Susceptibility of Sap Beetles (Coleoptera: Nitidulidae) to Entomopathogenic Nematodes

Nitidulid beetles (Coleoptera) are considered to be serious pests of date palms throughout the world. They attack ripe fruit, causing it to rot, and damage is reflected in both reduced yield and lower fruit quality. The present study was aimed at an evaluation of the susceptibility to different sap beetles to entomopathogenic nematodes. We further tested nematode efficacy in pots filled with soil infested by third instar larvae of the two beetle species. In Petri dish assay, mortality levels of Carpophilus humeralis and C. hemipterus exposed to Heterorhabditis sp. IS-5 strain indicated that the latter is less susceptible to nematode infection. Exposure of both sap beetle species to different nematode strains gave moderate levels of mortality (35-65%) with the heterorhabditid strains HP88, IS-5 and IS-25. The IS-12 strain of Heterorhabditis sp. showed poor virulence (<35% mortality) against larvae of C. humeralis as well as larvae and pupae of C. humipterus. The nematode species S. riobrave showed moderate virulence (35-65%) mortality to larvae and pupae of S. humeralis as well as to larvae of C. hemipterus . Exposure of C. hemeralis to different concentrations of Heterorhabditis sp. IS-5 in pots containing soil resulted in high mortality (>65%). In contrast, the lower concentrations (500 and 1000 nematodes/pot) caused low mortality (35%) of C. hemipterus . Other heterorhabditid strains caused 95-100% mortality of C. humeralis in pot assay. The HP88 strain of H. bacteriophora and the Tx strain of Steinernema riobrave showed poor effectiveness. Incubation of different nematode strains with the C. humeralis larvae at high temperature (32 C) resulted in an increase in insect mortality with the IS-12 and IS-21 strains. Reduced mortality was recorded with the HP88 strain treatment at the higher temperature. The IS-5 and IS-12 strains were equally effective in all three soil types tested, whereas the IS-19 strain was more effective in the Almog type soil than in the others.     

Evaluation of Entomopathogenic Nematodes for the Control of Mediterranean Fruit Fly (Diptera: Tephritidae)

The virulence of various entomopathogenic nematode (EPN) strains was evaluated against the Mediterranean fruit fly, C. capitata . The selected nematodes were assessed for their infectivity for the final larval stage of the insect host and under varying environmental conditions. Among 12 EPN strains tested, Steinernema riobrave Texas ( Sr TX) and Heterorhabditis sp. IS-5 (H IS-5), showed high activity and induced >80% mortality. Six EPN strains showed limited activity (>30% mortality), and four strains had no effect (<20% mortality). Sr TX was more effective than H IS-5. Mature C. capitata larvae were most susceptible to nematode infection during the first 4h after they began to emerge from their diet to pupate. Activity of the two nematode strains at a constant inoculation rate was dependent on insect larval density. The highest activity was recorded at 1.88 larvae cm -2 and decreased at higher larval densities. EPN activity was also directly related to nematode density. Maximal activity was shown at a density of 150 infective juveniles cm -2 . A similar activity pattern was also recorded with Sr TX in four different soil types. The persistence of this EPN in the soil extended over 5 days but there was no activity after 14 days. Except for a lower activity under cool conditions (17°C), temperatures ranging between 22 and 41°C, or moisture levels in the treated soil ranging between 3 and 20%, had no significant effect on nematode activity. Our results suggest that application of Sr TX against C. capitata may have potential for controlling C. capitata .     

Biocontrol Potential of Entomopathogenic Nematodes Against Winter Moths (Operophtera brumata and O. fagata) (Lepidoptera: Geometridae) Infesting Urban Trees

Infectivity and biocontrol potential of entomopathogenic nematodes against winter moths (Operophtera brumata and O. fagata)pupating in the soil were examined in laboratory, semi-field and field conditions. A pilot experiment conducted in the field showed that Steinernema feltiae was completely ineffective against pupae of these moths in the soil. Subsequent laboratory tests revealed that none of the tested species (i.e. S. feltiae, S. affinae, S. carpocapsae, Heterorhabditis megidis and H. bacteriophora) could colonise the pupae, while mature larvae descending to the soil for pupation and prepupae were highly susceptible to nematode infection. No differences were observed between O. brumata and O. fagata in susceptibility to nematodes. In laboratory experiments H. megidis applied at 1.5×10⁵infective juveniles (IJ) m^-2infected almost 100% of insects exposed for 6 days in the soil. It was significantly more infective than H. bacteriophora (73-77%) and Steinernema species (29-50%). H. megidis was also highly effective in semi-field conditions when applied at an even lower dose, i.e. 10⁵IJ m^-2. After a 45-day experiment, only 3% of insects descending for pupation survived in the soil pre-treated with this species. This was significantly less than in soil with S. feltiae (43%) and control treated with water only (59%). Very high efficacy of H. megidis and a relatively easy method for its field application through ground spraying gives some promise for environmentally safe and successful biological control of winter moths during their pupation in the soil. The low application rate required and recycling in the host could be additional advantages for economic and long lasting protection of high value trees, particularly those in urban parks and forests.     

Legislation on the Introduction of Exotic Entomopathogenic Nematodes into Australia and New Zealand

There are stringent requirements for the importation of all exotic organisms into Australia and New Zealand but since both countries have already permitted the importation and release of some species of both Heterorhabditis and Steinernema , the difficulties of the importation of entomopathogenic nematodes are reduced. In both countries, a series of authorities must be consulted before importation is permitted but only in New Zealand must entomopathogenic nematodes be registered before commercial trials and sales are allowed. Registration not only entails a thorough evaluation of the nematode species and its formulation for a wide range of possible harmful effects to humans, crops and the environment, but efficacy must be demonstrated for each species of nematode in each type of formulation against each pest.     

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.     

Insect Biocontrol with Non-endemic Entomopathogenic Nematodes (Steinernema and Heterorhabditis spp.): Conclusions and Recommendations of a Combined OECD and COST Workshop on Scientific and Regulatory Policy Issues

Fifteen invited experts from 10 Organisation for Economic Cooperation and Development (OECD) and European countries participating in the European Commission's Cooperation in the Field of Science and Technical Research (COST) Action 819, along with 12 other participants, met to review and debate the potential problems associated with the introduction and commercial use of non-indigenous nematodes for insect biological control. The consensus view of the participants was that entomopathogenic nematodes (EPNs) possess specific biological and ecological features, which make their use in biological control exceptionally safe. All the scientific evidence available supports the conclusion that EPNs are safe to the environment, as well as to production and application personnel, the general public and the consumers of agricultural products treated with them. Only a few potential, but very remote, risks could be identified. Therefore, it was recommended that EPNs should not be subject to any kind of registration. The introduction of non-indigenous nematode species, however, should be regulated. Species should be accurately identified, and details of the origin, known distribution, probable host range and safety to the user must be provided. In addition, an expert opinion, based on available information, of the possible impact on non-target organisms must be available.     

EVect of the Entomopathogenic Nematodes, Steinernema feltiae and S. carpocapsae , on the Potato Cyst Nematode, Globodera rostochiensis , in Pot Trials

Two pot experiments, one in a glasshouse and the other in an outdoor sand plunge, were conducted to examine the influence of the entomopathogenic nematodes, Steinernema feltiae and S. carpocapsae , on the invasion and development of the potato cyst nematode, Globodera rostochiensis . Of a total of eight diVerent treatments with entomopathogenic nematodes in the glasshouse trial, three reduced the invasion of G. rostochiensis and one reduced the numbers of new cysts that were produced compared with controls. In the outdoor experiment, seven of the 12 treatments gave a reduction in invasion but none resulted in changes in the numbers of cysts found at plant senescence. In general, invasion of G. rostochiensis juveniles was reduced more eVectively by S. carpocapsae than by S. feltiae , and was greatest in the outdoor trial where larger inocula of entomopathogenic nematodes were used. Overall, the results indicated that use of S. feltiae and S. carpocapsae is unlikely to provide a viable control strategy for G. rostochiensis .     

Effects of Entomopathogenic Nematodes on Control of a Mushroom-infesting Sciarid Fly and on Mushroom Production

The entomopathogenic nematodes Steinernema feltiae (Biosys strain #27) and Heterorhabditis heliothidis were evaluated for the larval control of a mushroom-infesting sciarid, Lycoriella mali, and for the effects of these nematodes on mushroom (Agaricus bisporus) production. In a series of small-scale mushroom crops, infective-stage H. heliothidis and S. feltiae were applied to the mushroom casing surface in the irrigation water or incorporated into the casing material at densities ranging from 28 to 1120 and 11 to 1120 nematodes cm-2 of casing surface respectively. The mortality of L. mali larvae ranged from 52 to 100% for H. heliothidis and 38 to 100% for S. feltiae. Both nematode species reduced mycelial coverage on the casing surface at primordia initiation. Neither mushroom strain (off-white or white hybrid) or method of application (incorporation into or irrigation onto the casing surface) altered the effect on mycelial coverage. The nematodes's negative effect on mycelial growth confounded the benefit of fly control. At high nematode densities (up to 1120 nematodes cm-2), damage-free mushroom yields for the first week of harvest were less than those from the untreated control. However, at lower nematode densities, at or below 140 cm-2, the nematodes had less effect on mushroom growth, and consequently, damage-free mushroom yields for the first week of harvest were frequently greater than those from the untreated control. In the absence of flies, the first-week mushroom yield generally declined with increasing nematode densities for both white and off-white mushroom hybrids. After 4 weeks of harvest, accumulated mushroom yields had nearly recovered from the earlier decline.     

Impact of Entomopathogenic Nematodes on Different Soil-dwelling Stages of Western Flower Thrips, Frankliniella occidentalis (Thysanoptera: Thripidae), in the Laboratory and Under Semi-field Conditions

The impact of entomopathogenic nematodes (EPN) on mortality of soil-dwelling stages of western flower thrips (WFT), Frankliniella occidentalis (Thysanoptera: Thripidae) with different insect stage combinations was studied in the laboratory and under semi-field conditions. In laboratory experiments, the efficacy of Steinernema feltiae strain Sylt (Rhabditida: Steinernematidae) at a concentration of 400 infective juveniles (IJs) cm -2 was tested against different proportions of soil-dwelling stages of WFT, i.e. late second instar larvae (L2), prepupae and pupae. Soil was used as the testing medium. S. feltiae significantly affected the mortality of all soil-dwelling life stages of WFT at all tested insect stage combinations. The proportion of late L2 in the population negatively correlated to EPN-induced mortality. WFT prepupa and pupa were similarly susceptible to S. feltiae and their proportion in the population did not affect the EPN-induced mortality under laboratory conditions. The highest mortality (80%) was recorded when the population consisted only of prepupae and/or pupae. In the semi-field study, the impact of S. feltiae , S. carpocapsae strain DD136 and Heterorhabditis bacteriophora strain HK3 (Rhabditida: Heterorhabditidae) ( H. bacteriophora ) at concentrations of 400 and 1000 IJs cm -2 was evaluated against WFT reared on green beans, Phaseolus vulgaris L., as host plant in pot experiments in a controlled climate chamber. All tested EPN strains at both dose rates significantly reduced the WFT populations. Up to 70% reduction of the WFT population was obtained at the higher EPN concentration.     

Current and Future Use of Nematodes in Biocontrol: Practice and Commercial Aspects with Regard to Regulatory Policy Issues

Constraints about the use of chemical insecticides have limited the availability of control measures against soil-borne insect pests. Entomopathogenic nematodes of the genera Steinernema and Heterorhabditis provide an environmentally safe and economically reasonable alternative. Their life cycle and current production, storage and formulation technology are described. An overview of their safety, use in integrated pest management and current market potential (US$10 million in 1994) is also given. The costs of research and development efforts and the scale-up of production technologies are discussed in relation to the current and future market potential. Large-scale, outdoor application will require additional scientific and technical progress in the areas of production, storage, formulation and application. Besides public funding, the current niche markets will need to provide the financial basis for further development, provided that regulatory conditions will not limit the exploitation of the nematodes' market potential. It is recommended that nematodes should be exempted from registration. Rules for risk assessment in the use of exotic nematodes should be internationally harmonized and related specifically to the biology and ecology of these nematodes. The volume of current markets would not justify the costs of registration procedures currently required for chemical or microbial insecticides or genetically engineered organisms. Regulatory policies should aim at supporting the further introduction of entomopathogenic nematodes as biocontrol agents.     

Efficacy and Persistence of Entomopathogenic Nematodes in Controlling Larval Populations of Thaumetopoea pityocampa (Lepidoptera: Thaumetopoeidae)

A 3-year study was conducted in a Pinus halepensis reforestation of Apulia Region (Southern Italy) injecting IJs (infective juveniles) of Steinernema feltiae , S. carpocapsae and Heterorhabditis bacteriophora in aqueous and gel suspensions (Idrosorb SR 2002 [Nigem ® ], and Compex) into the nests of Thaumetopoea pityocampa caterpillar. This study showed that the gel suspensions do not percolate and that slow release of water from the gels allowed nematodes to survive and complete their life cycle in the host. Results demonstrate the feasibility of reducing overwintering larval populations by injecting gel suspension of S. feltiae . We found no negative effects on the endoparasite Phryxe caudata .     

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.     

Fruit Bins Washed with Steinernema carpocapsae (Rhabditida: Steinernematidae) to Control Cydia pomonella (Lepidoptera: Tortricidae)

A bin washing apparatus (drencher) was used to treat late-instar codling moth, Cydia pomonella Linnaeus-infested wooden fruit bins with the entomopathogenic nematode Steinernema carpocapsae . Sentinel late-instar codling moth larvae were placed in strips of wood, made from dismantled bins, that were subsequently screwed into the corners of real bins before treatment. Pre-soaking the bins with water prior to treatment and covering the bins with a plastic tarp post-treatment, to maintain a high humidity, significantly increased sentinel codling moth mortality. Treatment for 1 min with 50 S. carpocapsae infective juveniles mL -1 water resulted in more than 80% mortality over 6 h in which the nematodes were continuously pumped through the drencher. The temperature of the water in the bin washer was ≤15°C and the chlorine in the city water was too low to harm the nematodes. The key disadvantage of washing the bins was the labour involved. It may be possible to reduce post-treatment holding time and increase codling moth mortality by holding the bins at a higher temperature post-treatment, however this parameter was not included in the study. The presence of the codling moth granulosis virus within the larvae did not influence the nematode-induced codling moth mortality over a range of concentrations and two temperatures in laboratory trials.     

Mortality of Immature Houseflies (Musca domestica L.) in Artificial Diet and Chicken Manure after Exposure to Encapsulated Entomopathogenic Nematodes (Rhabditida: Steinernematidae, Heterorhabditidae)

The entomopathogenic nematodes, Steinernema felitae (=bibionis) and Heterorhabditis megidis, were encapsulated in calcium alginate and their efficacy was tested against immature houseflies. Aliquots of capsules (15 ml) containing either 1 000 000, 500 000, 250 000 or 125 000 nematodes were added to 70-ml portions of grassmeal diet containing either eggs of first, second or third instar larvae. After 2 days, the treatment with 1 000 000 encapsulated S. feltiae (=bibionis) had killed 94% of housefly eggs and 90% of first instar larvae. By day 6, both of these mortalities had increased significantly (P 0.005) to 100%. By day 2, in the same medium, 1 000 000 encapsulated H. megidis had killed 71.4% of eggs and 90% of first instar larvae. This increased significantly (P 0.01) by day 6, to 99.2% and 100% respectively. Another experiment was carried out where immature houseflies were placed in chicken manure. The emergence of houseflies as adults was used to measure the effect of the encapsulated nematodes. Depending on the numbers of nematodes and the original stage of the housefly, the treatment with encapsulated S. feltiae resulted in 55-96% reduction in adult housefly emergence, whereas treatment with encapsulated H. megidis resulted in 35-98% reduction in emergence. Finally, when encapsulated nematodes were presented as a bait to adult houseflies, little infectivity was observed.     

Field Efficacy of Entomopathogenic Nematodes against the Sugar-beet Weevil Temnorhinus ( = Conorrhynchus ) mendicus Gyll. (Coleoptera: Curculionidae)

In 1992 and 1993, the field effectiveness of Heterorhabditis sp. (NL-HL81 strain), H. bacteriophora (HP 88 strain) and Steinernema carpocapsae ('All' strain) against the larvae of Temnorhinus mendicus Gyll. was assessed. The biological tests were compared with two chemical treatments (cypermethrin or deltamethrin) and one untreated control. In 1992, S. carpocapsae gave better results than Heterorhabditis sp. in reducing the percentage of infested roots, as compared with the untreated sample and the chemical one; similarly, the irrigated control gave the best results. In 1993, three concentrations of entomopathogenic nematodes (EPNs) were tested: 0.250 106 infective juveniles (IJs) m - 2, 0.125 106 IJs m - 2 and 0.075 106 IJs m - 2. The different numbers of EPNs did not give very different results from each other; however, H. bacteriophora at 0.075 106 IJs m - 2 was the least effective. In general, cypermethrin was more effective than deltamethrin, but one treatment with EPNs followed by irrigation was always more effective than two chemical applications.     

Influence of Salinity on Survival and Infectivity of Entomopathogenic: Nematodes

Exposure to NaC1, KCI, and CaCl₂ affected the entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema glaseri differently. Survival, virulence, and penetration efficiency of S. glaseri were not affected by these salts. At high concentrations, however, all three salts inhibited its ability to move through a soil column and locate and infect a susceptible host. Calcium chloride and KCl had no effect on H. bacteriophora survival, penetration efficiency, or movement through a soil column, but moderate concentrations of these salts enhanced H. bacteriophora virulence. NaCl, however, adversely affected each of these parameters at high salinities (>16 dS/m). Salt effects on S. glaseri are attributed solely to interference with nematode host-finding ability, whereas the NaCl effects on H. bacteriophora are attributed to its toxicity and possibly to interference with host-finding behavior.     

Comparison of the Efficacy of Entomopathogenic Nematodes for the Biological Control of the Mushroom Pests Lycoriella auripila (Sciaridae) and Megaselia halterata (Phoridae)

The efficacy of different species of entomopathogenic nematodes was tested against larvae of the mushroom phorid Megaselia halterata (Diptera: Phoridae) and the mushroom sciarid Lycoriella auripila (Diptera: Sciaridae). Sciarid larvae originating from infestations in casing soil during colonization by Agaricus bisporus were almost completely controlled by applications of Steinernema feltiae to the casing soil. When larvae originated from infestations in freshly spawned compost, they could be controlled by compost applications halfway through spawnrunning and by very early casing treatments. The control of phorids in compost was maximally 31% when nematodes were mixed within the infested compost at a concentration of 3 106 nematodes/m2. Only slightly higher reduction rates were obtained at higher concentrations. The control of phorids was more promising in the infested casing layer, in which S. carpocapsae was most successful. At concentrations of 6 and 15 106 nematodes/m2 this species obtained reduction rates of 65 and 73% respectively when it was applied 3 days after the end of the infestation period. These concentrations are, however, too high for practical application.     

Efficacy of the Entomopathogenic Nematodes Heterorhabditis megidis and Heterorhabditis bacteriophora for the control of grubs (Phyllopertha horticola and Aphodius contaminatus) in Golf Course Turf

Liquid culture-produced entomopathogenic nematodes, Heterorhabditis megidis and Heterorhabditis bacteriophora, were applied at 0.5 and 1.5 million dauer juveniles m-2 against Aphodius contaminatus and Phyllopertha horticola on a golf course. The reduction of A. contaminatus was found to be between 40 and 62%. P. horticola reduction reached 70% with H. megidis and 83% with H. bacteriophora. Turf damage caused by birds preying on the grubs was successfully prevented.     

Entomopathogenic Nematodes: Biodiversity, Geographical Distribution and the Convention on Biological Diversity

This paper addresses three major issues. Firstly, molecular taxonomy and its application to elucidate the biodiversity and biogeography of entomopathogenic nematodes is considered. Accurate identification is fundamental for understanding biodiversity, and because these nematodes are morphologically conservative, molecular techniques will provide the insights necessary to develop a robust, morphologically based taxonomy. Secondly, a review of the knowledge on their biogeography and habitat specificity, including a consideration of the limitations to the available data is given. Much of the information is presented in two tables which summarize the distributions of recognized species at continental and national levels. Thirdly, this paper provides a brief consideration of the Convention on Biological Diversity and its implications for future work with entomopathogenic nematodes and biological control.