In-vitro liquid culture of the entomopathogenic nematode,Steinernema colombiense,in orbitally shaken flasks

Steinernema colombiense, an entomopathogenic nematode species (EPN) was grown in two types of orbitally shaken flasks at 130?rpm and 28°C, containing 10 or 20?mL, respectively of a complex culture medium with an initial EPN-concentration of 1,000 Infective Juveniles (IJ)/mL. At the 10th day, the EPN-concentration was 58,771 individuals/mL with 87% of them in the IJ stage. No significant differences were found between the EPN growth kinetics in both types of flasks. The nematode-population growth was modelled by a re-parameterized Gompertz equation of three-parameters with best-fit values of 3.8 days for the lag time, 33.8 day^-1 for the maximum growth rate, and 57.3 (dimensionless) for the maximum asymptotic growth.     

Storage of osmotically treated entomopathogenic nematode Steinernema carpocapsae

The infective juveniles (IJs) of Steinernema carpocapsae‘All’ were osmotically stressed by a mixture of ionic (fortified artificial seawater) and non‐ionic (3.2 mol/L glycerol) solutions to establish a method for osmotic storage of entomopathogenic nematodes. Seven combinations (termed solution A to G) with different proportions of these two solutions were tested, with sterile extra pure water (sepH₂O, termed solution H) as a control. The mortality of the IJs at a concentration of 5 × 10⁵ IJ/mL in the solutions A to G, and H were 13.2%, 16.2%, 16.7%, 13.5%, 25.2%, 31.6%, 44.6%, and 1.0%, respectively, after 21 days storage at 25°C. Most of the IJs shrunk and stopped motility after 6–9 hours incubation at 25°C in solutions A to D. Based on the results, solutions A to D and H were chosen to further test the osmotic survival of the IJs at different IJ concentrations (5 × 10⁵, 2.5 × 10⁵, 2 000 IJ/mL) and incubation temperature (30°C, 25°C, 10°C). The resulting IJs were exposed to a high temperature assay (45°C for 4 h, HTA). Osmotically stressed IJs showed improved heat tolerance. The mortality of the IJs increased with the increasing concentrations of the test IJs and the storage temperatures after exposing to the HTA. More than 88.4%, 62.3% or 2.4% of the treated IJs died at the above three IJ concentrations, respectively. At the three IJ concentrations (2 000 IJs/mL, 2.5 × 10⁵ IJs/mL or 5 × 10⁵ IJs/mL), the highest mortality was recorded in solution D (11.6%, 85.9% or 98.0%, respectively), and the lowest mortality in solution B (2. 4%, 62.3% or 86.6%, respectively). No untreated IJs survived after the heat treatment. During 42 days storage at 10°C, the IJs mortality in the solutions A to D and H were 7.19%, 5.97%, 4.41%, 4.34%, and 4.34% respectively, and showed no significant differences. In conclusion, osmotic treatment of the IJs of S. carpocapsae‘All’ in a mixture of ionic and non‐ionic solutions enhances the heat tolerance. The mortality of the IJs after HTA increased with the increasing concentrations of the test IJs and the storage temperatures after exposure to the HTA. The result is promising for the osmotic storage of the entomopathogenic nematodes.     

Evaluation of hydraulic,pneumatic and mechanical agitation for the spray application of Steinernema carpocapsae (Rhabditida: Steinernematidae)

The application of entomopathogenic nematodes (EPN) is generally done using standard spray application techniques. However, in contrast to chemical pesticides, these biological antagonists must remain viable during and after the application process. For the application of EPN, a good agitation system is indispensable as the nematodes tend to sediment fast in a spray tank without agitation. Three agitation systems, viz. mechanical, pneumatic and hydraulic agitation were tested for their ability to keep Steinernema carpocapsae (Rhabditida: Steinernematidae) suspended in an undamaged way. Hydraulic agitation was tested using a centrifugal and a diaphragm pump. Nematode damage was quantified based on viability and infectivity of the EPN. The ability of the agitation system to keep the nematodes in suspension was examined by comparing the nematode concentration observed in the samples taken at different agitation times. Only the hydraulic agitation using the centrifugal pump damaged the nematodes. After 120 min of recirculation, only 19.3% of the nematodes survived. Infectivity was even reduced to 0%. An additional experiment revealed that the temperature rise, from 21.7 to 45.4°C, was responsible for the observed nematode damage. The concentration measurements showed that the pneumatic agitation was unstable. Agitation during 120 min using the other agitation systems resulted in a significant loss of nematodes at 15 cm above the spray tank bottom. In conclusion, mechanical and hydraulic agitation using a diaphragm pump can be recommended when S. carpocapsae is applied, although attention should be paid to possible nematode loss during application.     

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.     

小卷蛾线虫脱水休眠的形态与耗氧量

报道了小卷蛾线虫 Steinernema carpocapsae (BJ品系)在高渗液中脱水进入休眠的形态变化和耗氧量。结果表明：脱水线虫的形态变化与耗氧量相关,线虫在高渗液第30 h内,脱水程度越强,耗氧量越低。线虫脱水进入休眠从形态上分为螺旋、鞘壁分离和侧线弯曲三个阶段。在螺旋阶段脱水线虫代谢开始减慢,耗氧量较对照减少了14%。在鞘壁分离阶段脱水线虫开始进入休眠,耗氧量较对照减少了65%,加水后线虫在10 min内100%复苏。在侧线弯曲阶段Ⅰ脱水线虫进入了深度休眠,耗氧量较对照减少了79%,加水后在30 min内100%复苏。     

Survival of Steinernema masoodi and S. carpocapsae (Rhabditida: Steinernematidae) on pigeonpea and chickpea after foliar application

Abstract

The survival of Steinernema masoodi and S. carpocapsae (Rhabditida: Steinernematidae) was investigated after foliar application on pigeonpea and chickpea twigs, respectively, at flowering and fruiting stage. The concentration used was 2500 infective juveniles (IJs)/ml water for both the species. On pigeonpea, the mean number of IJs of S. masoodi found alive were 303.4, 158.4 and 51.6 after 0, 30 and 60 minutes of spray in evening hours whereas 236.1, 44.4 and 6.8 IJs were found alive when sprayed in morning hours, respectively. S. masoodi survival at 30 minutes post-spray in the morning was on par with 60 minutes post-spray in the evening hours. On chickpea, the mean numbers of IJs of S. carpocapsae were 165.4, 65.8, 4 and 0 at 0, 1, 2 and 3 h post-spray in the morning hours whereas in the evening spray, 159.4, 111.8, 83.8 and 11.4 IJs found alive at 0, 1, 2 and 3 h post-spray, respectively. Overall, nematode survival in the evening hours was higher compared to morning spray at a given time. Addition of glycerine and UV retardant improved the survival of nematode. Results indicated that survival rate of IJs decreased fast and viability remained up to 3 h and in evening hours very few nematodes remained alive. Serious attempts are needed to improve the survival of nematodes after foliar spray by adding efficient adjuvant, humectant, antidesiccant and/or UV retardant for the management of aerial insect pests.     

Effect of an entomopathogenic nematode,Steinernema carpocapsae on haemocyte profile and phenoloxidase activity of the Colorado potato beetle,Leptinotarsa decemlineata

The Colorado potato beetle (CPB), Leptinotarsa decemlineata Say is the most destructive insect pest of potato in many areas of the world. Little is known about the haemocyte types of the CPB and its plasma phenoloxidase (PO). In this regard, we investigated the haemocyte profile and PO of CPB and its immune response to the entomopathogenic nematode, Steinernema carpocapsae. Five types of haemocytes, the plasmatocytes (~67.4%), granulocytes (~23.5%), oenocytoids (~2.4%), spherulocytes (~0.25%) and prohaemocytes (~6.5%) were identified in fourth instar CPB larvae. Total haemocyte counts (THCs) were significantly reduced in nematode-injected insects compared with control groups (P < 0.05). Nematode cellular encapsulation observed in haemolymph of nematode-injected insects may partially explain decreased THCs. Plasma PO assay showed increased PO activity in nematode-injected insects compared with control groups (P < 0.05). Plasma PO assay on native polyacrylamide gel electrophoresis (PAGE) assay with L-3, 4-dihydroxyphenylalanine as substrate showed five bands (with molecular weights of approximately 200, 118, 68.5, 62.5 and 58.75 kDa).     

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.     

Efficacy of entomopathogenic nematode,Steinernema carpocapsae against the diamondback moth,Plutella xylostella (L.) in laboratory condition

In vitro studies were carried out on the diamondback moth, Plutella xylostella larvae using an insect entomopathogenic nematode isolate, Steinernema carpocapsae obtained from the Koppert company, the Netherlands. Larvae of P. xylostella were collected from cabbage farms around Mashhad city of Iran. During the study, the responses of larvae at 25?°C for three periods of 24, 48 and 72?h with different concentrations of 0, 5, 10, 20, 40, 80, 160 and 320 third instar larvae of nematode (infective stage?=?IJs) per insect into 10?cm Petri dishes containing filter paper soaked with 1?ml of nematodes suspension were compared. Maximum mortality caused by S. carpocapsae nematode was 88% at 24?h, and it was 100% at 48 and 72 h. With increasing nematode population level and exposure time (ET in hour), mortality of P. xylostella larvae was increased. Based on probit analysis, LC₅₀ values of S. carpocapsae nematode in three test periods were 45.61, 12.02 and 40.80 IJs per insect, respectively. Initial ANOVA was performed for S. carpocapsae nematode. The effect of both nematode population levels (IJ) and ET on third instar larvae of the diamondback moth, P. xylostella and interaction between IJ and ET were significant. In general, it is recommended to apply this nematode in suitable condition for controlling diamondback moth.     

Behavioural response of the entomopathogenic nematodes Steinernema carpocapsae and Heterorhabditis bacteriophora to oxamyl

Infective juveniles of the entomopathogenic nematode Steinernema carpocapsae show a low level of locomotory activity that is presumed to limit their usefulness as biological insecticides. A 30 μg ml^-1 solution of the carbamate pesticide oxamyl reduced the proportion of nonmobile nematodes by nearly two thirds to 35%, while stimulating a 7.5-fold increase in sinusoidal movement. This increase in activity did not result in a corresponding increase in host-finding. Oxamyl treatment did not enhance infective juvenile pathogenicity to Galleria mellonella larvae. At higher concentrations, oxamyl caused aberrant nematode movement and partial paralysis. Heterorhabditis bacteriophora infective juveniles maintain a high level of locomotory activity. Treatment with 30 μg ml^-1 oxamyl increased the proportion of sinusoidal over nonsinusoidal movements, but infective juvenile host-finding and pathogenicity were significantly reduced. Higher rates impaired movement and induced complete paralysis. We conclude that oxamyl is incompatible with S. carpocapsae and H. bacteriophora. The concept of chemically activating infective juveniles to increased locomotory activity and thereby achieving enhanced efficacy is inconsistent with our results.     

Kinetics of infective juvenile production of the entomopathogenic nematode Steinernema carpocapsae in submerged monoxenic culture

The effects of culture medium formulations on the kinetics of infective juvenile (IJ) production of the entomopathogenic nematode Steinernema carpocapsae in submerged monoxenic culture, were studied at the cylindrical-bottle scale using six culture media containing agave juice from Agave spp. among other ingredients. The IJ production kinetics was well modelled through a re-parameterised 3-parameter Gompertz model with kinetic parameters: IJ-lag phase lambda (IJ) (day), maximum IJ-stage production rate m (max )(day(-1)), and IJ-multiplication factor (C (IJ)/C (IJ,0))(max)(-). The variation of lambda (IJ) was not very important within fermentations (10.3-16.2 days); nonetheless, important effects were observed on m (max) (32.8-241.2 days(-1)) and (C (IJ)/C (IJ,0))(max) (66(-) to 611.4(-)). Particularly, maximum values of m (max) and (C (IJ)/C (IJ,0))(max) were obtained in medium A4 (0.276 l l(-1) agave juice, 17 g l(-1) yeast extract, 12 g l(-1) dried egg yolk, 0.025 l l(-1) corn oil). Also, the maximum IJ concentration (249,444 per ml) was achieved in A4-fermentations.     

昆虫病原线虫液体培养研究概况

综述昆虫病原线虫液体培养的研究概况。包括液体培养的研究历史,不同培养基组分对昆虫病原线虫产量的影响,营养源的选择,培养参数在昆虫病原线虫液体培养过程中的作用,感染期昆虫病原线虫脂肪酸含量,以及昆虫病原线虫应用前景等。     

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.     

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.     

Tank-Mix compatibility of the entomopathogenic nematodes, Heterorhabditis bacteriophora and Steinernema carpocapsae, with selected chemical pesticides used in turfgrass

We evaluated the compatibility of two entomopathogenic nematodes, Heterorhabditis bacteriophora HP88 and Steinernema carpocapsae All strain with selected pesticide formulations used in turfgrass in tank-mixes under laboratory conditions. The nematodes were exposed to the recommended rates of pesticides applied in either 100, 300, or 500 L/ha tank-mix volumes in 24-well plates at room temperature for 3 h and infective juveniles (IJ) viability determined, and then tested against Galleria mellonella larvae at 22-26°C for 96 h to assess IJ pathogenicity. We found that S. carpocapsae viability was not affected by any of the pesticides, while aluminum tris and trichlorfon significantly reduced S. carpocapsae pathogenicity at all concentrations. Thiamethoxam and trichlorfon significantly reduced H. bacteriophora viability, while halofenozide, aluminum tris, trichlorfon, and carbaryl significantly reduced H. bacteriophora pathogenicity. Imidacloprid, at the recommended rate 330-440 g AI/ha, significantly increased H. bacteriophora pathogenicity at 500 and 300 L/ha application volume. The integration of these nematode pesticide combinations in turf pest management programs is discussed.     

Evolution of culture broth rheological properties during propagation of the entomopathogenic nematode Steinernema carpocapsae,in submerged monoxenic culture

This article presents the evolution of culture broth rheological properties during monoxenic cultures of Steinernema carpocapsae in cylindrical bottles agitated orbitally. Rheological properties were evaluated in simple-shear flow conditions and were well-modeled by the Ostwald-de Waele model. Rheological properties varied from slightly dilatant, n = 1.2 (-), to moderately pseudoplastic flow behavior, n = 0.6 (-). Nematode concentrations increased from 750 +/- 190 to 130 900 +/- 6900 nematodes/mL, and the apparent viscosity (eta(a)) evolved from 4.5 +/- 0.7 to 46.6 +/- 3.2 mPa.s during the fermentations. Rheological behavior did not appear to be strongly influenced by nematode number and/or its stage of development; however, the release of substances from the decomposition of nematode cadavers appeared to be of great importance. Among the different developmental stages of the nematodes, only juveniles of the first stage (J1) were highly susceptible to the shearing conditions tested (shear stress, tau(r)()(theta), from 0.9 to 3.5 Pa during periods of 80-100 min), resulting in the viability loss of 85% of J1 nematodes.     

Steinernema kraussei,an indigenous nematode found in coniferous forests: efficacy and field persistence against Hylobius abietis

1 More than 100 ha of forest restocking sites in Northern Britain are treated annually with the entomopathogenic nematode Steinernema carpocapsae to control larvae of the large pine weevil, Hylobius abietis. However, data on geographical distribution, thermal niche breadth and foraging strategy suggest that S. carpocapsae may be a poor choice for this application. 2 We undertook laboratory and field experiments that compared S. carpocapsae with Steinernema kraussei for efficacy towards H. abietis. Steinernema kraussei is indigenous to Northern Britain, active at low temperatures and is thought to adopt a ‘cruise’ foraging strategy particularly suitable for finding subterranean sedentary insects such as H. abietis. Conversely, S. carpocapsae has not been recorded in Northern Britain, prefers warmer temperatures and is thought to remain at the soil surface adopting an ambush foraging strategy. 3 There were no differences in the ability of the two nematodes to infect and kill larvae of H. abietis in laboratory experiments and, in two field experiments, both species were equally effective both in terms of parasitizing larvae within the stumps and reducing emergence of adult H. abietis. In one experiment, we monitored persistence of nematodes for 1 year after application and found S. kraussei persistence was much greater than that of S. carpocapsae. 4 The greater persistence of S. kraussei suggests that this nematode is better adapted to conditions in Northern Britain, but this may potentially represent a greater threat to nontarget insects. 5 Our data suggest that there would be little benefit associated with substituting S. kraussei for S. carpocapsae in integrated pest management systems for H. abietis.     

Modulation of immune responses of Rhynchophorus ferrugineus (Insecta: Coleoptera) induced by the entomopathogenic nematode Steinernema carpocapsae (Nematoda: Rhabditida)

Aim of this study was to investigate relationships between the red palm weevil (RPW) Rhynchophorus ferrugineus (Olivier) and the entomopathogenic nematode Steinernema carpocapsae (EPN); particularly, the work was focused on the immune response of the insect host in naive larvae and after infection with the EPN. Two main immunological processes have been addressed: the activity and modulation of host prophenoloxidase‐phenoloxidase (proPO) system, involved in melanization of not‐self and hemocytes recognition processes responsible for not‐self encapsulation. Moreover, immune depressive and immune evasive strategies of the parasite have been investigated. Our results suggest that RPW possess an efficient immune system, however in the early phase of infection, S. carpocapsae induces a strong inhibition of the host proPO system. In addition, host cell‐mediated mechanisms of encapsulation, are completely avoided by the parasite, the elusive strategies of S. carpocapsae seem to be related to the structure of its body‐surface, since induced alterations of the parasite cuticle resulted in the loss of its mimetic properties. S. carpocapsae before the release of its symbiotic bacteria, depress and elude RPW immune defenses, with the aim to arrange a favorable environment for its bacteria responsible of the septicemic death of the insect target.     

Effects of Crop Residue on the Persistence of Steinernema carpocapsae

We determined the effects of crop residue on the persistence of an entomopathogenic nematode, Steinernema carpocapsae. During 2 consecutive years, nematodes were applied at rates of 2.5 × 10₄ and 1.0 × 10⁵ infective juveniles/m² to small field plots planted with corn. Nematode persistence was monitored by exposing Galleria mellonella larvae to soil samples from plots with and without crop residue (approximately 75% coverage of soybean stubble). Persistence of S. carpocapsae was significantly greater in crop residue plots than in plots without residue. In crop residue plots that received the higher rate of nematode application, larval mortality did not significantly decrease during the study period (3 to 5 days) and remained above 85%. In nematode-treated plots without crop residue, however, larval mortality fell from over 96% to below 11% and 35% in the first and second trials, respectively. The increased crop residue may have benefited nematode persistence through protection from desiccation or ultraviolet light. We conclude that increased ground cover in cropping systems (e.g., due to reduced tillage) may lead to increased insect pest suppression with entomopathogenic nematodes.     

Infectivity of entomopathogenic nematode Steinernema carpocapsae Pocheon strain (Nematoda: Steinernematidae) on the green peach aphid Myzus persicae (Hemiptera: Aphididae) and its parasitoids

Infectivity of entomopathogenic nematode (EPN) Steinernema carpocapsae Pocheon strain on the green peach aphid Myzus persicae and its parasitic wasps (e.g., Aphidius colemani, Aphidius gifuensis and Diaeretiella rapae) was evaluated under laboratory conditions. Infective juveniles (IJs) of S. carpocapsae Pocheon strain had low infectivity against nymph and adult stages of M. persicae, showing 2% and 6.7% of mortality, respectively. Application of the EPNs had little effect on mummies caused by the three parasitoid species, allowing them to remain intact. No IJ invaded the host, regardless of EPN application rate. The parasitoid emergence from mummies ranged from 80% to 85% in the presence of EPN while 79–86% was recorded in the absence of EPN. However, the presence of the IJs reduced oviposition by the three parasitoid species, decreasing the rate up to 59% when the nematodes were applied before parasitoid release, while little difference in oviposition was observed when nematodes were applied after parasitoid release.