Field Test Using the Nematode Phasmarhabditis hermaphrodita for Biocontrol of Slugs in Spain

An experiment was carried out between May and July 1999 in Galicia (North-West Spain) to test the capacity of the nematode Phasmarhabditis hermaphrodita to protect field grown lettuces from slug damage in our field conditions. The experiment compared a single dose of nematodes (3 ×109 ha -1) with mini-pellets containing 5% metaldehyde, applied at the recommended field rate (3 g pellets m -2), and untreated plots. Slug damage for each lettuce head was estimated on six dates during the first 4 weeks after planting. At harvest, each lettuce head was weighed, scored as marketable or not by weight and external aspect, and inspected for slugs. Metaldehyde significantly reduced slug damage to lettuce plants from the first day after planting to the third week. Nematodes significantly reduced slug damage from the second to the third week. At harvest, 6 weeks after planting, the mean weight of the lettuce heads and the number of marketable heads in the nematode plots were as good as in the metaldehyde plots, and both treatments were significantly better than the untreated plots. The number of slugs within the harvested plants was significantly reduced only with the metaldehyde treatment.     

Targeting Biocontrol with the Slug-Parasitic Nematode Phasmarhabditis hermaphrodita in Slug Feeding Areas: a Model Study

The nematode Phasmarhabditis hermaphrodita was applied to soil in an outdoor miniplot experiment to protect Chinese cabbage seedlings from damage by the field slug Deroceras reticulatum. The aim was to investigate the possibility of reducing the numbers of nematodes applied by only partially spraying soil in the area where slug control was needed. Nematodes sprayed as overall applications were compared with band applications along plant rows and spot applications around individual plants, in plots with nine or 18 plants. Band and spot applications were applied at two rates, designated the full rate (same number of nematodes per plot as in the overall application) and the area rate (same number of nematodes per unit area comprising 43% (band) and 18% (spot) of the overall application). In plots with 18 plants, where spot-treated plant alternated with untreated plants, no significant difference in damage was found between spot-treated plants and untreated plants. This indicates that slugs were not repelled from nematode-treated areas and that any effects in reducing slug damage were not due to repellency. All nematode treatments resulted in a significant reduction in the mean level of slug damage to seedlings from six or more days after treatment. However, there were significant interactions between nematode treatment, the number of plants per plot, the position of plants within plots (edge or middle) and time after treatment. The effect of time after treatment was modelled. The log time to 50% reduction in slug damage (t 50 ) was related to the area treated and the dose applied. In plots with band or spot treatments at the full dose, there was a relatively small increase in t 50 with declining area treated. In plots treated with band or spot treatments at the area dose, t 50 increased consistently with declining relative area treated. The final level of damage, expressed as a percentage of damage on untreated plots (P), was influenced by both the dose and area treated. Final damage was greatest on spoti treated plots where half the plants were untreated. We conclude that partial treatment of soil around all plants to be protected from slug damage is a potentially valuable method of reducing the overall nematode dose required for control of slug damage, provided that some damage can be tolerated.     

The rhabditid nematode Phasmarhabditis hermaphrodita as a potential biological control agent for slugs

A nematode, Phasmarhabditis hermaphrodita, known to be associated with slugs but not previously thought to be parasitic, was shown to be a parasite capable of killing the pest slug Deroceras reticulatum. The parasite infects slugs in the area beneath the mantle surrounding the shell, causing a disease with characteristic symptoms, particularly swelling of the mantle. Infection leads to death of the slug, usually between seven and 21 days afterwards. The nematode then spreads and multiplies in the cadaver. In an experiment where individual D. reticulatum were exposed to different numbers of P. hermaphrodita, a significant positive relationship was found between nematode dose and slug mortality. In two experiments on host range, the nematode was found to infect and kill all pest slug species tested: Deroceras caruanae, Arion distinctus, Arion silvaticus, Arion intermedius, Arion ater, Tandonia sowerbyi and T. budapestensis, in addition to D. reticulatum.     

Non-Susceptibility of Earthworm Eisenia fetida to the Rhabditid Nematode Phasmarhabditis hermaphrodita, a Biocontrol Agent of Slugs

The rhabditid nematode Phasmarhabditis hermaphrodita is a lethal parasite for slugs that is sold commercially in Europe under the trade name Nemaslug™. We evaluated the effects of P. hermaphrodita on the earthworm Eisenia fetida (Savigny). Adults of E. fetida were exposed in one-liter glass beakers to P. hermaphrodita at three concentrations (1×, 10× and 50× of the field recommended rate of 3×10⁹ billion nematodes/ha) during a 14-day period in an artificial soil substrate. The average body weight, burrowing behavior, mortality and other clinical signals of the earthworms were recorded at 0, 7, and 14 days after exposure to the treatments. In addition, injured earthworms (posterior ends removed) were exposed to the 10× field recommended rate of the commercial formulation. Neither intact nor injured E. fetida showed susceptibility to the slug-parasitic nematode P. hermaphrodita during the 14 days of exposure even at concentrations 10 and 50 times higher than the label dose. However, the worms in the attenuated control (autoclaved formulation) had higher mortality and lost less weight compared to the other treatments. Under the conditions of the test, we conclude that the use of the commercially available strain of P. hermaphrodita is safe to E. fetida.     

Susceptibility of North American Native and Non-native Slugs (Mollusca: Gastropoda) to Phasmarhabditis hermaphrodita (Nematoda: Rhabditidae)

Phasmarhabditis hermaphrodita caused significant mortality of the two native species, Deroceras laeve and Leidyula floridana, and of one introduced species D. reticulatum, but not of the other three introduced species, Arion hortensis, A. subfuscus and Limax maximus. Even the juvenile stages of A. subfuscus and L. maximus showed no mortality in nematode treatments. However, treatments with nematodes resulted in rapid and strong feeding inhibition in all six species. Surviving slugs resumed feeding when fresh food was provided. This study expands the host range of P. hermaphrodita to include a new family Vaginulidae and demonstrates the inability of P. hermaphrodita to cause mortality of A. hortensis, A. subfuscus and L. maximus. These three species use feeding suspension as an evasive behavior to escape nematode infection.     

Overall Versus Band Application of the Nematode Phasmarhabditis hermaphrodita With and Without Incorporation into Soil, for Biological Control of Slugs in Winter Wheat

In two concurrent field experiments, the effects of three types of soil cultivation and two patterns of nematode application were studied in order to investigate their effects on damage to winter wheat by slugs (assessed at Zadoks Growth Stage 12). In experiment 1, infective juveniles (IJs) of the nematode Phasmarhabditis hermaphrodita were applied to soil as an overall spray or as a band spray (8-cm wide), centred on the drill rows (16.7-cm apart). Nematodes were either left undisturbed on the soil surface or harrowed into the soil immediately after application. The control provided by nematodes was compared with that provided by metaldehyde and methiocarb pellets broadcast at the recommended rate immediately after drilling. In this experiment, winter wheat on plots treated with IJs showed significantly less slug damage than on wheat plots treated with metaldehyde or methiocarb pellets or untreated plots. There was no significant difference in plant damage between plots treated with band and overall spray applications of IJs, nor was there any significant difference between plots with and without harrowing. There was also no significant difference between untreated plots and plots treated with metaldehyde or methiocarb pellets, probably because rainfall shortly after treatment rendered the pellets ineffective. In experiment 2, nematodes were applied as an overall spray or plots were not treated with nematodes before soil was cultivated with tines, Roterra or Dutzi cultivators. Nematode application before soil cultivation using tines or Roterra reduced the number of plants damaged significantly. However, nematodes applied before Dutzi cultivation appeared to be rendered ineffective. Damage to winter wheat was lowest in plots that had been sprayed with nematodes and subsequently cultivated with tines or Roterra.     

Influences of preceding cover crops on slug damage and biological control using Phasmarhabditis hermaphrodita

In a replicated field experiment, ryegrass, vetch and red clover were grown or the soil was kept bare over a 2–month period in summer to compare the effects of these treatments on slug damage to the following crop (Chinese cabbage) and on the efficacy of nematodes (Phasmarhabditis hermaphrodita) applied as biological control agents to the soil at planting time to protect this crop. Slug damage was significantly (c. two times) greater after red clover or vetch than after ryegrass. Damage on plots without cover crop was intermediate and not significantly different from either extreme. Slug damage was reduced by about one‐third by the nematode treatment. The preceding cover crop did not influence nematode efficacy. Numbers of slugs on harvested plants (mainly Deroceras reticulatum and Deroceras panormitanum) were influenced by an interaction between cover crop and nematode treatment. On subplots without nematodes, more slugs were recorded with than without a preceding cover crop. No such differences were found on nematode‐treated subplots. Soil samples were collected at intervals from 0–99 days after nematode treatment to monitor nematode survival and infectivity in bioassays with D. reticulatum. No significant effects of cover crops were detected in bioassays. Moreover, there were no significant effects of nematodes on slug survival. Their effects on slug food consumption were mostly insignificant and any effects were transient and not consistent. However, significantly more slug cadavers contained nematodes when slugs were exposed to nematode‐treated soil. The implications of these results are discussed.     

Biological control of slugs in winter wheat using the rhabditid nematode Phasmarhabditis hermaphrodita

A field experiment on winter wheat in autumn 1991 investigated the effect of the rhabditid nematode, Phasmarhabditis hermaphrodita, applied to soil at five dose rates (10⁸ - 10¹⁰ infective larvae ha^-1) immediately after seed sowing, on slug populations and damage to seeds and seedlings. The nematode was compared with methiocarb pellets broadcast at recommended field rate immediately after drilling and no molluscicide treatment. Slug damage to wheat seeds and seedlings was assessed 6 and 13 wk after drilling. Seedling survival increased and slug grazing damage to seedlings declined linearly with increasing log nematode dose. These two measures of slug damage were combined to give an index of undamaged plant equivalents, which also increased linearly with increasing log nematode dose. ANOVA showed that, after 6 wk, there were significantly more undamaged plant equivalents on plots treated with the two highest nematode doses (3 × 10⁹ and 1 × 10¹⁰ ha^-1) than on untreated plots, but the number of undamaged plant equivalents on methiocarb-treated plots was not significantly greater than that on untreated plots. Slug populations were assessed by refuge trapping and soil sampling. Deroceras reticulatum was the commonest of several species of slugs recorded. During the first 4 wk after sowing, significantly more slugs were found under refuge traps on plots treated with certain doses of P. hermaphrodita than under traps on untreated plots and more showed signs of nematode infection than expected from the prevalence of infection in slugs from soil samples, suggesting that the presence of P. hermaphrodita altered slug behaviour. Application of P. hermaphrodita had no significant impact on numbers or biomass of slugs in soil during a 27 wk period after treatment, except after 5 wk when slug numbers were inversely related to log nematode dose. However, by this time, numbers in soil samples from untreated plots had declined to levels similar to those in plots treated with the highest dose of nematodes. During the first 5 wk after treatment, c. 20% of slugs in soil samples from untreated plots showed symptoms of nematode infection. It is suggested that this represented the background level of infection in the experimental field rather than spread of infection from treated plots. The apparent lack of impact of P. hermaphrodita on slug numbers and biomass in soil suggests that its efficacy in protecting wheat from slug damage was through inhibition of feeding by infected slugs.     

Susceptibility and immune response of Deroceras reticulatum, Milax gagates and Limax pseudoflavus exposed to the slug parasitic nematode Phasmarhabditis hermaphrodita

We exposed three slug species (Deroceras reticulatum (Müller), Milax gagates (Draparnaud) and Limax pseudoflavus L.) to the parasitic nematode Phasmarhabditis hermaphrodita Schneider. P. hermaphrodita was able to cause mortality and feeding inhibition to both D. reticulatum and M. gagates but did not negatively affect L. pseudoflavus. On dissection of surviving L. pseudoflavus large numbers of P. hermaphrodita were found encapsulated in the shell of the slug. We found that by increasing shell size, the slug was able to trap invading nematodes, which could be an immune response to P. hermaphrodita invasion. This is the first report of a slug defense mechanism to inhibit P. hermaphrodita.     

Feeding Activity and Survival of Slugs,Deroceras reticulatum,Exposed to the Rhabditid Nematode,Phasmarhabditis hermaphrodita: A Model of Dose Response

The slug, Deroceras reticulatum (Stylommatophora: Limacidae), was exposed to different concentrations of infective dauer juveniles of the rhabditid nematode Phasmarhabditis hermaphrodita, in a two-stage bioassay, at 10°C. Slugs were exposed in groups of 10 or 12 to nematodes in plastic boxes filled with soil aggregates for 3 or 5 days and then transferred individually to petri dishes each containing a disk of Chinese cabbage leaf as food. Subsequently, slug food consumption and survival were measured for 10 to 13 days. Models were developed to describe the way that exposure to the nematode caused inhibition of slug feeding followed by death. Both effects were related to nematode concentrations and time after exposure to the nematode. Following exposure to high concentrations (300,000 dauer juveniles per box), slugs were killed rapidly, within a few days after the end of the exposure period. Following exposure to low concentrations of nematodes (7000 or 15,000 per box), substantial numbers of slugs survived until the end of the bioassay, but feeding activity by these slugs was strongly inhibited. It is suggested that inhibition of slug feeding is important for the success of this nematode as a biocontrol agent.     

Susceptibility of indigenous UK earthworms and an invasive pest flatworm to the slug parasitic nematode Phasmarhabditis hermaphrodita

We exposed five earthworm species, one flatworm species and the susceptible slug Deroceras reticulatum to the recommended field application rate and five times this rate of Phasmarhabditis hermaphrodita. P. hermaphrodita caused significant mortality to D. reticulatum at the recommended rate and five times the recommended rate. Survival of the earthworms and the flatworm was not different from the untreated controls. P. hermaphrodita is a safe biological control agent causing no harm to earthworms and could not be used to kill Arthurdendyus triangulatus.     

Behavioural avoidance by slugs and snails of the parasitic nematode Phasmarhabditis hermaphrodita

The nematode Phasmarhabditis hermaphrodita has been developed as a biological control agent for slugs and snails. Slugs avoid areas where P. hermaphrodita is present. We investigated whether behavioural avoidance of P. hermaphrodita is a common feature of slugs and snails by exposing eight species to P. hermaphrodita. We showed that slugs generally avoided P. hermaphrodita, whereas snails did not. We also showed that slugs specifically avoided the commercial strain and a natural isolate of P. hermaphrodita and were not deterred by other nematodes such as Steinernema kraussei or Turbatrix aceti. We also showed that slugs avoided the dauer stage of P. hermaphrodita and not mixed-stage cultures. Furthermore, slugs do not avoid dead P. hermaphrodita or exudates from live nematodes. Taken together, we have unravelled further factors that are essential for slugs to avoid P. hermaphrodita in soil, which could have important implications for the biological control of slugs and snails.     

Low Temperatures Suppress Growth of the Ciliate Parasite, Tetrahymena rostrata , and Pathogenicity to Field Slugs, Deroceras reticulatum

The slug parasitic ciliate protozoan, Tetrahymena rostrata , was isolated from the slug Deroceras reticulatum and used in bioassays against D. reticulatum at 10 and 17oC and in in vitro growth experiments at a range of temperatures. The parasite reproduced, and significantly reduced the life span of juvenile D. reticulatum at 17oC, but not at 10oC.     

First record for the slug parasitic nematode,Phasmarhabditis hermaphrodita (Schneider) in Egypt

The rhabditid nematode, Phasmarhabditis hermaphrodita is a lethal parasite of certain terrestrial gastropods and has been shown as a biocontrol agent under laboratory and field conditions. In Egypt, P. hermaphrodita was isolated for the first time from different species of terrestrial gastropods found associated with various crops at Aga and Mansoura districts of Dakhalia Governorate during the year 2000/2001. Females and dauer larvae (IJs) were described and illustrated based on the light microscope. Males are not found as this species seemed to be protandrous. PCR analysis confirmed nematode identification. The Egyptian isolate of P. hermaphrodita was found to be shorter and lower in width than the British isolate. V%, a, b and c parameters showed detectable variations between two isolates with values of 54%, 17.7, 4.28, 13.7 in the Egyptian isolate, and 51%, 19.5, 7.2 and 15.8 for the British isolate, respectively.     

Size-specific susceptibility of the pest slugs Deroceras reticulatum and Arion lusitanicus to the nematode biocontrol agent Phasmarhabditis hermaphrodita

The nematode Phasmarhabditis hermaphrodita is a commercially available biocontrol agent against slugs. This product is especially interesting for use in organic farming, where products containing metaldehyde or carbamates cannot be used for controlling pest slugs. We investigated the potential of P. hermaphrodita for the control of the pest slugs Deroceras reticulatum and Arion lusitanicus. These two species are the most harmful slug pests in Switzerland. At different times of the year, we collected slug specimens of different weight and assessed their susceptibility to P. hermaphrodita in the laboratory. Batches of five slugs were subjected to five different doses of nematodes plus an untreated control and replicated three times. During six weeks, feeding and survival of the slugs were recorded. D. reticulatum was strongly affected by increasing nematode doses, irrespective of the slugs' body weight. In small specimens of A. lusitanicus, feeding and survival were strongly affected by the nematodes, while larger specimens remained almost unaffected. Because A. lusitanicus has an asynchronous development in Switzerland, it seems difficult to control the entire population with a single nematode application. To what extent nematodes will be used in practice for slug control depends on their effectivity against the pest slugs of major importance, on the longevity of the molluscicidal effect and on the price of nematodes.     

The effects of the slug biological control agent, Phasmarhabditis hermaphrodita (Nematoda), on non-target aquatic molluscs

The nematode Phasmarhabditis hermaphrodita is used as a commercial biological control agent of slugs in the UK. Although it is known to affect other terrestrial mollusc species, its effects on freshwater molluscs are not known. The present study investigated the effects of P. hermaphrodita on the survival of juvenile Lymnaea stagnalis and Physa fontinalis, two common freshwater snails, at 'spray tank' concentration and a 50% diluted 'spray tank' concentration over a 14-day period. Survival of L. stagnalis was significantly reduced at both application levels but P. fontinalis suffered no mortalities over the experimental period. The possible differential mechanisms of pathology between the two host species are discussed.     

Mass cultivation and storage of the rhabditid nematode Phasmarhabditis hermaphrodita,a biocontrol agent for slugs

The rhabditid nematode Phasmarhabditis hermaphrodita (a parasite capable of killing pest slugs) was grown in vitro, in association with a mixed bacterial flora on foam chips impregnated with a kidney‐based nutrient medium in aerated bags, to provide sufficient numbers for laboratory and field experiments. The feasibility of producing nematodes in liquid culture was investigated using 250 ml flasks. Baffled flasks containing 25 or 50 ml or liquid were found to be better than baffled flasks containing 100 ml or unbaffled flasks. Inoculum densities ranging from 50 to 330 ml^‐1 did not affect final yield. Dauer larvae in aerated water died rapidly at temperatures of 26–35°C. Survival was progressively better at 22°C and 15°C, and best at 5°C or 10°C.     

Daily variation in the number of slugs under refuge traps

Daily variation in the number of slugs under and around 25 cm × 25 cm pad‐traps was recorded in plots of crop cultures. Grey field slugs Deroceras reticulatum and garden slugs (Arion distinctus and Arion hortensis) left traps at dusk and came back at the end of the night. The time of day at which the number of trapped slugs was maximal did not occur in the middle of the day, when slugs were inactive, but in the early morning and in the evening, a few hours before dusk. This was due to a rise in temperature under the traps in the middle of the day. However, the grey field slugs stayed under traps throughout the morning, when temperatures under the traps reached values more favourable for rest.     

Hirsutella rhossiliensisand Verticillium chlamydosporium as Biocontrol Agents of the Root-knot Nematode Meloidogyne hapla on Lettuce

Hirsutella rhossiliensis and Verticillium chlamydosporium infected second-stage juveniles (J2) and eggs of Meloidogyne hapla, respectively, in petri dishes and in organic soil in pots planted to lettuce in the greenhouse. In vitro, H. rhossiliensis produced 78 to 124 spores/infected J2 of M. hapla. The number of J2 in roots of lettuce seedlings decreased exponentially with increasing numbers of vegetative colonies of H. rhossiliensis in the soil. At an infestation of 8 M. hapla eggs/cm³ soil, 1.9 colonies of H. rhossiliensis/cm³ soil were needed for a 50% decrease in J2 penetration of lettuce roots. Egg-mass colonization with V. chlamydosporium varied from 16% to 43% when soil was infested with 8 M. hapla eggs and treated with 5,000 or 10,000 chlamydospores of V. chlamydosporium/cm³ soil. This treatment resulted in fewer J2 entering roots of bioassay lettuce seedlings planted in the infested soils after harvesting the first lettuce plants 7 weeks after infestation with M. hapla. Hirsutella rhossiliensis (0 to 4.3 colonies/cm3 soil), V. chlamydosporium (500 to 10,000 chlamydospores/cm3 soil), or their combination, added to organic soils with 8 M. hapla eggs/cm³ soil, generally did not affect lettuce weight, root galling, or egg production of M. hapla. However, when lettuce was replanted in a mix of infested and uninfested soil (1:3 and 1:7, v:v), egg production was lower in soils with V. chlamydosporium than in soils without the fungus. Both fungi have potential to reduce the M. hapla population, but at densities below 8 eggs/cm³ soil.     

Low temperature survival in different life stages of the Iberian slug, Arion lusitanicus

The slug Arion lusitanicus Mabille (Gastropoda: Pulmonata: Arionidae) is an invasive species which has spread to most parts of Europe. The area of origin is unknown, but A. lusitanicus seems to cope well with the local conditions in the countries to which it has migrated. It spreads rapidly, occurs often in high densities and has become a serious pest in most European countries. Therefore there is an urgent need for better knowledge of the ecophysiology of A. lusitanicus, such as the influence of climatic conditions, in order to develop prognostic models and strategies for novel pest management practises.The aim of our study was to investigate the influence of subzero temperatures in relation to winter survival. A. lusitanicus is shown to be freeze-tolerant in some life stages. Most juveniles and some adult slugs survived being frozen at −1.3 °C for 3 days, but none of the slugs survived freezing at −3 °C. The eggs survived subzero temperatures (down to −2 °C) probably by supercooling. Juveniles and adults may also survive in a supercooled state (down to −3 °C) but are generally poor supercoolers. Therefore, the winter survival of A. lusitanicus depends to a high degree on migration to habitats protected from low winter temperatures, e.g. under plant litter, buried in the soil or in compost heaps.