining soil samples, using larvae of Tenebrio molitor (Coleoptera: Tenebrionidae) as a bait. Results showed that EPNs, either applied as a suspension or as microgranules, were able to persist in the soil of all field sites. EPNs could be detected for at least 6 weeks in the soil. Persistence levels were higher in sandy soil types than in clay or silty soils. The applied EPN formulation had no effect on the persistence level of EPNs in the soil, and the presence or absence of the host insects had a minor effect. It can be concluded that EPNs applied with sowing of maize persist long enough to potentially control the later hatching larvae of the maize pest D. v. virgifera.

In a series of laboratory experiments, acclimated pupae of Tuta absoluta were exposed to various constant low temperatures in order to estimate their maximum survival times (Kaplan–Meier, Lt99.99). A Weibull function was fitted to the data points, describing maximum survival time as a function of temperature. In another experiment at −6°C, the progress of mortality increasing with exposure time was identified. These values were fitted by a sigmoidal function converging asymptotically to 100% mortality for very long exposure times. Analysing mortality data from the maximum survival experiment by a generalized linear model showed a significant common slope parameter (p < .001) that reveals parallelism of the survival curves at each temperature if a log time axis is used. These curves appear stretched (time scaled) if plotted with a nonlogarithmic time axis. By combining these mathematical relations, it was possible to calculate a species‐specific ‘mortality surface’ which exhibits mortalities, depending on temperature and duration of exposure. In order to accumulate hourly mortalities for courses of varying temperatures, an algorithm was developed which yields mortality values from that surface taking into account the attained mortality level. In validation experiments, recorded mortalities were compared against modelled mortalities. Prediction of mortality was partially supported by the model, but pupae experiencing intensely fluctuating temperatures showed decreased mortality, probably caused by rapid cold hardening during exposure. Despite this observation, mortality data converged to distinct levels very close to 100% depending on the intensity of temperature fluctuations that were characteristic for different types of experiments. The highest mortality limit occurred at intensely fluctuating temperatures in laboratory experiments. This constituted a benchmark that was not reached under various field conditions. Thus, it was possible to identify temperature limits for the extinction of field populations of Tuta absoluta pupae.