Martin J. Downes

Dispersal Behaviour and Transmission Strategies of the Entomopathogenic Nematodes Heterorhabditis and Steinernema

Entomopathogenic nematodes of the Heterorhabditidae and Steinernematidae appear to be capable of long-distance dispersal and local migration. Their transmission strategies include both highly active seek-and-destroy behaviours and ambusher strategies, and they may be sensitive to sex-related factors in their own populations. Their host-finding abilities are poorly understood, despite the fact that these abilities are fundamental to their success as biocontrol agents in soil. Like the vast numbers of exotic hymenopterans and other natural enemies that have been released for biological control over the past century, they may be used in their ecologically competent wild-type form. On the other hand, because they are applied inundatively, they may be tailored, by breeding or transformation, to their intended purpose and to ecological incompetence, improving both their efficacy and their ecological safety.

ESTABLISHMENT, PERSISTENCE, AND INTROGRESSION OF ENTOMOPATHOGENIC NEMATODES IN A FOREST ECOSYSTEM

Entomopathogenic nematodes (EPN) are currently marketed worldwide for use in inundative biological control, where the applied natural enemy population (rather than its offspring) is expected to reduce insect numbers. Unlike classical biological control, in inundative control natural enemy establishment is not crucial in order to achieve pest suppression. Field trials in Irish forestry provided the opportunity to test predictions regarding the establishment of two exotic (Steinernema carpocapsae and Heterorhabditis megidis) and two indigenous (Steinernema feltiae and Heterorhabditis downesi) species. Nematodes were inundatively applied to pine stumps to control populations of pine weevil, Hylobius abietis, on three clearcut sites, and their persistence and spread monitored for up to five years. All species were recovered three years after application but only S. feltiae was recovered in years 4 and 5. Limited horizontal dispersal to 20 cm (but not 100 cm) was observed, but the majority of nematodes were recovered close to the area of application. Steinernema feltiae was also recovered from nearby stumps to which it had not been applied, indicating possible phoretic dispersal by weevils or other stump-associated fauna. EPN were not recovered from stumps outside the treated area, suggesting that such dispersal is quite localized. Two strains of S. feltiae (Irish and exotic) were applied. Amplified fragment length polymorphism (AFLP) analysis on 11 populations isolated from soil four years later showed that all had a much closer affinity to the applied Irish strain, suggesting persistence of this genotype and extinction of the exotic one. Some strains were clustered close together, and this is interpreted in the light of possible population genetic scenarios. The findings from the field study confirm predictions based on background knowledge of the species and demonstrate the importance of medium-term studies, as a 3-year study would have overestimated the risk of establishment of exotic species. Short-term persistence and spread of S. carpocapsae, S. feltiae, and H. downesi was also studied in pine forest mesocosms. Similar trends to field results, such as limited horizontal dispersal, even vertical distribution, and more abundant recovery of S. feltiae than of other species, point to the utility of mesocosm studies as a predictive tool.

Occurrence and distribution of the entomopathogenic nematodes Steinernema spp. and Heterorhabditis indica in Indonesia

Soil samples from 79 sites on five islands of Indonesia were baited with insects for the recovery of entomopathogenic nematodes. Heterorhabditis and Steinernema were equally prevalent, and were recovered from 11.7% of samples representing 20.3% of sites sampled. Both genera were recovered from coastal sites only. Entomopathogenic nematodes were more prevalent on the Moluccan islands of Ambon and Seram than on Java or Bali. They were not detected on Sulawesi, where non-coastal sites only were sampled. RFLP analysis was used in the identification of nematode isolates. Heterorhabditis indica was the only heterorhabditid identified. Two RFLP types of Steinernema were identified.

Molecular characterisation of Heterorhabditis indica isolates from India, Kenya, Indonesia and Cuba

Summary - Isolates of Heterorhabditis were identiA½ ed as H. indica using the following molecular diagnostic features: hybridisation to a H. indica speciA½ c satellite DNA probe; AluI and MboI restriction proA½ les of the rDNA ITS PCR product and the AluI proA½ le of the rDNA IGS PCR product. The Kenyan isolates represent a distinct subgroup of H. indica. These isolates lacked one of the two Hinf I restriction sites which are present in the rDNA ITS product of all the other isolates tested and they also differed from other H. indica isolates in their rDNA IGS HaeIII restriction proA½ le. The Indian isolates are interfertile. The Kenyan isolates are interfertile but only one Kenyan isolate, Ki3, produced viable progeny when crossed with H. indica LN2. The four Indonesian isolates are interfertile, but only one Indonesian isolate (INA H1) produced viable hybrids when crossed with H. indica LN2. INA H1 was also interfertile with the Kenyan isolate Ki3.

Control of Black Vine Weevil larvae Otiorhynchus sulcatus (Fabricius) (Coleoptera:Curculionidae) in grow bags outdoors with nematodes.

Abstract  1 Outdoor trials were carried out during 2001–02 on strawberries grown in commercial growing bags naturally infested with black vine weevil larvae (BVW) Otiorhynchus sulcatus in Co. Wexford, Ireland.

Effect of Timber Condition on Parasitization of Pine Weevil ( Hylobius abietis L.) Larvae by Entomopathogenic Nematodes under Laboratory Conditions

The large pine weevil ( Hylobius abietis L.) is one of the most important pests in coniferous reforestation in Europe. Larvae develop in the stumps of recently felled trees; the emerging adults feed on the bark of seedlings and may kill them. The ability of the entomopathogenic nematodes Heterorhabditis megidis and Steinernema carpocapsae to invade pine weevil larvae in Sitka spruce ( Picea sitchensis ) buried in moist sand was evaluated. Overall, four times as many H. megidis as S. carpocapsae invaded pine weevil larvae. The two species of nematode differed in their response to timber condition. The number of S. carpocapsae invading pine weevil larvae was twice as high in billets inoculated with the wood-rotting fungus Phlebiopsis gigantea as in fresh timber, while the number of H. megidis invading was reduced by 25%. Invasion into non-feeding insects (larvae of the wax moth Galleria mellonella ) contained in timber disks was also affected by timber quality, indicating that nematode behaviour was affected directly by the physical or chemical condition of the timber, though trophically mediated effects may also have been involved.

Facilitating co-existence by tracking gene dispersal in conventional potato systems with microsatellite markers

Based on international findings, Irish co-existence guidelines for the cultivation of GM potato stipulate that an isolation distance of 20 m is required to minimize the spread of transgenic pollen in accordance with required labeling thresholds. As potato tolerant to Phytophthora infestans is the most applicable GM crop from an Irish context, we tested the efficacy of this isolation distance under Irish environmental conditions using the conventional variety Désirée as a pollen donor and the male-sterile variety British Queen as a pollen receptor. Gene flow was determined by scoring for berry presence on receptor plants and confirmed using a microsatellite marker system designed to assess paternity in F(1) seedlings. 99.1% of seedlings recovered were identified as having Désirée paternity. Whereas 19.9% (140/708) of total berries formed on receptor plants occurred at a distance of 21 m from the pollen source, only 4 of these berries bore viable true potato seed (TPS), from which 23 TPS germinated. TPS-bearing berry formation was negatively correlated with distance from the pollen source, and although overall distribution of berries and seeds was non-random across the plot, no significant correlation was evident with respect to wind direction. Microsatellite markers were also used to confirm that the foraging beetle Meligethes aeneus is a vector for the transmission of potato pollen, but a more detailed statistical analysis of this dataset was limited by inclement weather during the trial. To conclude, we recommend that a two-tiered system be established in regard to establishing isolation distances for the experimental trial and commercial cultivation of GM potato in Ireland, and that responsible crop management be adopted to minimize the establishment of TPS-derived volunteers, which we have noted will emerge through a rotation as a result of pollen-mediated gene flow.

The effect of temperature on hatch and activity of second-stage juveniles of the root-knot nematode, Meloidogyne minor, an emerging pest in north-west Europe

{null=Meloidogyne minor is a serious threat to turfgrass in north-west Europe, and has a broad host range that includes other economically important plants. The species was described only recently and little is known about its biology. This study examines the effect of temperature on hatch and motility of second-stage juveniles (J2), and records hatching from egg masses collected from golf greens in different seasons. Eggs were present throughout the year and a high percentage hatch (46-88%) was recorded when they were incubated at 20°C. When egg masses were incubated at constant temperatures, J2 hatched between 15 and 25°C, with limited hatch ( Meloidogyne minor is a serious threat to turfgrass in north-west Europe, and has a broad host range that includes other economically important plants. The species was described only recently and little is known about its biology. This study examines the effect of temperature on hatch and motility of second-stage juveniles (J2), and records hatching from egg masses collected from golf greens in different seasons. Eggs were present throughout the year and a high percentage hatch (46-88%) was recorded when they were incubated at 20°C. When egg masses were incubated at constant temperatures, J2 hatched between 15 and 25°C, with limited hatch (<1%) at 10 and 30°C. The percentage hatch was lower at 15°C (43%) than at 20-25°C (63-76%). J2 hatched fastest at 23°C, with an average duration to hatching of 7 days compared to 17 days at 15°C. The range of temperatures at which J2 was active was broader than that at which they hatched. J2 were active from 4-30°C, with greatest activity between 15 and 25°C. The addition of grass root extract temporarily increased J2 activity at 10-20°C, but not at lower temperatures. }

Intraspecific variation among isolates of the entomopathogenic nematode Steinernema feltiae from Bull Island, Ireland

The application of large numbers of entomopathogenic nematodes (EPN) to control insect pests of agriculture is likely to have an impact on the local EPN fauna, yet little is known about the intraspecific relationships between EPN populations, particularly with regard to phylogeny and outbreeding. Here we assess the fitness, with regards to fecundity, host insect mortality and time taken to produce progeny, of isolates of Steinernema feltiae from Bull Island, Ireland. Exon-primed, intron-crossing (EPIC) PCR was used to examine intraspecific phylogenies between S. feltiae isolates, and identified up to three possible colonisation events of Bull Island. EPIC-PCR grouped two isolates, 33.D.(2) and 59.F.(2), separately from the remaining ten S. feltiae isolates These same two isolates consistently performed poorly in all fitness assessments. Following the crossbreeding of all isolates in Galleria mellonella , the number of host cadavers exhibiting emerging infective juveniles was significantly fewer than expected and there were significant differences between isolates in the number of days until progeny were observed. Host insect mortality varied between 40 and 87%. Such intraspecific variation may be a result of adaptation to different microhabitats of Bull Island, which in turn may be accentuated by laboratory culture practices.

Correlation between survival in water and persistence of infectivity in soil of Heterorhabditis spp. isolates

Persistence in soil of ten isolates of three species of Heterorhabditis was monitored by baiting the soil with insects and recording their mortality. Infective juveniles (IJ) of the same ten isolates were also incubated in tap water and their survival recorded. Both tests were conducted in the laboratory at 20°C. Inter- and intraspecific differences in persistence were detected: H. bacteriophora HI was the most persistent isolate in both media. There was no clear division between H. megidis (North West European Group) and H. downesi , but isolates of H. downesi tended to be less persistent than those of H. megidis . Nematodes persisted longer in soil than in water: after 180 days in water, all IJ were dead in seven of the ten isolates, whereas all isolates still killed insects after 265 days in soil. Persistence of isolates in soil (indicated by LT 50 , the time that nematode-infested soil retained the ability to kill 50% of the bait insects) was correlated with their survival in water (represented by ST 50 , the time at which 50% of the IJ were still alive), with r 2 = 0.84, indicating that similar factors were responsible for the reduction in each parameter.