Production technology for entomopathogenic nematodes and their bacterial symbionts

Entomopathogenic nematodes (genera Steinernema and Heterorhabditis) kill insects with the aid of mutualistic bacteria. The nematode–bacteria complex is mass produced for use as biopesticides using in vivo or in vitro methods, i.e., solid or liquid fermentation. In vivo production (culture in live insect hosts) is low technology, has low startup costs, and resulting nematode quality is high, yet cost efficiency is low. In vitro solid culture, i.e., growing the nematodes and bacteria on crumbled polyurethane foam, offers an intermediate level of technology and costs. In vivo production and solid culture may be improved through innovations in mechanization and streamlining. In vitro liquid culture is the most cost-efficient production method but requires the largest startup capital and nematode quality may be reduced. Liquid culture may be improved through progress in media development, nematode recovery, and bioreactor design. A variety of formulations is available to facilitate nematode storage and application. Journal of Industrial Microbiology & Biotechnology (2002) 28, 137–146 DOI: 10.1038/sj/jim/7000230 Received 16 August 2001/ Accepted in revised form 10 November 2001     

生物杀虫剂-昆虫病原线虫的培养技术

昆虫病原线虫是新型的生物杀虫剂,对钻蛀及土栖性害虫防效较好,具有部分替代化学农药的潜力。昆虫病原线虫的产业化培养是昆虫病原线虫商业化应用的基础。本文介绍了目前世界上常用的昆虫病原线虫的培养方法,包括活体培养、半固体培养和液体培养的技术,这些技术的应用有助于昆虫病原线虫的种质保存及培养,为拓展昆虫病原线虫的产业化生产和应用奠定了基础。     

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.     

Synergistic effect of the herbicides glyphosate and MCPA on survival of entomopathogenic nematodes

The survival and infectivity of the infective juveniles of two species of entomopathogenic nematodes, Steinernema feltiae (Rhabditida: Steinernematidae) Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae), were determined after exposure for 72 h to two concentrations of the herbicides glyphosate and MCPA, as well as to the combination of the two herbicides (glyphosate + MCPA). For all herbicide treatments, concentrations and exposure times, S. feltiae was more tolerant to the herbicides than H. bacteriophora. The exposure of entomopathogenic nematodes to glyphosate + MCPA caused significantly higher mortality (26.33–57.33%) than glyphosate (0.67–15%) or MCPA (2.33–19%) alone. These results confirm the synergistic effect of the glyphosate + MCPA combination on the mortality in these nematodes. Nematode infectivity of Galleria mellonella larvae in response to the herbicides presence was evaluated in Petri dish assays containing sterile sand. Nematode infectivity was not significantly reduced by exposure to herbicides in S. feltiae but H. bacteriophora was less tolerant. Synergistic effect was obtained in the nematode mortality test but no synergistic effect was observed in the nematode infectivity assay. Our results suggest that possible synergistic effects of agrochemicals on survival of nematodes should be tested before mixing with entomopathogenic nematodes.     

Assessment of establishment and persistence of entomopathogenic nematodes for biological control of western corn rootworm

Abstract:  The use of entomopathogenic nematodes (EPN) is potentially one ecological approach to control the invasive alien western corn rootworm ( Diabrotica virgifera virgifera LeConte, Col., Chrysomelidae) in Europe. This study investigated the establishment and the short- and long-term persistence of Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae), Heterorhabditis megidis Poinar, Jackson and Klein (Rh., Heterorhabditidae) and Steinernema feltiae Filipjev (Rh., Steinernematidae) in three maize fields in southern Hungary, using the insect-baiting technique. All three EPN species equally established and persisted in maize fields. The timing of application (April or June) did not influence the establishment of EPN species. EPNs persisted for 2–5 months, i.e. they survived up to and throughout D. v. virgifera larval occurrence in the soil. Results demonstrate that D. v. virgifera larvae can potentially be controlled by EPNs during the same year of EPN application but no long-term control effect is expected under intensive maize cultivation practices.     

Application methods of entomopathogenic nematodes for control of Mahanarva spectabilis (Hemiptera: Cercopidae)

The pathogenicity of entomopathogenic nematodes (EPNs) against nymphs of the pasture spittlebug Mahanarva spectabilis (Distant, 1909) (Hemiptera: Cercopidae) was studied under four application methods. Nymphs of the fourth or fifth instar were placed on the roots of elephant grass plants and submitted to four EPN isolates (Steinernema carpocapsae, S. feltiae, S. riobravis and Heterorhabditis amazonensis RSC1), at two concentrations (2000 and 4000 infective juveniles/mL), with four application methods (pipetting, spraying on the nymphs after froth formation, spraying before froth formation and placement of infected host cadaver method). There was no significant difference in the nymph mortality in function of the concentration and/or the isolate used. However, the efficacy was influenced by the application method, with the most efficient being sprayed on nymphs after froth formation and infected host cadavers. Steinernema riobravis applied by spraying on nymphs with froth, at a concentration of 2000 EPNs/mL, and H. amazonensis RSC1 applied by infected host cadavers caused 71% of the nymphs to die. The use of infected host cadavers and spraying in an aqueous solution on nymphs after froth formation were found to be the most efficient methods to control Mahanarva spectabilis.     

Host density effects on efficacy of entomopathogenic nematodes against white grub (Coleoptera: Scarabaeidae) species

We tested the effect of host density on entomopathogenic nematode efficacy in 1-L pots with grass and soil. In four experiments, combinations ranged from somewhat resistant hosts (oriental beetle, Anomala orientalis, or northern masked chafer, Cyclocephala borealis, with Heterorhabditis bacteriophora) over more susceptible hosts (Japanese beetle, Popillia japonica, with Steinernema glaseri) to a highly susceptible host (P. japonica and S. scarabaei). In each experiment, four larval densities were exposed to two nematode rates over a 14-day period. A significant effect of host density on nematode efficacy occurred only in the A. orientalis–H. bacteriophora combination, but there was no clear trend in the data. This suggests that an exhaustion of available nematode populations to less lethal levels by high host numbers was counteracted by other factors such as increased chances for nematode-host contact and increased host susceptibility due to stress via reduced food resources and increased aggression between larvae.     

Specific oxygen uptake of the entomopathogenic nematode,Steinernema carpocapsae CABA01, in submerged culture

The specific oxygen uptake rate (qO₂) of stages of the entomopathogenic nematode (EPN) Steinernema carpocapsae CABA01 in liquid culture was measured. Nematodes were grown into previously pasteurised culture broths of their symbiotic bacterium, Xenorhabdus nematophila, in orbitally agitated flask cultures (V_L = 125 mL) at N = 150 rpm and T = 25°C. The basal medium contained 3% (w/v) soy trypticase broth and 0.5% (w/v) yeast extract. The EPNs developed from the egg stage to the adult stage exhibiting qO₂ values of 1.92, 5.48, 0.48, 0.28 and 0.0014 [10^?1 mmolO₂/(g_{nematode-wet base} h)] for the egg-Juvenile 1 (J1), J2, J3, J4 and the adult stages, respectively.     

昆虫病原线虫对腰果细蛾的致病力测定

室内测定了Steinernema carpocapsae All、S.carpocapsae Biosys、S.glaseri NC52、S.longicaudum X-7、Heterorhabditis bacteriophora H06和H.indica LN2共6个昆虫病原线虫品系对腰果细蛾Acrocercops syngramma Meyrick幼虫的致病力。结果表明,线虫S.carpocapsae All、Biosys品系与线虫S.glaseri NC52对腰果细蛾幼虫的致死效果显著高于其它线虫,但供试的6个线虫品系均未能在腰果细蛾幼虫体内繁殖下一代,说明供试线虫在腰果细蛾幼虫体内难以繁殖。选择小卷蛾斯氏线虫S.carpocapsae All进一步研究其对腰果细蛾的致病力,结果发现,All线虫对腰果细蛾3龄幼虫处理24 h后的致死中浓度LC50为19.88 IJs/虫。腰果细蛾各龄幼虫和蛹对小卷蛾斯氏线虫S.carpocapsae All均表现出高度的敏感性,但不同幼虫龄期和处理时间敏感程度不同。小卷蛾斯氏线虫S.carpocapsae All对不同龄期腰果细蛾幼虫和蛹均表现出较强的致病力。     

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.     

Survey of entomopathogenic nematodes and associate bacteria in Thailand and their potential to control Aedes aegypti

Aedes aegypti is an insect vector that transmits several viruses affecting humans worldwide. Entomopathogenic nematodes (EPNs) and their symbiotic bacteria are organisms with the potential to control many insects. In this study, we did a survey aimed to identify EPNs and their symbiotic bacteria and evaluate the larvicidal activity of bacteria against Ae. aegypti. We collected 540 soil samples from 108 sites in Phitsanulok Province, lower northern Thailand. Baiting techniques and White traps were used to isolate EPNs from soil samples. By sequencing of 28S rDNA and internal transcribed spacer regions, 51 EPN isolates were identified as Steinernema surkhetense (35 isolates), Heterorhabditis indica (14 isolates) and Heterorhabditis sp. SGmg3 (two isolates). Based on sequencing of a partial region of the recA gene, 35 isolates of Xenorhabdus were identified as Xenorhabdus stockiae, and 20 Photorhabdus isolates were identified as Photorhabdus luminescens subsp. akhurstii (10 isolates), P. luminescens subsp. hainanensis (seven isolates) and P. asymbiotica subsp. australis (three isolates). Screening for larvicidal activity of bacteria against Ae. aegypti was performed in the laboratory. Xenorhabdus WB5.4 and Xenorhabdus WB12.5, which were closely related to X. stockiae, resulted in high mortality of Ae. aegypti (99.99% and 70%, respectively) at 96 hr after exposure. Comparing with control groups, mortality of Ae. aegypti larvae was low (1.11%–6.67%) after exposure for 24–96 hr. Our findings showed the potential of X. stockiae for controlling Ae. aegypti. Further studies are needed to elucidate the mechanisms through which these bacteria kill Ae. aegypti larvae.     

Evaluation of the virulence of entomopathogenic nematodes as a biological control agents against Gryllotalpa gryllotalpa (Gryllotalpidae)

The European mole cricket Gryllotalpa gryllotalpa Linnaeus (Orthoptera: Gryllotalpidae) is a key pest of several crops in different regions of the world. The mole crickets usually damage seedlings, feeding aboveground on foliage or stem tissue and belowground on roots and tubers. In biological control, entomopathogenic nematodes (EPNs) are used against many insect pests inhabiting the soil as safe alternatives to chemical pesticides. The present study aimed to evaluate the virulence of the EPNs Heterorhabditis bacteriophora strain (HP88) and Steinernema carpocapsae strain (All) against the adults of European mole cricket G. gryllotalpa under laboratory and field conditions. Three concentrations of each of the EPNs H. bacteriophora and S. carpocapsae were tested against G. gryllotalpa. Infected adults of the pest with nematodes, at the concentration of 2000/cup, attained a mortality rate of (96%), when treated with S. carpocapsae and (72%) when treated with H. bacteriophora 8 days post-treatments. Mortality percentage of G. gryllotalpa adults increased as the concentration of the nematodes increased. Adults' mortality of G. gryllotalpa reached 100% when treated with the combination of the 2 nematode's strains at all concentrations 4 days post-treatments. At the 3 tested concentrations, 500, 1,000 and 2,000 IJs/adults, H. bacteriophora showed a higher reproductive rate in vivo than S. carpocapsae. A single adult of G. gryllotalpa treated with H. bacteriophora produced the average of 29,972, 74,768 and 140,474 IJs/adult, at the 3 concentrations, respectively. Under field application, a combination of EPNs successfully reduced the loss of the cabbage seedlings and the population's number of the mole crickets after 3 applications in the 2 seasons of (2019 and 2020). Therefore, EPNs as biological control agents can be recommended against the mole cricket G. gryllotalpa in vegetable crops as s pest management practice in an integrated pest management programme.     

The first record of entomopathogenic nematodes (Rhabiditiae: Steinernematidae and Heterorhabditidae) in natural ecosystems in Lebanon: A biogeographic approach in the Mediterranean region

A survey of entomopathogenic nematodes in Lebanon was conducted for the first time during 2008-2009. Samples were collected on the coastal strip and in nine vegetation types extending from the coastal line to 3088 m above sea level. Wooded and herbaceous ecosystems were considered for sampling purposes. A total of 570 samples were taken, out of which 1% were positive for entomopathogenic nematodes. Approximately, 15.8% out of the 19 sites sampled revealed entomopathogenic nematodes presence (representing three samples). Two entomopathogenic nematodes species Heterorhabditis bacteriophora and Steinernema feltiae were recovered, and identification of their symbiotic bacteria revealed the presence of a Xenorhabdus bovienii, Photorhabdus temperata subsp. thracensis, Photorhabdus luminescens subsp. kayaii and Photorhabdus luminescens subsp. Laumondii.     

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。     

Biological control potential of native entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) against Spodoptera cilium (Lepidoptera: Noctuidae) in turfgrass

In laboratory studies, we demonstrated that five native entomopathogenic nematode species/isolates caused 100% mortality of Spodoptera cilium larvae, a soil surface-feeding pest of turfgrass. At 25 infective juveniles/cm² applied to sod, two selected Turkish species, Steinernema carpocapsae and Heterorhabditis bacteriophora (Sarigerme isolate), averaged 77% and 29% larval mortality, respectively.     

Efficacy and persistence of entomopathogenic nematodes for black cutworm control in turfgrass

Field experiments were conducted in turf maintained under golf course fairway conditions in May, June, and August 2009 and in August and September 2010 to evaluate the ability of entomopathogenic nematodes to control larval populations of the black cutworm, Agrotis ipsilon, on golf courses. Commercial products containing the entomopathogenic nematodes Heterorhabditis bacteriophora, Steinernema carpocapsae, S. feltiae, and S. riobrave were applied at 1.0 or 2.5×10⁹ infective juveniles per ha against fourth-instar black cutworms. Larval mortality and turf damage were evaluated at 4 and/or 7 days after treatment (DAT). Steinernema carpocapsae was the best performing species due to a combination of high control rates (average 83%), most consistent results (70–90% range), high speed of kill (average 68% at 4 DAT), and prevention of significant turf damage despite very high larval densities at 0 DAT. Efficacy of S. feltiae and H. bacteriophora was often similar to that of S. carpocapsae but overall less consistent. Short-term persistence of the nematodes was evaluated in four turfgrass sites maintained under golf course putting green, fairway, or rough conditions in June and August 2009 by baiting soil samples at 0, 4, 7, and 14 DAT. Relative to recovery immediately after application, at least 50% of S. feltiae and 25% of S. carpocapsae consistently persisted up to 4 days in one of the greens and up to 7 days in some trials. Our finding suggests that S. carpocapsae and S. feltiae may provide adequate black cutworm control in golf course turf under moderate summer temperatures.     

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.     

Isolation and virulence of entomopathogenic fungi against larvae of hazelnut weevil Curculio nucum (Coleoptera,Curculionidae) and the effects of combining Metarhizium anisopliae with entomopathogenic nematodes in the laboratory

An approach to ensure effective pest biocontrol would be to select native isolates of biological control agents (BCAs). A survey to isolate entomopathogenic fungi (EPF) from a hazelnut growing area has been carried out. EPF were recovered from 133 of the 295 soil samples. The main species isolated were Metarhizium anisopliae sensu lato (36%) and Beauveria bassiana sensu lato (42.8%). With regard to controlling abiotic factors, altitude had an effect on the distribution of B. bassiana, but not on M. anisopliae. Cropping system did not have an effect on the occurrence of these EPF, while pH appeared as a predictive variable for both. In addition, we tested the virulence of six of these isolates: three M. anisopliae and three of B. bassiana against larvae of Curculio nucum L. The highest larval mortality (reaching 80%) was due to M. anisopliae (strain 34) when applied in simultaneous combination with four entomopathogenic nematode species: Steinernema carpocapsae (strain B14), Steinernema feltiae (strain D114), Steinernema. sp. (strain D122) and Heterorhabditis bacteriophora (strain DG46). The effect of nematodes was greater and no antagonistic or synergistic effects were observed.     

Efficacy of entomopathogenic nematodes against potato tuber moth,Phthorimaea operculella (Lepidoptera: Gelechiidae) under laboratory conditions

The susceptibility of potato tuber moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae) to native and commercial strains of entomopathogenic nematodes (EPNs) was studied under laboratory conditions. Native strains of EPNs were collected from northeastern Iran and characterised as Steinernema feltiae and Heterorhabditis bacteriophora (FUM 7) using classic methods as well as analysis of internal transcribed spacer (ITS) and D2/D3 sequences of 28S genes. Plate assays were performed to evaluate the efficiency of five EPN strains belonging to four species including Steinernema carpocapsae (commercial strain), S. feltiae, Steinernem glaseri and H. bacteriophora (FUM 7 and commercial strains). This initial assessment with 0, 75, 150, 250, 375 and 500 IJs/ml concentrations showed that S. carpocapsae and H. bacteriophora caused the highest mortality in both larval and prepupal stages of P. operculella, PTM. Thereafter, these three strains (i.e. S. carpocapsae, H. bacteriophora FUM 7 and the commercial strains) were selected for complementary assays to determine the effects of soil type (loamy, loamy–sandy and sandy) on the virulence of EPNs against the second (L2) and fourth instar (L4) larvae as well as prepupa. A soil column assay was conducted using 500 and 2000 IJs in 2-ml distilled water. Mortality in the L2 larvae was not affected by the EPN strain or soil type, while there was a significant interactive effect of nematode strains and soil type on larval mortality. The results also showed that EPN strains have higher efficiency in lighter soils and caused higher mortality on early larvae than that in loamy soil. In L4 larvae, mortality of PTM was significantly influenced by nematode strain and applied concentrations of infective juveniles. The larval mortality induced by S. carpocapsae was higher than those caused either by a commercial or the FUM 7 strain of H. bacteriophora. Prepupa were the most susceptible stage.