Enhanced cold tolerance of the entomopathogenic nematode Steinernema feltiae through genetic selection

Cold sensitivity of entomopathogenic nematodes severely restricts their biological control potential in some environments. We selected the SN strain of Steinernema feltiae together with its bacterial symbiont, Xenorhabdus bovenii, for improved cold tolerance by repeated passage through the wax moth Galleria mellonella larvae at 15°C. Nematode virulence (total insect mortality and speed of kill) and establishment (initiation of nematode development following penetration) were evaluated after six (= 12–24 generations) and 12 passages (= 24–36 generations). Cold selection enhanced nematode virulence at the cooler temperatures. Virulence measured as total insect-mortality at 8°C improved by 5.3- and 6.6-fold after six and 12 passages, respectively. Only small improvements (1.2–1.5-fold) were observed in speed of kill. Nematode establishment improved at all temperatures after 12 passages; the highest increase of 9-fold was observed at 8°C. Our results lend support to the hypotheses that functional traits along a continuously distributed environmental variable are genetically correlated and that the area under the fitness function is not always constant. Trade-offs in percentage mortality and speed of kill by the selected nematodes were observed at the warmer extreme after six passages of selection only. The implications of rapid changes in thermal sensitivity for economic mass-production of nematodes are discussed.     

Critical factors required by the nematode Steinernema feltiae for the control of the leafminers Liriomyza huidobrensis, Liriomyza bryoniae and Chromatomyia syngenesiae

The critical factors required by entomopathogenic nematodes for the control of dipteran leafminers were investigated and compared. The foliar application of Steinernema feltiae and Heterorhabditis sp. (strain UK 211) caused a significant reduction in the numbers of dipteran leafminer larvae. There was no significant difference in efficacy between these nematode species against Liriomyza huidobrensis at 20°C and 85–90% r.h. Application of S. feltiae to all three larval instars of L. huidobrensis significantly reduced larval survival, with application to the second instar being the most effective at 20 ± 2°C and 80 ± 10% r.h. Humidities of >90% significantly enhanced the efficacy of S. feltiae. When S. feltiae was applied to second instar larvae, it was equally effective throughout the temperature range 10–30°C. At 20°C and >90% r.h., sufficient nematodes were able to enter the leaf tissues within the first 12 h after application to reduce larval survival to c. 15%. A comparison between L. huidobrensis, L. bryoniue and Chromatomyia syngenesiue indicated that the efficacy of S. feltiae was affected by the same critical factors for all three species.     

Efficacy of the entomopathogenic nematode, Steinernema feltiae, against Bemisia tabaci in relation to plant species

Abstract:  The use of infective juveniles (IJs) of the entomopathogenic nematode, Steinernema feltiae , to control the immature stages of the tobacco whitefly, Bemisia tabaci (Gennadius) (Hom., Aleyrodidae), on a range of host plants was investigated. Foliar applications of S. feltiae (10 000 IJs/ml) were made to tomato ( Lycopersicon esculentum ), cucumber ( Cucumis sativus ), poinsettia ( Euphorbia pulcherrima ), chrysanthemum ( Dendranthema spp.) and verbena ( Verbena hybrida ) infested with second instar B. tabaci , to determine whether efficacy was influenced by plant species. The effect of the adjuvants Agral, Triton X-100, methylcellulose, glycerol and spraying oil on the level of pest mortality was also assessed using two selected host plants, tomato and verbena. Following nematode application B. tabaci mortalities of 32, 28, 22 and 22% were recorded on tomato, cucumber, verbena and chrysanthemum, respectively, but a lower mean mortality was noted for whitefly feeding on poinsettia (10%). Mortality of B. tabaci on tomato and verbena was significantly increased by the addition of either Triton X-100 or Agral to the spray suspension. The use of Triton X-100 raised the mortality level to 63 and 37% on tomato and verbena, respectively, while 50 and 27% mortality followed the use of Agral on the two hosts. With the exception of glycerol no phytotoxic effects were observed by the adjuvants when applied to all five host plant species used in this study. The potential for use of the entomopathogenic nematode, S. feltiae , as a non-chemical alternative control measure for B. tabaci immatures is discussed.     

芜菁夜蛾线虫和嗜菌异小杆线虫寄生特性的比较

对2种昆虫病原线虫(芫菁夜蛾线虫Steinernema feltiae Filipjev和嗜菌异小杆线虫Heterorhaditis bacteriphora Poinar)的生物学特性进行研究。结果表明:在25℃下,在大蜡螟Galleria mellonella L.幼虫体内完成1个侵染周期芫菁夜蛾线虫Otio品系需220～230h,嗜菌异小杆线虫15-2品系需200～210h。15-2品系比Otio品系具有较强的繁殖力。2种线虫侵染大蜡螟幼虫的温度范围均为20～30℃,在此温度范围内,Otio品系的侵染力和运动能力均强于15-2品系。当土壤含水量(w/w)为5%～15%,线虫的侵染活性最高,随湿度降低线虫的侵染活性明显降低。     

Comparative study of the effect of selected agrochemical products on Steinernema feltiae (Rhabditida: Steinernematidae)

The effect of three neurotoxic insecticides, three photosynthetic inhibitor herbicides and three enzymatic inhibitor herbicides on infective juveniles (IJs) of Steinernema feltiae Rioja (native) and ENTONEM® (commercial) strains were evaluated after a 48-h exposure at field tank concentrations and overnight treatment in mQ-water, using Spodoptera littoralis as target. Nematode survival was not affected by acetyl-cholinesterase inhibitors chlorpyrifos and pirimicarb, although chlorpyrifos seriously reduced their virulence. Both nematode strains showed differential sensitivity to cypermethrin, which affects the sodium channels of the nerve membrane, with the ENTONEM® strain being more tolerant than Rioja strain. However, these chemicals showed a strong sublethal effect on the nematode reproductive potential, limiting seriously their possible recycling in the field. Herbicides showed differential toxic effects on nematode survival. The commercial strain was tolerant to enzymatic inhibitor herbicides, whereas tribenuron and chlorsulfuron reduced Rioja strain survival. However, photosynthetic inhibitor herbicides severely affected survival of both nematode strains, with the Rioja strain being more sensitive. Sublethal effects on both nematode strains were observed only after exposition to terbutryn+chlortoluron+triasulfuron, increasing the time to kill insect larvae. These results are useful to optimize EPN dosages and to estimate their field recycling.     

Phoresy of the entomopathogenic nematode Steinernema feltiae by the earthworm Eisenia fetida

The free-living stage of entomopathogenic nematodes occurs in soil, and is an environmental-friendly alternative for biological control. However, their dispersal capability is limited. Earthworms improve soil characteristics, changing soil structure and influencing many edaphic organisms. Thus, earthworms could be used as vectors to introduce/disperse beneficial organisms. Nevertheless this interaction has not been studied in detail. This study presents the infectivity results of Steinernema feltiae after passing through the Eisenia fetida gut. Although entomopathogenic nematodes have no deleterious effects on earthworms, their passage through E. fetida gut seriously affected their mobility and virulence.     

Field efficacy of the entomopathogenic nematode Steinernema feltiae against the Mediterranean flat-headed rootborer Capnodis tenebrionis

The Mediterranean flat-headed rootborer, Capnodis tenebrionis (L.) (Coleoptera: Buprestidae), is an economically important pest of stone fruit and seed fruit in Mediterranean areas. The potential control of the entomopathogenic nematode Steinernema feltiae (Filipjev) (strain Bpa), isolated from a dead C. tenebrionis larva, was tested in a cherry tree orchard in Ullastrell, Barcelona (Spain). Nematode infective juveniles (IJs) were applied by drench and injection. In both the treatments, a rate of 1 million IJs was applied per tree every week during 4 or 8 weeks, with a total dose of 4 × 10⁶ IJs/tree and 8 × 10⁶ IJs/tree. Number, stage and localization of insects in each tree trunk were recorded. In both the experiments, S. feltiae significantly reduced the population of C. tenebrionis providing control ranging from 88.3% to 97%. No significant differences were recorded between the different treatments. Persistence of nematodes was recorded until 6 weeks after application. Results indicate that the application of S. feltiae (Bpa) provides adequate control of C. tenebrionis in cherry trees.     

Population growth kinetics of the nematode, Steinernema feltiae, in submerged monoxenic culture

Monoxenic cultures of the nematode, Steinernema feltiae, were carried out on two complex liquid media: P1, mainly soybean flour/egg yolk/yeast extract, and P2, mainly egg yolk/yeast extract. Up to 140 000–200 000 nematodes ml^–1 were produced within 7 days, and more than 95% of the final population was in the infective juvenile stage. The total nematode concentration growth curve had a sigmoidal shape. Nematode population growth kinetics were modelled using a re-parameterised Gompertz model. Yeast extract concentration appeared to be a key factor for obtaining high nematode concentrations.     

Storage temperature influences desiccation and ultra violet radiation tolerance of entomopathogenic nematodes

1. The effect of cold (5 °C) and warm (35 °C) storage on desiccation tolerance of cold-adapted Steinernema feltiae, intermediate S. carpocapsae and warm-adapted S. riobrave was evaluated at 5 and 35 °C.     

Enhanced biological control potential of the entomopathogenic nematode,Steinernema carpocapsae,applied with a protective gel formulation

Entomopathogenic nematodes (EPNs) are adapted to subterranean environments, and are prone to damage by UV light and desiccation. EPNs, Steinernema carpocapsae, combined with a protective gel, and anti-UV ingredients, have potential for above-ground pest management. We (1) ascertained whether the gel could provide protection to EPNs at low concentrations when applied in direct sun, (2) determined if other ingredients added to the gel increased efficacy, and (3) quantified retention and survival of EPNs applied to foliage with the gel. EPNs in 1% protective gel caused higher host mortality (60%) than other treatments (2–37%). UV protection provided by titanium dioxide (TD) and octyl methoxycinnamate with 1% protective gel solution was tested outdoors; these formulations resulted in higher host mortality (43% and 25%) than other treatments (2–7%). After 8?h in the greenhouse, 0.25% protective gel solution had the highest percentage of live EPNs on leaves. The gel at low concentration protects EPNs, and addition of TD enhanced the protective properties of the formulation. It is important to continually improve options for different growing systems and insect pest behaviour. TD added to a low-concentration formulation of the protective gel makes this application technique more viable for growers to use.     

Selective breeding of Steinernema feltiae (Filipjev) (Nematoda: Steinernematidae) for improved efficacy in control of a mushroom fly,Lycoriella solani Winnertz (Diptera: Sciaridae)

A method of selecting a Steinernema feltiae strain that is effective against a mushroom fly, Lycoriella solani, is described in detail. The pest control efficacy of the selected nematode strain was evaluated and compared with the efficacy of two unselected strains. The selection procedure was designed to give preference to nematode individuals with the greatest ability (1) to search effectively for the target insect larvae in their natural habitat, (2) to infect them shortly after application and (3) to reproduce in their haemocoel. Thirty‐four rounds of selection achieved a 4‐fold improvement in nematode ability to find and parasitize third‐ and fourth‐instar larvae of the pest in the mushroom substrate. In 24‐h laboratory experiments, mortality of the insect caused by nematode juveniles rose from 22.5%, recorded for the original unselected isolate, to 92.5% for the selected strain. In a 51‐day experiment conducted on a mixed age mushroom house population of L. solani, the enhanced pest control ability of the selected strain was detected shortly after nematode application and remained high throughout the experimental period. During the first 4 weeks of the trial the selected nematode strain was significantly better than both unselected strains and caused 91.1–92.7% reduction of the fly emergence from the mushroom substrate. No difference was observed between the efficacy of the selected nematodes applied at 1 × 10⁶ and 3 ×10⁶ infective juveniles per m², while the unselected strains performed significantly better at the higher concentration. All the nematodes examined showed good persistence in the mushroom casing apparently due to recycling in the insect host.     

Comparative effects of Steinernema feltiae (Nematoda: Steinernematidae) and insecticides on yield and cropping of the mushroom Agaricus bisporus

In a mushroom crop (Agaricus bisporus) affected by a very low level of sciarid fly (Lycoriella auripila) infestation, the effects of an indigenous isolate of insect-parasitic nematode (Steinernema feltiae) and of two commonly used insecticides (diazinon and diflubenzuron) were studied. When compared with untreated plots, nematodes applied to the casing had no adverse effects on mushroom yields whereas insecticides decreased yields. At a rate of 3 × 10⁶ infective juveniles per tray (surface area = 0.56 m²), S. feltiae elicited increases of 28.5% and 19% in the mean total numbers and weights of mushrooms respectively. Treatment only with diflubenzuron resulted in 14.6% and 6% reductions in mean total numbers and weights of mushrooms, respectively; treatment with both diazinon and diflubenzuron caused 18.5% and 9.4% losses. Application of nematodes generally reduced the mean weight per mushroom whereas insecticides increased it; nematodes delayed the onset of mushroom production (first flush) whereas diflubenzuron delayed the third and fourth flushes. Nematode contamination of sporophores was minimal when S. feltiae was applied at casing. Although their numbers declined with time, the nematodes persisted, in the casing layer, throughout the cropping period of seven weeks. It is concluded that yield benefits associated with nematode application can result mainly from nematode effects on A. bisporus and not solely from suppression of a damaging pest population.     

Migratory behaviour and desiccation tolerance of protostrongylid nematode first-stage larvae

Migration of first-stage larvae (L1) from faeces to soil is a crucial stage in the life-history of protostrongylids transmitted via land snails. Migration of Muellerius cf. capillaris and a Cystocaulus sp. L1 from fresh Nubian ibex (Capra ibex nubiana) faeces (48–50% water content, W.C.) to substrate soils (at 100% r.h., 26°C) was measured experimentally using dry (3 ± 1% W.C.), wet (31 ± 0.43% W.C.) and flooded (48.4 ± 2.45% W.C.) soils. The highest migration rates (90.4 ± 1.6% migration) in both species occurred on flooded soils when the faecal pellet W.C. reached 90%. The next highest migration rates (43.2 ± 3.6% migration, at 60% faecal W.C.) were on the wet soils and no migration occurred on dry soil or dry-substrate papers. Migration rates did not differ significantly (P > 0.05) between species. Active Theba pisana were not infected by M. cf. capillaris L1 on dry infested soils, but were infected following rehydration of the same soils. By day 10, L1 of M. cf. capillaris demonstrated lower survival rates in water and in 97% and 76% r.h. (74.5%, 15.2% and 1.9%, respectively) than the Cystocaulus sp. (97.5%, 43.8%, 43.3%) and Protostrongylus sp. (97.9%, 43.2%, 23.8%, P < 0.05). All three nematodes had a remarkably high survival rate (> 99% overall survival, by day 10) when exposed directly to 0% r.h. at 23°C, Results demonstrate the ability of L1 to survive extreme desiccation through anhydrobiosis. Migration of L1 from facces to soil can take place only during rains which coincide, with peak activity of land snails in desert habitat.     

Effects of Application Rates of Steinernema feltiae (Nematoda: Steinernematidae) on Biological Control of the Mushroom Fly Lycoriella auripila (Diptera: Sciaridae)

The potential of Steinernema feltiae for the biological control of Lycoriella auripila was tested in commercial mushroom‐growing conditions. The nematodes, applied at rates of 1.5, 3, 6 or 12 x 10 ⁶ infective juveniles per 34 kg tray of spawn‐run compost, were mixed into the casing material before it was spread over the compost surface. When compared with untreated control trays, any rate of nematode application significantly reduced fly emergence. Insecticides significantly reduced mushroom yields; nematodes significantly increased them. At a rate of 3 x 10 ⁶ infectives/tray S. feltiae elicited mean total increases in the weight and numbers of mushrooms produced of 8% and 11% respectively. The nematodes also reduced the incidence of mushrooms spoiled by tunnelling sciarid larvae. The early decline in the numbers of nematodes persisting in casing was a trend that was reversed later, when evidence was obtained that S. feltiae was recycling in insects that had been killed. When applied at a rate of 3 ‐10⁶infectives/tray of compost S. feltiae should provide reliable and cost‐effective biological control of L. auripila.     

Effects of host desiccation on development, survival, and infectivity of entomopathogenic nematode Steinernema carpocapsae

This study investigates the effect of host desiccation on entomopathogenic nematode (EPN) development, emergence, infectivity, and cross-protection against secondary environmental stress. Galleria mellonella hosts infected with the EPN Steinernema carpocapsae A10 were allowed to dehydrate in an environmental chamber for up to 56 days at 23 degrees C achieving a weight loss of approximately 86% by day 44 post-infection. Host carcasses were rehydrated on water-saturated filter paper in White traps to collect emergent infective juveniles (IJ) at specific time intervals. Populations were counted with an apparent peak coinciding with desiccated hosts rehydrated at 24-day post-infection. Desiccation-stressed IJ populations from each time interval were tested for infectivity, and cross-resistance to secondary temperature and pH stresses and were found to have significant increases in both infectivity and protection from extremes of temperature and pH compared with controls. Total aqueous soluble protein profiles from control and desiccation-stressed IJs were analyzed using 10% SDS Laemmli gels. Several novel proteins were over-expressed in EPN from hosts subjected to desiccation suggesting the induction and expression of stress response genes.     

The compatibility of the entomopathogenic nematode,Steinernema feltiae, and chemical insecticidesfor the control of the South American leafminer, Liriomyza huidobrensis

The compatibility of infectivejuveniles (IJs) of entomopathogenic nematodes, Steinernema feltiae, and chemical insecticides tocontrol larval stages of the South American leafminer,Liriomyza huidobrensis, was investigated.Initially the effect of direct IJ exposure to 5insecticides (abamectin, deltamethrin, dimethoate,heptenophos and trichlorfon) for 24 hours was testedagainst Galleria mellonella in a standard sandtube bioassay. Trichlorfon and dimethoate did notreduce the nematodes ability to locate and infect G. mellonella larvae to an unacceptable level. However,nematode infectivity was significantly reducedfollowing exposure to abamectin, deltamethrin andheptenophos. Secondly, IJ infectivity for L.huidobrensis in the presence of dry pesticideresidues on foliage was tested. No significantdetrimental effects on the level of control of L. huidobrensis was recorded when compared with theeffect of nematodes applied to residue free foliage.The integration of these agents into a pest managementprogramme is discussed.     

Genetic Variability among Strains of the Entomopathogenic Nematode Steinernema feltiae

A systematic program of genetic improvement was initiated by assessing the phenotypic variation of Steinernema feltiae strains for two traits assumed to limit efficacy: ultraviolet tolerance and host-finding ability. All of the strains assayed showed both low ultraviolet tolerance and poor host-finding ability, indicating that the likelihood of improving these traits through more extensive population sampling is remote. Limited genetic variation was detected among the strains for tolerance to ultraviolet, suggesting that selective breeding for increased tolerance would be inefficient. By contrast, highly significant phenotypic differences were found with regard to host-finding ability, suggesting that this trait would be responsive to selection. A genetically heterogeneous population was constructed by round-robin mating of 10 strains; it will serve as the foundation population for selective breeding.     

Comparative assessment of the efficacy of entomopathogenic nematode species at reducing western corn rootworm larvae and root damage in maize

The western corn rootworm (Diabrotica virgifera virgifera LeConte, Coleoptera: Chrysomelidae) is an invasive maize (Zea mays L.) pest in Europe. Crop yield is significantly impacted by the feeding of all three larval instars on maize roots, making them prime targets for control measures. Therefore, the control efficacy of three entomopathogenic nematodes (EPNs), Steinernema feltiae (Filipjev), Heterorhabditis bacteriophora Poinar, and H. megidis Poinar, Jackson and Klein (Nematoda: Rhabditida), was studied in four field plot experiments in southern Hungary in 2005 and 2006. All EPN species significantly reduced D. v. virgifera independently, whether applied as a row spray with a solid stream into the soil at sowing or onto the soil along maize rows in June. When applied at maize sowing, H. bacteriophora was more effective at reducing D. v. virgifera (81%) than H. megidis (49%) and S. feltiae (36%). When applied in June, H. bacteriophora and H. megidis were more effective at reducing D. v. virgifera (around 70%) than S. feltiae (32%). All tested EPN species significantly reduced damage on maize roots independently, whether they were applied at sowing or in June. Damage, however, was not totally prevented. The use of H. bacteriophora for the development of a biological control product for inundative releases against D. v. virgifera larvae is suggested.