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.     

Hybridisation and genetic selection for improving desiccation tolerance of the entomopathogenic nematode Steinernema feltiae

Shelf life of biological control products based on the entomopathogenic nematode Steinernema feltiae is rather limited. In order to prolong shelf life, the metabolism of nematodes during storage must be reduced, either by low temperature or by means of desiccation of the third stage dauer juveniles (DJs). Tolerance to desiccation is limited in S. feltiae. Their tolerance can be increased by an adaptation to moderate desiccation conditions. The objective of this study was to screen for tolerant strains among wild type populations of S. feltiae, hybridise most tolerant strains and further improve desiccation tolerance by subjection of the hybrid strain to genetic selection. Dehydrating conditions, measured as water activity (a _w-values), were produced by treating DJs with different concentrations of the polymer polyethylene glycol 600. Significant variation was recorded among 24 S. feltiae strains. The mean tolerated water activity survived by 50% of the population (WA₅₀) ranged from 0.78 to 0.93 when nematodes were not adapted to desiccation stress and from 0.66 to 0.88 when preadapted to desiccation stress. The six most desiccation tolerant strains of non-adapted and adapted nematode populations were crossed. Preadapted tolerance of hybrids was superior to the tolerance of parental strains, whereas non-adapted tolerance was not increased. The most tolerant hybrid had a WA₅₀ when preadapted of 0.67 and 0.86 when not adapted. The tolerance was lost after few reproductive cycles in the insect Galleria mellonella but was recovered again after six selection cycles with exposure to increasing stress conditions. Virulence and reproduction potential was not negatively affected by the selection. Stabilisation of the selection progress will be a major challenge to enable commercial exploitation of the genetic improvement.     

Ecological characterization of Steinernema scarabaei,a scarab-adapted entomopathogenic nematode from New Jersey

This study describes the basic ecological characteristics of the entomopathogenic nematode Steinernema scarabaei (Rhabditida: Steinernematidae) that was originally isolated from epizootics in scarab populations in New Jersey turfgrass areas. Under laboratory conditions, S. scarabaei infected a limited range of insect species and appeared best adapted to scarab larvae as hosts. It uses a widely ranging foraging strategy with a low attachment rate to mobile hosts on the soil surface but with excellent infection of sedentary host placed at >or=2 cm soil depth. It has a wide thermal activity range with optimum infectivity from 17.5 to 25 degrees C. Because of its foraging strategy and adaptation to scarab larvae as hosts, S. scarabaei has outstanding potential for the control of scarab pests.     

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.     

Present and future prospects for entomopathogenic nematode products

Entomopathogenic nematodes in the genera Steinernema and Heterorhabditis have emerged as excellent candidates for biological control of insect pests. Attributes making the nematodes ideal biological insecticides include their broad host range, high virulence, safety for nontarget organisms and high efficacy in favourable habitats. Progress achieved in liquid fermentation, formulation stability and application strategy has allowed nematode‐based products to become competitive with chemical insecticides in medium‐ and high‐value crops on the basis of cost/benefit ratio and ease of application. Further technological advancements are needed to expand and improve the market potential of the nematode‐based products.     

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.     

Field and tree distribution of Capnodis tenebrionis (Linnaeus, 1767) (Col., Buprestidae) adults in an apricot orchard in Italy

A study was conducted into the spatio–temporal distribution of adult Capnodis tenebrionis (Linnaeus, 1767) in an apricot orchard in Sicily (Italy) using inverse distance squared weighting (IDW), a spatial interpolation technique to create a distribution map of adults in the orchard. The study was conducted throughout the period of presence of the adult in the field and it investigated distribution on individual plants with regard to the observed height on the crown and exposure to the sun’s rays. The variation is gradual in the distribution of the buprestid in the field and the areas with the greatest exposition to the sun (south‐east) were marked by the highest densities. The presence of the pest was also found to be higher in those parts of the crown most exposed to the sun; moreover, the position in relation to the ground varied constantly and was increasingly linked to time and temperature. The data obtained confirmed the distinct thermal demands of the species and provided useful information on both sampling and control.     

Desiccation tolerance among different isolates of the entomopathogenic nematode Steinernema feltiae (Fillipjev)

Poor storage capacity is a major constraint limiting further expansion of the use of entomopathogenic nematodes. In order to prolong shelf life, a quiescent state of the dauer juveniles (DJs) should be induced. This can be attained by means of desiccation of DJs. In this study, 24 natural isolations of Steinernema feltiae were exposed to desiccation stress in non-ionic polyethylene glycol 600. The dehydrating conditions were measured as water activity, a(w)-value. Non-adapted and adapted DJs were tested separately under a series of dehydrating conditions. The mean tolerated a(w)-value (MW50) ranged from 0.85 for the isolate NEP1 to 0.95 for FIN1, ISR5 and ITA2 when not adapted to desiccation stress and from MW50 of 0.822 for CR1 to 0.98 for ISR6 when adapted to the stress conditions. CR1 tolerated the lowest desiccation stress at an a(w)-value for the most tolerant 10% of the population (MW10) at 0.65 when DJs had been adapted to stress. No significant differences were recorded between all isolates in non-adapted DJs populations MW10 was compared. No correlation between tolerance under non-adapted and adapted conditions were found. Most tolerant isolates will now be used for cross-breeding and subsequent genetic selection to enhance desiccation tolerance.     

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.     

Susceptibility of larvae of the mushroom fly Megaselia halterata to the entomopathogenic nematode Steinernema feltiae in bioassays

Bioassays were initially conducted in Petri-dishes to screen the efficacy of four Heterorhabiditis and Steinernema species against the mushroom phorid Megaselia halterata. Control rates of 61 to 70% control were obtained at a dosage of 1500 infective juveniles (IJs) per 30 larvae. In order to avoid stress-induced susceptibility, an improved bioassay system in micro-wells, filled with 0.6 ml of compost agar and 0.2 of compost colonized by Agaricus bisporus, was developed. In a screening of different species of Heterorhabiditis and Steinernema with applications of 30 IJs per phorid larva, a highest parasitization rate of 20% was obtained with S. feltiae. Bioassays were continued with S. feltiae in dosage-mortality assays in which larvae of the sciarid Lycoriella auripila and the phorid M. halterata were challenged. At the lowest dosage of 30 IJs per sciarid larva, 78% control was obtained. Increasing the dosage from 30 to 1000 led to only small increases of the phorid mortality. At 1000 IJs per larva a significant mortality of 18% was obtained. The nature of the substrate, compost or casing did not greatly influence the parasitization rates. The sex ratio of nematodes that were able to penetrate and establish in the phorid larvae appeared to be female-skewed. Males were only present at a mean of 19%. Low susceptibility of the phorid larva was ascribed to the inaccessibility of its small mouth opening.     

Developmental temperature effects on five geographic isolates of the entomopathogenic nematode Steinernema feltiae (Nematoda: Steinernematidae).

The development of five geographic isolates of Steinernema feltiae at 5, 8, 10, 15, 20, 25, and 28 degrees C in wax moth, Galleria mellonella, larvae was examined. The isolates were from Mediterranean (Sinop from Turkey, SN from France, and Monterey from California), subtropical (Rafaela from Argentina), and tropical (MG-14 from Hawaii) regions. All isolates caused 100% mortality of wax moth larvae and developed and produced progeny between 8 and 25 degrees C. At 28 degrees C, mortality was 100%, but no progeny was observed. The highest infective juvenile production was observed at 15 degrees C for all isolates. In general, the tropical isolate, MG-14, had the lowest production of infective juveniles. The time of emergence of the infective juveniles from the host cadaver showed some differences among isolates, with the Sinop isolate having the earliest emergence time from cadavers at 15 degrees C (10 days) and 20 degrees C (8 days). At 25 degrees C, the infective juveniles of the Sinop, SN, and Rafaela isolates emerged from the cadavers from 5 to 7 days. Time of host death by all isolates showed no differences at 8, 10, 15, 20, and 28 degrees C. At 25 degrees C for all isolates (except the MG-14), shorter times to host death were observed. Host death occurred at 12 days at 8 degrees C, 9 to 11 days at 10 degrees C, 4 to 5 days at 15 degrees C, 3 days at 20 degrees C, and 2 days at 25 and 28 degrees C. For penetration efficiency, the Sinop, SN, and Rafaela isolates penetrated their hosts at 5, 8, and 10 degrees C. Penetration of the infective juveniles was consistently high for all isolates at 15, 20, 25, and 28 degrees C, but it was significantly lower for the MG-14 isolate at 15, 25, and 28 degrees C. No progeny production occurred at 28 degrees C, but nematode penetration did occur with the MG-14 isolate having significantly lower penetration than the other isolates. When nematodes were produced at 8, 15, and 23 degrees C in wax moth larvae, all isolates had infective juveniles with longer body lengths at 8 degrees C followed by 15 and 23 degrees C. To further verify body length at the different temperatures, beet armyworm, Spodoptera exigua, larvae and dog-food agar medium were used, respectively, for in vivo and in vitro culture of the Sinop isolate. Infective juvenile body length showed the same trends, with the longest being at 8 degrees C and decreasing in length from 15 to 23 degrees C. The data suggest that quality of food for the nematode and temperature (that is, developmental time) influence the body length of the infective juvenile.     

Evaluation of entomopathogenic nematode, Steinernema feltiae against field population of mustard sawfly, Athalia lugens proxima (Klug) on radish

Mustard sawfly, A. lugens proxima, was found to be highly susceptible to entomopathogenic nematode, S. feltiae under laboratory condition. Application of three different doses of S. feltiae, viz. 1.1 x 10(3), 1.1 x 10(4) and 1.1 x 10(5) infective juveniles/ml, at weekly intervals, significantly reduced the field population of mustard sawfly on radish. The mean larval population of A. lugens proxima in all doses of nematode treated plots ranged from 0.42 to 0.48 larvae per plant as against 2.95 larvae / plant in untreated control plots. Similarly, the yield of radish in all the nematode treated plots was significantly higher by way of recording 2.80 to 2.87 tons/ha as compared to 1.63 tons/ha in the case of control.     

Field efficacy of entomopathogenic nematodes against the beetle Maladera matrida (Coleoptera: Scarabaeidae)

Single, double and triple releases of the entomopathogenic nematode Heterorhabditis bacteriophora Poinar, reduced the population of the beetle Maladera matrida Argaman, infesting peanuts (’Shulamit’ cv.) by 70, 75 and 93% respectively in microplot tests. Simultaneous and late (2 weeks after infestation) applications reduced beetle numbers by 63 and 79% respectively, in the microplots, while early application (2 weeks prior to infestation) did not reduce the beetle population. In a field trial, reductions in insect population and damage to the crop were achieved by early treatment with the nematode as well as by Heptachlor, leading to reductions in the insect population of 60 and 90% respectively, when recorded 4 weeks after nematode application. However, the nematode treatment did not maintain its effectiveness for a longer period and pest damage increased to the same level as the untreated control after 7 weeks. When the nematodes were applied at different concentrations (0.25–1.0 x 10⁶ infective juveniles (IJs) m^‐2) their effectiveness was not related to the concentration level. The only significant (P < 0.05) reduction in insect levels was recorded in the treatment with 0.5 X 10⁶ IJs m^‐2. In a second field trial, both H. bacteriophora and Steinernema glaseri reduced insect populations significantly (P < 0.05) by approximately 50% in comparison to the control. In the third trial, treatment with H. bacteriophora resulted in a decrease in insect population of 90% while treatment with S. carpocapsae reduced the grub numbers by 40% in comparison to the control. A differential susceptibility of various grub developmental stages was recorded in the field. The small grubs (I‐4 mm long, lst‐2nd larval stage) were not affected by the nematode treatments while the numbers of medium and large size grubs were reduced by 2‐ and 3‐fold respectively in the various tests. Nematodes were recovered by ‘nematode traps’ containing Galleria mellonella larvae from treated field plots 78 days after application. The implications of the results from the present studies on the use of entomopathogenic nematodes are discussed in relation to the development of an integrated pest management programme.     

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 sweetpotato whitefly Bemisia tabaci (Homoptera: Aleyrodidae) under laboratory and glasshouse conditions

The potential of using the entomopathogenic nematode Steinernema feltiae to control the sweetpotato whitefly Bemisia tabaci (Gennadius) has been established in previous laboratory studies. However, laboratory studies can overestimate the level of control achieved by biocontrol agents in the glasshouse. Glasshouse trials are therefore required to confirm laboratory results before full-scale commercial development is considered. Under both controlled laboratory and glasshouse conditions high mortality of second instar B. tabaci (>90% and >80%, respectively) was recorded after application of S. feltiae. The efficacy of the biocontrol agent at various application rates was also investigated, where halving the rate of S. feltiae application caused no significant reduction in B. tabaci mortality on tomato foliage. Steinernema feltiae has shown much potential for incorporation into integrated pest management strategies for the control of B. tabaci.     

A new bioassay method reveals pathogenicity of Metarhizium anisopliae and Beauveria bassiana against early stages of Capnodis tenebrionis (Coleoptera; Buprestidae)

We used a newly developed bioassay method to demonstrate for the first time the potential of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae to be used for the control of neonate larvae of Capnodis tenebrionis, a major threat to stone-fruit orchards in several countries. Four B. bassiana and four M. anisopliae isolates were all pathogenic for neonate larvae of C. tenebrionis; mortality rates 10 days after inoculation by dipping in a suspension with 10(8)conidia/ml varied from 23.5% to 100%. Three of the four M. anisopliae isolates caused 100% mortality. In most cases, postmortem hyphal growth and sporulation of M. anisopliae or B. bassiana was observed covering the larvae in their galleries. The eight isolates were also evaluated for pathogenicity to C. tenebrionis eggs at the same dosage. Only two B. bassiana isolates caused significant egg hatching reduction of 84.5% and 94.5%. Our results indicate that M. anisopliae and B. bassiana may be considered as promising for a new approach to prevent larval infestations by C. tenebrionis.     

Control of the larvae ofConorhynchus mendicus (Col.: curculionidae) bySteinernema carpocapsae andSteinernema feltiae (nematoda,steinernematidae) in the gharb area (Morocco)

Two entomopathogenic nematodes,Steirnernema carpocapsae andS. feltiae were evaluated in the laboratory and under field conditions against larvae ofConorhynchus mendicus, a pest of sugar beet in the Gharb area of Morocco. Nematodes were applied in an artificially infested field using a knapsack sprayer either during the day or in the evening after sunset. The number of insect cadavers recovered from both soil and sugar beet plants was greater in the treated plots compared with the untreated ones. The percent reduction in live larvae was significantly greater in theS. carpocapsae treated plots (89.5% and 59.5% for night an day application respectively) compared with those treated withS. feltiae (68.5% and 28% respectively). The efficacy of entomopathogenic nematodes was therefore increased by application at night particularly in the case ofS. carpocapsae where control was maintained for 45 days.     

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.