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A technique for the mass propagation of the DD 136 nematode
... Two methods are used to mass produce EPNs: (i) in vivo (Dutky et al., 1964) and (ii) in vitro (Glaser, 1940). in vitro processes are conducted in artificial media; in vivo processes use insects as the bioreactors (Sharma et al., 2011). Bioreactors are used to create EPNs on a small-and large-scale basis in a number of North American, European, and Asian countries (Shapiro-Ilan and Gaugler, 2002). ...
... Using a micropipette, larvae are infected with about forty thousand live and active IJs of Steinernema feliae MK 256355 kept in 1 ml of sterilised distilled water. Such ten sets are prepared, and each petri dish is covered with its respective lid and placed in a BOD incubator at 20°C (Dutky et al., 1964). The petri dishes are taken out of the BOD incubator after three days. ...
... (Blinova & Ivanova, 1987;Flanders et al., 1996). (Dutky et al., 1964;Blinova & Ivanova, 1987;Shapiro et al., 1999). When adopting a host, simplicity of culture and infection are crucial considerations in addition to yield (Blinova & Ivanova, 1987;Shapiro-Ilan & Gaugler, 2002). ...
Entomopathogenic nematodes (EPNs), Heterorhabditis, and Steinernema are effective biocontrol agents for insect pests. In vitro and in vivo methods are used for mass production of EPNs in large quantities. The In vivo technique, wherein insect hosts are used for EPN production, requires low technology and low startup costs, but the production of nematodes by this method is of high quality and is generally used for laboratory purposes or in small areas such as kitchen gardens. On the other hand, the production of EPN by the In vitro technique requires artificial diets, and nematodes produced in masses by this method are generally for application in large areas. EPNs are to be easily transported, stored, and used. They can be formulated in aqueous suspension, synthetic sponge, clay, gel, and infected cadavers. Nonetheless, the development of novel formulations and the search for improved additives boost EPNs' viability and infectivity while preserving their virulence, all of which contribute to their eventual commercialisation.
... JIs por larva (POINAR, 1979), podendo chegar até a 200.000 JIs (DUTKY et al., 1964). Outros hospedeiros alternativos são conhecidos para este fim, entre eles Spodoptera frugiperda (Smith), (Lepidoptera: Noctuidae), Amyelios transitella (Walker) e Tenebrio spp. ...
... Segundo DUTKY et al. (1964) e ADAMS & NGUYEN (2002, a principal causa da redução na produção dos JIs deve-se, principalmente, ao tamanho do hospedeiro, já que insetos de tamanhos maiores favorecem ciclos mais longos e um maior numero de gerações, por existir uma reserva maior de nutrientes. GOUGE & HAGUE (1994) encontraram, em seus resultados, que o tempo de emergência dos JIs é dependente também do tamanho do hospedeiro; em hospedeiros menores como larvas de Bradysia paupera (Diptera: Sciaridae), a emergência de nematóides ocor-reu até o 6º dia, ao contrário de um hospedeiro como G. mellonella, para o qual a emergência pode se prolongar em até duas semanas. ...
... Estes resultados registram produções aproximadas às encontradas por DUTKY et al. (1964), com 160.000 JIs para Steinernema carpocapsae "Mexican strain" em G. mellonella, e com as registradas por MOLINA & L ÓPEZ (2001) para S. carpocapsae, com 149.258 ...
Entomopathogenic nematodes (EPNs) can be an effective tool in Integrated Pest Management programs (IPM). However, in vivo production for laboratory bioassays is still difficult, requiring tests with new host insects and different infection techniques aiming to improve the production. Thus, this work was aimed at evaluating the in vivo multiplication of 3 EPN species: Steinernema carpocapsae, Steinernema glaseri, and Steinernema arenarium, in last instar larvae of Galleria mellonella, Tenebrio molitor, Spodoptera frugiperda and Bombyx mori with three infection systems (injection, topic simple and topic complex). A 3x4 factored (3 infection systems and 4 hosts) randomized experimental design was used. Each treatment had 6 replicates (larvae). The largest productions took place in the first 3 days of IJ emergence. The treatments S. carpocapsae and S. arenarium with simple topic method in G. mellonella yielded the largest productions of IJs/larva, with 302,124 and 149,213 IJs/larva, respectively. S. arenarium was the only species to multiply by injection in G. mellonella, although with low production (1,076 IJs/larva), being a rather inefficient system for production of these species. An alternative host for in vivo production is T. molitor, since the treatment S. arenarium by topic complex yielded high production (103,059 IJs/larva). S. glaseri did not multiply by any infection system in T. molitor and B. mori. The multiplication of this species with the simple topic system in G. mellonella achieved a total production of 132,065 IJs/larva. The simple topic and complex systems presented the largest productions per larva. T. molitor for the species S. arenarium and B. mori for S. carpocapsae presented great possibilities as alternative hosts for in vivo production.
KEY WORDS:
Microbial control; multiplication; entomonematodes; insect pest.
... Moreover, wide distribution, easy culturing, and the development of a higher number of EPNs make wax moth the most preferable host. Various protocols for nematodes infection, inoculation, and isolation have been demonstrated by many researchers (Dutky et al. 1964;Howell 1979;Woodring and Kaya 1988;Flanders et al. 1996;Finnegan et al. 1999). ...
... Multiple authors have reported various methods for culturing EPNs (Dutky et al. 1964;Kaya and Stock 1997;Poinar 1979). The in-vivo process of EPN-mass production is very simple as it does not require any modern or latest technology, just a live infected insect host, a surrogate host (to which IJs from the infected host are to be transmitted), trays and shelves are required. ...
... Under in vivo process, per larvae yield of IJs depends on the selection of nematode and host species (ranging from 0.5 Â 105 to 4 Â 105). Host size is directly proportional to the yield of specific EPN species (Flanders et al. 1996;Blinova and Ivanova 1987), but the same is not true for infection susceptibility (Blinova and Ivanova 1987;Dutky et al. 1964). In vivo production is not effective for a large scale because it involves high costs due to extensive labor, material (insects), and equipment. ...
Entomopathogenic fungi are microorganisms capable of infecting and killing arthropods and therefore have a great potential in pest management. As the extensive use of synthetic pesticides has led to increased resistance in insects, decreased natural enemies, and had negative impacts on environmental and human health, the search for eco-friendly control agents is urgent. Entomopathogenic fungi are promising alternatives in this regard and are attracting global attention, with increasing efforts and financial investments being made for the development, commercialization and use of fungus-based control products. Despite scientific and technological advances, there is still a need for studies to expand the number of species applicable in pest management and improve their performance in the field. There is also a need to increase user awareness regarding their correct use with the aim to establish their widespread adoption and market potential. This chapter covers the main taxonomic groups that comprise entomopathogenic fungi, their modes of action to establish insect infection and spread, and the insect’s defense mechanisms against these fungi. Furthermore, techniques of fungal isolation, selection, and production are discussed. The usage status, challenges, and prospects of mycoinsecticides are also addressed, highlighting their application potential for sustainable agricultural production.
... Moreover, wide distribution, easy culturing, and the development of a higher number of EPNs make wax moth the most preferable host. Various protocols for nematodes infection, inoculation, and isolation have been demonstrated by many researchers (Dutky et al. 1964;Howell 1979;Woodring and Kaya 1988;Flanders et al. 1996;Finnegan et al. 1999). ...
... Multiple authors have reported various methods for culturing EPNs (Dutky et al. 1964;Kaya and Stock 1997;Poinar 1979). The in-vivo process of EPN-mass production is very simple as it does not require any modern or latest technology, just a live infected insect host, a surrogate host (to which IJs from the infected host are to be transmitted), trays and shelves are required. ...
... Under in vivo process, per larvae yield of IJs depends on the selection of nematode and host species (ranging from 0.5 Â 105 to 4 Â 105). Host size is directly proportional to the yield of specific EPN species (Flanders et al. 1996;Blinova and Ivanova 1987), but the same is not true for infection susceptibility (Blinova and Ivanova 1987;Dutky et al. 1964). In vivo production is not effective for a large scale because it involves high costs due to extensive labor, material (insects), and equipment. ...
Biopesticides, using living microbial bodies and their bio-active composites against insects, are potential replacements for synthetic insecticides for safer and modern food production systems. Entomopathogenic bacteria (EPB) are important biological control agents of insect pests since the last century. Though bacterial species have been documented to be used against insects for developing symbiotic relationships, only a few of them are identified as entomopathogens. Most of these are members of the family Bacillaceae, Enterobacteriaceae, Pseudomonadaceae, Clostridiaceae, and Neisseriaceae. More than 100 bacterial species have been reported to infect various arthropods. Bacillus thuringiensis (Bt), B. sphaericus, B. cereus, and B. popilliae are the most appreciated microbial pest control agents. However, new bacterial species also need to be explored for their entomopathogenic role and materialized as new biopesticide products. The commercial biopesticides based on novel EPBs with improved genetic materials must be a part of future research for effective integrated pest management programs. This present chapter highlights the classification, infection, replication, transmission mechanisms, and important EPB in integrated pest management.
... Moreover, wide distribution, easy culturing, and the development of a higher number of EPNs make wax moth the most preferable host. Various protocols for nematodes infection, inoculation, and isolation have been demonstrated by many researchers (Dutky et al. 1964;Howell 1979;Woodring and Kaya 1988;Flanders et al. 1996;Finnegan et al. 1999). ...
... Multiple authors have reported various methods for culturing EPNs (Dutky et al. 1964;Kaya and Stock 1997;Poinar 1979). The in-vivo process of EPN-mass production is very simple as it does not require any modern or latest technology, just a live infected insect host, a surrogate host (to which IJs from the infected host are to be transmitted), trays and shelves are required. ...
... Under in vivo process, per larvae yield of IJs depends on the selection of nematode and host species (ranging from 0.5 Â 105 to 4 Â 105). Host size is directly proportional to the yield of specific EPN species (Flanders et al. 1996;Blinova and Ivanova 1987), but the same is not true for infection susceptibility (Blinova and Ivanova 1987;Dutky et al. 1964). In vivo production is not effective for a large scale because it involves high costs due to extensive labor, material (insects), and equipment. ...
The successful control of many insect-pests makes entomopathogenic nematodes (EPNs) among one of the best biocontrol agents for insect pests. Moreover, the ability of EPNs to seek out their hosts and kill them in those habitats where chemicals fail makes them even more attractive. The EPNs-bacterial mutualistic association helps them kill their hosts in a relatively shorter period than other necromenic or parasitic nematode associations. In addition to this end-user safety, hotspot application which allows minimizing treated area, natural enemies’ safety, withholding period absence, and environmental protection are a few of many advantages over chemical pesticides. Two important genera of EPNs, i.e., Heterorhabditid and Steinernematids, are associated with symbiotic bacteria Photorhabdus and Xenorhabdus, respectively, while bacterial symbiont of neosteinernamatids is yet to be described. About 21 species of Heterorhabditis and 100 species of Steinernema have been isolated and identified worldwide. With the increasing environmental concerns and low efficacy of synthetic pesticides, agriculturists and researchers have a growing interest in finding alternatives to synthetic pesticides. Several EPNs can be widely used in place of synthetic pesticides in agro-ecosystem. There is still a need to improve several aspects of EPNs, such as efficacy and efficiency, reduced costs, mass production, and formulation technology. Furthermore, their potential for recycling in the host population beckons them to be further exploited for sustainable pest control. This chapter will emphasize the use and potential of EPNs as an integral part of integrated pest management. To aid with understanding the potential of EPNs, this chapter will also provide an overview of ecology and biology, mass production, application strategies, and integration with other management tools.
... Moreover, wide distribution, easy culturing, and the development of a higher number of EPNs make wax moth the most preferable host. Various protocols for nematodes infection, inoculation, and isolation have been demonstrated by many researchers (Dutky et al. 1964;Howell 1979;Woodring and Kaya 1988;Flanders et al. 1996;Finnegan et al. 1999). ...
... Multiple authors have reported various methods for culturing EPNs (Dutky et al. 1964;Kaya and Stock 1997;Poinar 1979). The in-vivo process of EPN-mass production is very simple as it does not require any modern or latest technology, just a live infected insect host, a surrogate host (to which IJs from the infected host are to be transmitted), trays and shelves are required. ...
... Under in vivo process, per larvae yield of IJs depends on the selection of nematode and host species (ranging from 0.5 Â 105 to 4 Â 105). Host size is directly proportional to the yield of specific EPN species (Flanders et al. 1996;Blinova and Ivanova 1987), but the same is not true for infection susceptibility (Blinova and Ivanova 1987;Dutky et al. 1964). In vivo production is not effective for a large scale because it involves high costs due to extensive labor, material (insects), and equipment. ...
Modern agricultural production is dominated by the use of synthetic chemical pesticides, which account for 95% of the global market share of total pesticide use. However, this over-reliance on synthetic pesticides adversely affects and interferes with the functioning of the ecosystem. Neem (Azadirachta indica), a botanical biopesticide widely known for its bactericidal, fungicidal, insecticidal, herbicidal, and nematicidal properties, offers an eco-friendly alternative to synthetic pesticides. To date, more than 200 bioactive compounds have been extracted from neem, and several commercial formulations have been developed and registered as broad-spectrum biopesticides. More advanced strategies in the use of neem as a botanical biopesticide have been developed with a focus on developing more innovative and effective approaches. This chapter also covers current advancement on neem bioactive ingredients, their efficacy and extraction methods. In addition, stability of the bioactive compounds and environmental, health and safety issues are discussed.
... Moreover, wide distribution, easy culturing, and the development of a higher number of EPNs make wax moth the most preferable host. Various protocols for nematodes infection, inoculation, and isolation have been demonstrated by many researchers (Dutky et al. 1964;Howell 1979;Woodring and Kaya 1988;Flanders et al. 1996;Finnegan et al. 1999). ...
... Multiple authors have reported various methods for culturing EPNs (Dutky et al. 1964;Kaya and Stock 1997;Poinar 1979). The in-vivo process of EPN-mass production is very simple as it does not require any modern or latest technology, just a live infected insect host, a surrogate host (to which IJs from the infected host are to be transmitted), trays and shelves are required. ...
... Under in vivo process, per larvae yield of IJs depends on the selection of nematode and host species (ranging from 0.5 Â 105 to 4 Â 105). Host size is directly proportional to the yield of specific EPN species (Flanders et al. 1996;Blinova and Ivanova 1987), but the same is not true for infection susceptibility (Blinova and Ivanova 1987;Dutky et al. 1964). In vivo production is not effective for a large scale because it involves high costs due to extensive labor, material (insects), and equipment. ...
The growing concern over potential hazards from chemical pesticide safety among consumers and potential harm to the environment has culminated in consideration of natural management strategies of pests. Because they are complementary to most crop production systems, biopesticides based on plants can be integrated into pest management systems. Plant essential oils (EOs) can replace the more persistent non-natural pesticides in protecting the environment from the accumulation of chemicals reduce resistance and increase crop productivity. In addition, they possess low mammalian toxicity, broad-spectrum activity, and degrade rapidly in foodstuffs. In addition to exhibiting distinctive properties compared with synthetic pesticides, including high levels of pest toxicity and reduced toxicity toward non-target organisms, EOs possess contact, feeding deterrence, fumigant toxicity, oviposition, and repellent properties. In this chapter, we review the sources of EOs, their insecticidal activities, constituents, and mode of action and discuss their synergism and formulation with encapsulation for producing nanoinsecticidal products.
... EPNs can be cultivated effectively using either in vivo or in vitro approaches in laboratory environments [48] . The advancement of mass production techniques for EPNs has evolved from the initial large-scale in vitro solid media production method established by Glaser [49] , to the in vivo production techniques developed by Dutky et al. [50] , followed by the three-dimensional solid media in vitro methods introduced by Bedding [51,52] , and ultimately to the in vitro liquid fermentation production technique proposed by Friedman [53] . ...
... This approach is based on the White trap method, which takes advantage of the natural migration of IJs from an infected host cadaver into an adjacent water layer for subsequent harvesting. Over the years, this method has been developed, refined, and adapted by numerous researchers [50,54,55,56,57,58] . Gaugler et al. [59] introduced the LOTEK system, which functions independently of nematode migration to a reservoir. ...
Entomopathogenic nematodes (EPNs) represent a promising group of biological control agents that offer a sustainable alternative to synthetic chemical pesticides in managing insect pests. These nematodes exploit a unique symbiotic relationship with pathogenic bacteria, which not only enhances their virulence but also facilitates efficient insect host utilization. Their application methods encompass a range of horticultural equipment and formulation strategies, optimizing their deployment in agricultural settings. Factors such as environmental conditions, interactions with competing biotic entities, and host specificity significantly influence EPN effectiveness and persistence. Despite their potential, widespread adoption of EPNs is hampered by several challenges, including high production costs, limited shelf life, and sensitivity to adverse environmental factors. Innovations in formulation— such as water-dispersible granules and encapsulated nematodes—aim to improve storage and efficacy. Genetic advancements are also critical; enhancing the longevity of infective juveniles, increasing symbiotic bacteria retention, and developing resilience against environmental stressors can substantially elevate the performance of EPNs in pest management. Comprehensive genomic studies of EPNs may unveil the genetic determinants associated with beneficial traits, leading to the discovery of novel strains adept at thriving under diverse climatic conditions. Ultimately, enhancing the practical application of EPNs in integrated pest management strategies could significantly contribute to sustainable agricultural practices, reducing reliance on chemical insecticides while fostering ecological balance. The holistic understanding of EPN biology and their ecological interactions will facilitate their integration into effective pest control programs.
... In 20 cm diameter petri dishes lined with filter paper, ten 5th instar larvae of G. mellonella were individually inoculated with approximately 1 x 10 3 infective juveniles (IJs) of the EPN strain in 0.5 ml of sterilized distilled water, following the protocol by Kaya and Stock (1997) [12] . The dishes were covered with an inverted petri bottom and stored in a BOD incubator (Dutky et al., 1964) [9] . After 2-3 days, nematode-infected deceased larvae were transferred to a modified White trap (White, 1927) [21] . ...
... In 20 cm diameter petri dishes lined with filter paper, ten 5th instar larvae of G. mellonella were individually inoculated with approximately 1 x 10 3 infective juveniles (IJs) of the EPN strain in 0.5 ml of sterilized distilled water, following the protocol by Kaya and Stock (1997) [12] . The dishes were covered with an inverted petri bottom and stored in a BOD incubator (Dutky et al., 1964) [9] . After 2-3 days, nematode-infected deceased larvae were transferred to a modified White trap (White, 1927) [21] . ...
... These bacto-helminthic complexes were successfully commercialised due to their high effectiveness against a wide range of insect pests (Lacey et al., 2015;Shapiro-Ilan et al., 2018). The mass culture of these nematodes has evolved from the in vitro solid media production by Glaser (1940a), to in vivo production by Dutky et al. (1964), arriving at the three-dimensional solid media in vitro process by Bedding (1981Bedding ( , 1984 and in vitro liquid fermentation production method by Friedman (1990). The in vivo mass-rearing nematodes have been reported by various authors (Dutky et al., 1964;Flanders et al., 1996;Kaya & Stock, 1997;Lindegren et al.,1993;Woodring & Kaya, 1988). ...
... The mass culture of these nematodes has evolved from the in vitro solid media production by Glaser (1940a), to in vivo production by Dutky et al. (1964), arriving at the three-dimensional solid media in vitro process by Bedding (1981Bedding ( , 1984 and in vitro liquid fermentation production method by Friedman (1990). The in vivo mass-rearing nematodes have been reported by various authors (Dutky et al., 1964;Flanders et al., 1996;Kaya & Stock, 1997;Lindegren et al.,1993;Woodring & Kaya, 1988). It is based on the White trap (White, 1927), in which nematode-infected hosts are placed above a water reservoir. ...
The amenability to axenic culture in vitro on solid medium including MacConkey, soybean casein, nutrient broth and egg yolk medium of Oscheius tipulae, Caenorhabditis elegans, Acrobeloides tricornis, Acrobeloides bodenheimeri and Teratorhabditis synpapillata were tested using Petri dishes. These nematodes were incapable of growing in MacConkey, soybean casein and nutrient broth, except for C. elegans which was able to multiply in soybean casein medium. While all nematodes used were successfully mass cultured using egg yolk medium at 25°C, results revealed that the highest yield of nematodes was observed with O. tipulae (TC2 and OC2), attaining 176 and 150 k nematodes per plate, respectively, after 10 days post-inoculation. In contrast, the lowest yield was recorded for T. synpapillata. The medium was optimised using different egg yolk concentrations (0, 5, 10, 20 and 40 g/L) and was tested for four nematode species O. tipulae, A. bodenheimeri, A. tricornis and T. synpapillata. The results showed that the egg yolk concentrations and the kinetic of nematode multiplication were positively correlated. Additionally, egg yolk liquid medium was applied to the mass culture of nematodes. Oscheius tipulae, A. bodenheimeri and A. tricornis were capable of reproduction in Petri dishes containing egg yolk liquid medium, at 25°C. Contrarily, T. synpapillata was unable to grow on the same liquid medium. The highest nematode yields of O. tipulae and A. bodenheimeri were attained 10,266 and 4110 nematodes in average, respectively, at 16 days post-inoculation. The maximum multiplication of A. tricornis was recorded at 20 days post-inoculation with 8430 nematodes.
... This method includes the natural migration of IJs (infective juveniles) far away from the infected host cadaver into a surrounding water layer, from where it should be harvested (Fig.4). This method was formulated (White, 1927) reformed and shortly modified by several workers (Dutky et al. 1964, Poinar, 1979, Woodring and Kaya, 1988). ...
... In vivo production byDutky et al. (1964). ...
Entomopathogenic nematodes (EPNs) are helpful nematodes that
parasitize agricultural insects, especially lepidopterans and coleopterans, and
can be employed as biopesticides against a wide range of insect pests. The
outstanding properties of EPNs have sparked a lot of economic interest in
nematodes as biological insecticides and they're seen as potential alternatives
to chemicals in Integrated Pest Management (IPM). A key component in the
effectiveness of Entomopathogenic Nematodes as biopesticides is their ability
to mass-produce them. Despite the fact that these nematodes were developed
more than 70 years ago, they are now commercially produced utilizing three
separate culture methods: in-vivo, in-vitro solid, and liquid culture
... In vivo production is suitable for laboratory work and/or small-scale field test because it requires relatively simple technology, low capital outlay, and produces of high quality IJs. Depending on nematode species and size, and host size, a single insect larva yields on average 20,000 to 500,000 IJs (Dutky et al., 1964;Bedding, 2006;Shapiro-Ilan et al., 2012, 2016a. However, host density and inoculation rate can affect IJ yields (Han et al., 1992(Han et al., , 1993Shapiro-Ilan et al., 2002). ...
... Dimensions and size of tray can be adjusted according to one's needs. This method guarantees >95 infection of hosts which is 15% more compared to the pipette method (Dutky et al., 1964). ...
Broad spectrum chemical pesticides are harmful to humans and other nontarget organisms. Biological control, which entails the use of natural enemies, is a viable alternative. Isolation, identification, and application of biocontrol agents such as the entomopathogenic nematodes (EPNs), Steinernema and Heterorhabditis and their symbiotic bacteria, Xenorhabdus and Photorhabdus have increased substantially over the last four decades, and the trend continues with advancement of molecular techniques. Yet, there is a need for a simple hands-on guide for their proper identification, classification, and handling, especially for researchers and users who are not totally familiar with these biocontrol agents. Thus, this manual is intended to provide a practical guide for students and researchers interested in or wanting to focus on these organisms. The manual describes the general biology and bionomics of these nematode/bacterium complexes and explains various basic standard protocols and methods frequently used in research and field laboratories ranging from isolation to application methods. Methods for rearing the insects, Galleria mellonella and Tenebrio molitor, which are routinely used in bioassays and recovery of EPNs, are also included.
... La técnica propuesta resulta de la conjugación de métodos clásicos para la extracción de nematodos del suelo (Dalmasso , 1968 ; Canavess y Jensen, 1955) y de la técnica de cría de Neoaplectana carpocapsae Weiser (Dutky et al., 1964). ...
A technique is described that allows rapid detection of the presence of infesting larvae of entomophagous nematodes in soil. This technique is the result of the conjugation of classical procedures in the extraction of nematodes from soil and the production of entomophages in the laboratory. The auxiliary host used is G. mellonelia. The totality of the results are obtained on the fourth day after the beginning of the experiment, having on the second day more than 50% of the information. The described technique is compared with that devised by Bedding and Akhurst using soil artificially infested with N. carpocapsae in known concentrations.
... The nematode then feeds on the bacteria, matures into an adult, and reproduces, generating a large number of nematodes. In fact, more than 100,000 infective-stage larvae can be found in the cadaver of a single wax moth larva (Dutky et al., 1964). ...
Entomopathogens are microorganisms that specifically target insects, are crucial in sustainable pest and disease management. This document reviews the roles and mechanisms of entomopathogens, including fungi, bacteria, viruses, nematodes and protozoa, in controlling insect pests and plant pathogens. Entomopathogenic fungi like Beauveria bassiana and Metarhizium anisopliae are prominent biological control agents, disrupting insect physiology through mechanisms such as cuticle penetration and immune evasion, while also inducing plant resistance against certain pathogens. Entomopathogenic bacteria, including Bacillus thuringiensis and Pseudomonas fluorescens, offer targeted pest control by producing toxins or antimicrobial compounds that impair pathogen growth and enhance plant health. Viruses such as Nuclear Polyhedrosis Viruses (NPVs) and Granulosis Viruses are effective in managing pest populations, particularly lepidopteran species, by causing lethal infections through ingestion. Entomopathogenic nematodes, like Steinernema and Heterorhabditis, utilize symbiotic bacteria to kill insects and influence soil microbial communities, although their application is limited. Protozoan pathogens, while slower acting, affect various insects by reducing feeding and reproduction rates. The document highlights commercial formulations and field applications of these agents, their efficacy, and challenges. Overall, entomopathogens represent a promising approach to integrated pest management, offering environmentally friendly alternatives to chemical pesticides and contributing to crop protection and disease control.
... Corn leaves (Zea mays) were used to raise the larvae in glass jars. Dutky et al. (1964) reported that G. mellonella larvae in their last instar were infected using the recently isolated EPN. Two weeks following their harvest, the infected juveniles (IJs) that came out of White traps were kept to use. ...
Background
The fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), is a polyphagous insect pest species that travel great distances every summer to disperse. They mostly feed on maize and over 350 other crops. In this investigation, two entomopathogens: Beauveria bassiana and Heterorhabditis taysearae , were utilized to test the efficacy of each against S. frugiperda separately and then as a group by inoculating H. taysearae two days after fungal infection.
Results
Evaluations were done on mortality percentage, infective juvenile (IJ) production, and conidia production. The LT 50 continually reduced with increases in nematode and fungus concentrations. For the LC 50 value, the H. taysearae isolate was 289 IJs/larva after 96 h of treatment, while for B. bassiana isolate, it was 10 ⁶ CFU/ml after 144 h of treatment. Dual infections with B. bassiana and H. taysearae had a beneficial effect on pest mortality, resulting in 83% mortality, and caused a significant increase in conidia production while utilizing the nematode or fungal separately caused decrease in mortality (63 and 73%, respectively).
Conclusions
The study’s findings indicated a quicker time to death and suggested that combining a moderately pathogenic fungal isolate with nematodes could raise the mortality rate. The mutually beneficial relationship between B. bassiana and H. taysearae controls S. frugiperda .
... In vivo production the process of cultivating culturing a specific entomopathogenic nematode in live insect hosts is seemingly simple but requires minimal technology and involves using a surrogate lab-or in-house-reared insect hosts including Corcyra cephalonica, crickets, Galleria mellonella and Tenebrio molitor, and finally harvesting the nematodes in bulk from the host cadavers after completion of their multiplication. Several authors (White, 1927;Dutky et al., 1964;Poinar, 1979;Woodring & Kaya, 1988;Lindegren et al., 1993;Flanders et al., 1996;Kaya & Stock, 1997;Shapiro-Ilan et al., 2012) have reported and reviewed in vivo production techniques for culturing EPN ( Table 5). ...
Global pesticide usage is 3.5 million tonnes at an average of 1.81kg/ha, while Indian usage is at 55,000 metric tonnes (2023) with an average of 0.517kg/ha. Compared to the pesticide market, the Indian biopesticide market remains small- cumulative annual biopesticide production at 9000 metric tonnes and a growth rate of 3-5% in consumption which is projected to reach a CGR of 8-10% by 2030. The utilization of biopesticides amounts to approximately 9% of overall pesticide use and is projected to increase to 50% of the total pesticide market by 2050. Among several microbial biocontrol agents, Entomopathogenic Nematodes (EPN) has been realised to be dependable IPM component against several insect pests. EPNs are soil-inhabiting beneficial nematodes that parasitize and kill insect pests, with immense potential for ecological services making them valuable tools in IPM. Worldwide, the demand for the development of EPN-containing products is mounting with several companies involved in their production, distribution and sales. India’s estimated demand for EPN is 24,000 metric tonnes, while the current production is 1800 metric tonnes from 25-30 firms. In India and other developing countries, the current EPN production and supply chain are in their infancy and operate as a cottage industry. The market is flourishing with products that are spurious, expensive, and unregulated due to the wide gap between demand and availability of EPN products. The authors present an overview of the status and prospects of EPN as an IPM component, contemporary and futuristic issues for the transformation of the upcoming EPN industry to a self-reliant, self-sufficient and profitable enterprise and accomplish better uptake of EPN individually or in IPM.
... The same procedure was adopted for the other EPN strain used in the study. Both the petri dishes were placed in B.O.D. incubator at 20 ± 2 °C (Dutky et al. 1964). After three days, the petri dishes were taken out from B.O.D. incubator and the dead larvae obtained from each plate were placed separately on white trap (White 1927) and incubated at 20 ± 2 °C in a B.O.D. incubator for the production of IJs. ...
Efficacy of two Indian isolates of entomopathogenic nematodes (EPNs) viz., Heterorhabditis bacteriophora MK256358 and Steinernema feltiae MK256355 were tested in laboratory against the larvae of cabbage butterfly, Pieris brassicae. Larval mortality was found directly proportional to initial inoculum level of infective juveniles (IJs). Susceptibility of larvae varied with respect to their variable size. H. bacteriophora MK256358 @ 25 IJs/larva caused 100% mortality to 3rd instar larvae at 72 h but @ 75 IJs/larva, the same mortality was achieved in 48 h. S. feltiae MK256355 @ 100 IJs/larva caused 100% mortality to 3rd instar larvae of P. brassicae at 48 h. H. bacteriophora MK256358 @ 25 and 100 IJs/larva resulted in 100% mortality to 4th and 5th instar larvae, respectively at 72 h, however S. feltiae MK256355 was unable to cause 100% mortality to either 4th or 5th instar larvae at any inoculum level or time period used in the study. LD50 and LT50 values of H. bacteriophora MK256358 were lower than S. feltiae MK256355 indicating that less nematode dose and time is required to kill 50% pest population. Reproduction capacity of nematode within the host was directly proportional to individual larval size and nematode inoculum level and for H. bacteriophora MK256358, it was higher and statistically significant (P ≤ 0.05) from S. feltiae MK256355. Our experimental findings open new avenues for utilization of EPNs against P. brassicae and set the basis for safe insect pest management programme.
... (EBN1e), and H. bacteriophora (EBN10k) isolated and identified by Atwa (2003). Method of Dutky et al. (1964) was used for cultured EPNs on last instar larvae of the greater wax moth, Galleria mellonella L. (Lepidoptera: Pyralidae). White trap technique as described by White (1927) was used for harvesting nematodes progeny (infective juveniles "IJs") at 25±2 o C. A stock suspension of the IJs in sterilized water was stored at 10 o C for 2 weeks until used. ...
Spodoptera littoralis (Boisd.) is a major plant pest that causes substantial economic losses worldwide especially in vegetable crops. Entomopathogenic nematodes (EPNs) in the families Steinernematidae and Heterorhabditidae are generally considered beneficial organisms which can serve within integrated pest management (IPM) in agroecosystem. The effect of EPNs Steinernema sp. (EBN1e), and Heterorhabdities bacteriophora (EBN10k) that exposed to 11 different chemical pesticides on the cotton leafworm larvae was determined under laboratory condition. Generally, Steinernema sp. (EBN1e strain) exposed to different tested chemical pesticides was more affected on fifth instar larvae of S. littoralis than H. bacteriophora (EBN10k strain). More than 90% of fifth instar larvae of S. littoralis was killed after exposure to EBN1e strain treated with all used chemical pesticides except EPN1e strain treated with chlorfluazuron, thiocyclam and benomyl which caused 75.45, 80.65 and 81.5 % mortality of cotton leafworm fifth larvae respectively. In otherness, the mortality of fifth instar larvae of S. littoralis required to Heterorahbdities strain was less than Steinernema strain treated with different tested chemicals insecticides. There were significant differences in mortality of S. littoralis fifth instar larvae between EBN1e and EBN10k nematodes strains exposed to different chemical insecticides. The EBN1e strain was highly virulent than EBN10k in all treatments. In general, there was significant difference in mortality rates of cotton leafworm larvae required to EPNs concentrations (500IJs and 1000IJs) exposed to different chemicals and between exposure times.
... Test nematode Heterorhabditis bacteriosphora was multiplied in vivo on greater wax moth, Galleria mellonella (Linn.) larvae at 25 o C, using the method described by Dutky et al. (1964). The infective juveniles (IJs) of the nematode were stored in tissue culture flasks (100 mL capacity) at 10 o C before use. ...
Entotomopathogenic nematode, Heterorhabittis bacteriophora possess tremendous potential for biological control of Plutella xylostella (L.) commonly known as the diamond back moth (DBM), major pest of cabbage and cauliflower. Five parameters viz., incubation dose, host body weight, incubation temperature, host food plant and host feeding status were taken to determine their effect on the propagation of H. bacteriophora in final instar of P. xylostella. The number of infective juveniles (IJs) produced increased with the increase in the inoculation dose up to 15 (IJs) per larva. Further increase in dose adversely affected the nematode progeny production because of overcrowding. A positive correlation between host body weight and number of IJs/mg body weight was observed (r=0.8343). Optima temperature for H. bacteriophora was found to be 25oC and the range was 15oC to 35oC. There was no significant difference in nematode progeny production and host food plant viz., cabbage, cauliflower, knolknol and mustard. Similarly IJs production per mg body weight was not significantly different in starved and fed larvae (host feeding status).
... Hundred larvae were kept in 50 cm diameter petri dish, lined with a filter paper and inoculated with approximately 10,000 IJs of S. feltiae contained in 1 ml of sterilized distilled water. The petri dishes were placed in BOD incubator at 20 °C (Dutky et al., 1964) upto 3 days for incubation and the infected C. cephalonica larvae were taken out from the petri dishes and the cadavers were placed on white traps (White 1927) again in BOD incubator at 20 °C. After 7-10 days, IJs moved from the C. cephalonica cadavers to the water reservoir of white trap were collected in a clean beaker, allowed to settle for one hour and the supernatant was decanted. ...
The reproductive potential of native strain of Steinernema feltiae MN044868 at different concentrations (50, 100, 150 and 200%) was evaluated against 4th and 5th instar larvae of cabbage butterfly (P.brassicae). The results revealed that reproduction rate was highest at concentration of 50 IJs as compared to 100, 150 and 200IJs. Application of S. feltiae MN044868 @ 50 IJs/larvae resulted in the highest progeny production (0.403 x 106 ) in 5th instar larva followed by 100 IJs/larvae (0.390 x 106 ), 150 IJs/larvae(0.320 x 106 ) and 200 IJs/ larvae (0.317 x 106 ) whereas in case of 4th instar larvae, lowest progeny production0.365 x 106 was recorded at 50IJs/larvae, followed by100 IJs/larvae (0.334 x 106 ), 150 IJs/larvae(0.310 x 106 ) and 200 IJs/larvae (0.304 x 106 ), respectively
... Hundred larvae were kept in 50 cm diameter petri dish, lined with a filter paper and inoculated with approximately 10,000 IJs of S. feltiae contained in 1 ml of sterilized distilled water. The petri dishes were placed in BOD incubator at 20 °C (Dutky et al., 1964) upto 3 days for incubation and the infected C. cephalonica larvae were taken out from the petri dishes and the cadavers were placed on white traps (White 1927) again in BOD incubator at 20 °C. After 7-10 days, IJs moved from the C. cephalonica cadavers to the water reservoir of white trap were collected in a clean beaker, allowed to settle for one hour and the supernatant was decanted. ...
The reproductive potential of native strain of Steinernema feltiae MN044868 at different concentrations (50, 100, 150 and 200%) was evaluatedagainst 4 th and 5 th instar larvae of cabbage butterfly (P.brassicae). The results revealed that reproduction rate was highest at concentration of 50 IJs as compared to 100, 150 and 200IJs. Application of S. feltiae MN044868 @ 50 IJs/larvae resulted in the highest progeny production (0.403 x 10 6) in 5 th instar larva followed by 100 IJs/larvae (0.390 x 10 6), 150 IJs/larvae(0.320 x 10 6) and 200 IJs/ larvae (0.317 x 10 6) whereas in case of 4 th instar larvae, lowest progeny production0.365 x 10 6 was recorded at 50IJs/larvae, followed by100 IJs/larvae (0.334 x 10 6), 150 IJs/larvae(0.310 x 10 6) and 200 IJs/larvae (0.304 x 10 6), respectively.
... In the tests, two strains of EPNs, Heterorhabditis Bacteriophora (HP88) and Steinernema carpocapsae (AT4), were obtained from the laboratory of Center nematode, Faculty of Agriculture, Cairo University and were reared in vivo on full-grown larvae of the greater wax moth, G. mellonella. [15]. G. mellonella larvae were grown in an insect rearing laboratory on old bee wax at 28±2 Cº and 65% Relative humidity. ...
... All entomopathogenic nematodes were reared on last instar larvae of Gallaria J. Mod. Res.2 (2023) 18-23 mellonella L. (Lepidoptera: Pyralidade) [17]. Larvae of G. mellonella were reared on old bee wax at 28±2 ºC and relative humidity of 65±5 % in the insect-rearing laboratory. ...
... We used both local [9] and foreign identifiers [10] The tests were repeated 3-3 times. Experimental nematodes were cultured according to the Datka method [11]. Mortality Insect was recorded on days 3, 5, and 7 according to the Abbott method [12]. ...
Hazelnuts occupy one of the most important places in agriculture in Georgia. It accounts for 6.4% of total exports. In recent years, hazelnut crops have declined sharply due to exposure to pests. In the fight against plant pests, it is important to use environmentally friendly biological preparations that are harmless to humans. These are the natural enemies of pests-entomopathogenic nematodes (EPNs). Therefore, the aim of our study was to study nematodofauna in soils with hazelnuts in the regions of Guria, Samegrelo and Kakheti, detect and identify nematodes EPN, and select highly virulent, effective forms against hazelnut pests from identified nematodes. Nematodes identified as a result of investigation of material taken from hazelnut soils belong to 6 orders (Dorilamida, Mononchida, Areolaimida, Enoplida, Tilenchida, Rhabditida), 17 families and 25 genera. Of the 30 identified forms, 11 were identified to species. By way of life, saprobiotic, free-living, parasitic, and entomopathogenic nematodes were identified. Experiments on test insects (Galleria mellonella and Tenebrio molitor) in the laboratory showed that of the order Rhabditida: genus Steinernema sp., Parasitorhabditis sp. and Phasmarabditis sp. nematodes, are pathogenic. We used these EPNs against hazelnut pests-Halyomorpha halys, Myzocallis coryli and Lymantria dispar. According to the results of experiments, the most effective of the pathogenic forms we identified against pests was Steinernema sp., His action resulted in mortality of H. halys-94.6% ± 2.33, M. coryli-72.2% ±1.00 and L. dispar-98.4% ± 2.00.
... The reproduction rate of IJs varied among different stages of pest and nematode species (Boff et al., 2000;Shapiro-Ilan et al., 2016). In general, IJ yield per cadaver tends to increase with increasing host size (Dutky et al., 1964;Boff et al., 2000), while IJ yield per unit body weight didn't show that tendency (Karunakar et al., 2000). In current study, the highest IJ yield per unit body weight was found from 3rd instar larvae, while it decreased from 6th instar larvae and the lest was found from pupae. ...
... Several writers have published on how to cultivate entomopathogenic nematodes in insect hosts (Dutky et al., 1964, Poinar 1979; Kaya and Stock, 1997). In vivo production is a straightforward procedure that uses trays, shelves, and a surrogate host-typically the larvae of instar of the greater wax moth, Galleria mellonella, is the most frequently used insect host for in vivo laboratory and commercial entomopathogenic nematode production due to its high susceptibility to most nematodes, ease of rearing, widespread availability, and capacity to produce high yields (Woodring and Kaya, 1988). ...
... Los resultados mostraron que por cada miligramo de larva de P. vorax se obtuvo 1073 IJs para el aislamiento CC01, 1 078 IJs para el aislamiento CC03. Dutky et al., (1964) obtuvieron 1 110 IJs por mg de larva de G. mellonella. Wang et al., (1995) reportaron una producción de 3 091 IJs de S. carpocapsae por mg de larva de G. mellonella inoculados con 0 IJs. ...
RESUMEN El objetivo de esta investigación fue la caracterización etológica de las poblaciones de nematodos entomopatógenos del género Heterorhabditis como candidatos para el control biológico del gusano blanco (Premnotrypes vorax), plaga de importancia económica en los cultivos de la papa (Solanum tuberosum). Se evaluó el potencial reproductivo, la capacidad de desplazamiento, la humedad relativa y el rango de hospederos sobre larvas del V instar de P. vorax. En el potencial de reproducción determinó que una larva de P. vorax con un peso promedio de 62 mg puede producir 66,5x10 3 IJs del aislamiento CC01 y 6,5x10 3 IJs del aislamiento CC03. Se estableció que el 45,6% de los IJs se desplazaron hasta 5 cm de profundidad, el 30,6 % hasta 10 cm y el 16,1% hasta 15 cm. La humedad relativa del 15% mostró ser la más óptima para el desplazamiento de los NEPs de los aislamientos CC01 y CC03 con 24,9 IJs/larva de Galleria mellonella. El 11% de humedad presentó un promedio de 13,8 IJs/larva y el 5% de humedad mostró 9,3 IJs/larva; lo que demuestra que a mayor porcentaje de humedad del sustrato ingresan un mayor número de IJs por larva, y lo opuesto, cuando el sustrato tiende a ser más seco inhibe la acción y desplazamiento de los NEPs. Los dos aislamientos mostraron ser altamente patogénicos también para otras plagas como Tecia solanivora y Symestrichema tangolias.
... bacteriophora juveniles around a million were harvested while hosts infected by S. carpocapsae gave numbers which are less 4 folds than H. bacteriophora (Tourtois et al., 2017). In vivo production of S. carpocapsae and H. bacteriophora on G. mellonella is approximately 200,000 IJs per host (Dutky et al., 1964;Selvan et al., 1993 andFlanders et al., 1996). EPNs were applied in an aqueous solution. ...
... Both species were reared in vivo on the full-grown larvae of the greater wax moth, G. mellonella. A Rearing of entomopathogenic nematode using larvae of G. mellonella as a host was performed according to the methods of Dutky et al. (1964). ...
The present study aimed to evaluate the influence of temperature on the ability of two entomopathogenic nematodes (EPNs), Heterorhabditis bacteriophora HP88 and Steinernema feltiae (Filipjev) California, to infect Ceratitis capitata (full-grown larvae and one and three days old pupae) under laboratory conditions. Results indicated that the temperature and EPNs concentration had a significant effect on the efficacy of nematode species. Full-grown larvae had the highest sensitivity compared to one and three days old pupae. The mortality percentages in the tested stages of C. capitata were increased as the concentration increased. The ability of H. bacteriophora was higher than of S. feltiae at all tested concentrations and temperatures. There were significant differences in terms of the number of IJs extracted from full-grown larvae and pupae of different treatments. Also, H. bacteriophora was the most reproductive, in vivo of full-grown larvae and pupae of C. capitata, at all tested concentrations. H. bacteriophora was superior to S. feltiae in reaching and killing the target host of C. capitata. Generally, H. bacteriophora had more virulent, high production and more ability to host finding than S. feltiae at all tested temperatures. Thus, the success of entomopathogenic nematode, H. bacteriophora as biological control agent against Ceratitis capitata seemes to be suitable for use in integrated pest control strategies.
... bacteriophora juveniles around a million were harvested while hosts infected by S. carpocapsae gave numbers which are less 4 folds than H. bacteriophora (Tourtois et al., 2017). In vivo production of S. carpocapsae and H. bacteriophora on G. mellonella is approximately 200,000 IJs per host (Dutky et al., 1964;Selvan et al., 1993 andFlanders et al., 1996). EPNs were applied in an aqueous solution. ...
... Heterorhabditis bacteriophora, isolated by Poinar (1975) and S. glaseri, isolated by Stuart and Gaugler, (1994). Both nematode species and strains were reared in vivo on the full-grown larvae of the greater wax moth, Galleria mellonella Linnaeus (Lepidoptera: Pyralidadae), following the methods of Dutky et al. (1964). ...
Application of entomopathogenic nematodes to control larvae of Temnorhynchus baal (Reiche and Saulcy) under laboratory conditions and in strawberry fields
Dalia Adly, Gehan M. Nouh and Aziza Eid Eid
Biological Control Department, Plant Protection Research Institute, Agricultural Research Center, Giza, Egypt.
Abstract
The white grub, Temnorhynchus baal, is a major pest of the strawberry crop in many Asian and African countries. The white grabs cause significant economic losses in the yield. The present study aims to evaluate the effectiveness of entomopathogenic nematodes (EPNs) on the third larval instar of T. baal under laboratory conditions. The study extended to compare the efficacy of a combination of EPNs (Steinernema glaseri+ Heterorhabditis bacteriophora) against a natural infestation of T. baal with an organophosphorus insecticide (Diazinon) in an Egyptian strawberry field. In the laboratory, five larvae of T. baal were placed in soil and infected with EPNs. At the highest concentration of 4000 IJs/larva, the larvae were more susceptible to infection with S. glaseri than H. bacteriophora, with mortality percentages of 96 and 88%, respectively. The LC50 of S. glaseri and H. bacteriophora was 937.44 and 1026.58 IJs/larvae, respectively. The mortality percentage was 96% and 100% when treated for five larvae/cup and one larva/cup, respectively, after being infected with a combination of S. glaseri+ H. bacteriophora. The mortality percentage was higher in the mixture than for each species studied individually. In the field, throughout the seasons, the percentage of wilted plants was 17.64, 66.09, and 83.95% in 2020 and 12.56, 67.87, and 75.62% in 2021, for the plots treated with a combination of EPNs, insecticides, and control, respectively. Present findings indicate that entomopathogenic nematodes are good alternatives to control the white grub, T. baal, in strawberry fields
... The entomopathogenic nematodes, Steinernema carpocapsae A11 strain and Heterorhabditis bacteriophora NC strain were reared in the laboratory of Plant Protection Department, Faculty of Agriculture, Minia University, on the host Galleria mellonella according to Dutky et al. (1964). ...
Date palms were applied under Aswan field condition in the fruiting season of 2017 with the
two entomopathogenic nematodes (EPNs), Steinernema carpocapsae and Heterorhabditis
bacteriophora against palm scarab beetles Phyllognathus excavates, the red palm weevil
Rhynchophorus ferrugineus and the frond palm borer Phonapate frontalis (Coleoptera ) as
well as the lesser date moth Batrachedra amydraula, date moth Ephestia calidella and the
pomegranate butterfly Viracola livia (Lepidoptera). Reduction in the infestation with
Phyllognathus excavates caused by S. carpocapsae and H. bacteriophora at the concentration
of 4000 IJs/ml after eight weeks were100 and 70.0 %, respectively. It was clear that S.
carpocapsae influence increased with the passing of the time in opposite to H. bacteriophora.
As for Rhynchophorus ferrugineus reductions in the infestation caused by S. carpocapsae and
H. bacteriophora at the concentration of 4000 IJs / ml after eight weeks were 91.0 and 34.2
%, respectively. It was also clear that S. carpocapsae influence increased gradually in the
course of the investigation time in opposite to H. bacteriophora. The tested EPNs S.
carpocapsae and H. bacteriophora reduced the infestation with Phonapate frontalis by 84.7
and 39.9%, respectively at the end of the investigation course (8 weeks after treatment). The
tested EPN S. carpocapsae and H. bacteriophora behaved with Lepidoptera insects
oppositely with Coleopteran whereas H. bacteriophora surpassed S. carpocapsae in reducing
infestation with Lepidoptera insects except with Ephestia calidella. For example, after eight
weeks from the application with 4000 IJs /ml of H. bacteriophora the reduction in infestation
with Batrachedra amydraula, Ephestia calidella and Viracola livia were 85.5, 79.9 and
78.2% opposite to 81.8, 89.4 and 70.4 % reduction caused by S. carpocapsae at the
concentration of 4000 IJs/ ml. with these insects, respectively.
Keywords: Date palm, insect pests, entomopathogenic nematodes (EPNs)
*Corresponding author: drhassanmhassan2000@yahoo.com
... IJ concentration-dependent factors also play an important role in entomopathogenic nematode production and can act directly by affecting the number of IJs produced by infected cadavers, or indirectly by influencing the longevity of juveniles (Selvan et al., 1993;Zervos et al., 1991). According to Poinar (1979), the nematodes production in the larvae of G. mellonella ranges from between 30,000 to 50,000 IJs/larvae but can also reach 2 × 10 5 (Dutky et al., 1964;Gaugler and RiChou, 2002). These values are comparable to those obtained for the average production of H. amazonensis (UEL 08) in G. mellonella, thereby indicating the potential utility of this isolate in pest control programs. ...
Entomopathogenic nematodes (EPNs) from Heterorhabditidae and Steinernematidae families are extensively used to control insect pests. In Brazil, however, relatively few studies have identified and characterized these entomopathogens. The objective of this study was to identify and characterize an EPN isolate obtained from soil samples collected in the state of Paraná, Brazil. An isolate (UEL 08) of Heterorhabditis was detected in a soil sample collected from a pasture area cultivated with Brachiaria grass in Londrina, state of Paraná, Brazil (23°34'311''S, 050°58'298''W), using the insect-baiting technique with Galleria mellonella larvae as hosts. The nematode was identified through morphometric studies and molecular analyses based on amplification of the rDNA ITS region. Although we identified certain morphometric differences compared with the original description, the molecular data indicated that the ITS sequence obtained for the UEL 08 isolate is identical to the reference sequence of H. amazonensis (DQ665222) and presented 100% similarity. Thus, the findings of our morphological and molecular studies confirmed that the isolated nematode is H. amazonensis, which is the first time this species has been registered in Paraná. Study of the biological characteristics of H. amazonensis (UEL 08) revealed that the isolate has two distinct life cycles - one short (216 h) and the other long (288 h) - and produces two generations in both cycles. We observed that H. amazonensis (UEL 8) was pathogenic and virulent to the three evaluated hosts, although with different virulence against these hosts. The larvae of G. mellonella and Alphitobius diaperinus were more susceptible than adult Dichelops (Diacereus) melacanthus, with 100%, 85%, and 46% mortality, respectively. Furthermore, an in vivo production assay revealed a mean daily yield of 3.4 × 103 infective juveniles/g host larvae.
... Meanwhile, S. carpocapsae Weiser was isolated by Weiser (1955). Nematode species were cultivated in vivo on the full-grown larvae of the greater wax moth Galleria mellonella Linnaeus (Lepidoptera: Pyralidae), according to the methods of (Dutky et al., 1964). Larvae of G. mellonella were collected from infested beehives and raised, following the technique of (Birah et al., 2008). ...
Laboratory Valuation of The Efficacy of Entomopathogenic Nematodes Against Some Insect Pests of the Potato crop (Solanum tuberosum L.)
Gehan M. Nouh
Biological Control Dept., Plant Protection Research Institute, Agriculture Research Center, Giza, Egypt
ABSTRACT
The potato crop (Solanum tuberosum L.) is one of Egypt's most fundamental food crops and is global. It is an onslaught by many insect pests that as the potato tuber moth, Phthorimaea operculella (Zeller), the hard black beetle, Pentodon bispinosus Kuster), and the Egyptian cotton leafworm, Spodoptera littoralis (Boisduval). The current research aims to study the efficacy of entomopathogenic nematodes (EPNs); Heterorhabditis bacteriophora, Steinernema glaseri, and Steinernema carpocapsae, against some major pest species that infest the potato crop. The experiment of P. operculella 4th larval instars and pupae treated with H. bacteriophora and S. carpocapsae gave the highest mortality compared with S. glaseri. The LC50 values of H. bacteriophora and S. carpocapsae were calculated in the 4th instar larvae of P. operculella as 127.1496 and 148.5994 IJs/larva, respectively. While the LC50 value of S. glaseri recorded, was 247.2687 IJs/larva. Treatment of P. bispinosus larvae S. glaseri showed the highest efficacy against the 2nd and 3rd instars larvae of P. bispinosus compared with H. bacteriophora and S. carpocapsae. The LC50 values of S. glaseri were 1073.640 and 1330.312 IJs/larva to combat the 2nd and 3rd instars larvae of P. bispinosus. In the experiment of S. littoralis on 3rd and 5th instar larvae, the H. bacteriophora and S. carpocapsae showed higher mortality than S. glaseri. LC50 values of H. bacteriophora and S. carpocapsae were (38.489 and 40.791) and (42.149 and 51.932) IJs/larva in the 3rd and 5th instar larvae of S. littoralis, respectively. While in the case of S. glaseri, correspondent LC50 values recorded were 70.106 and 95.295 IJs/larva. H. bacteriophora recorded the most elevated mortality in (the 4th larval instars and pupae of P. operculella) and (the 3rd and 5th instar larvae of S. littoralis), followed by S. carpocapsae. S. glaseri recorded the highest mortality (100%) of 2nd and 3rd instars larvae of P. bispinosus compared with H. bacteriophora and S. carpocapsae. The results also concluded that S. littoralis was the most sensitive to the use of EPNs, followed by P. operculella and then P. bispinosus. EPNs can be relied on as successful biocontrol agents against soil-dwelling pests and add nematodes to pest control programs.
... For in vivo production of bio-control agents, though it needs less investment and expertise, it is difficult to scale up the production, and hence achieving economic viability may be difficult [35]. Usually, the in vivo mass rearing of EPNs is carried out by using the White trap method devised in 1927 by White and later it was modified and reconstructed [36]. genera by this method with the advantage of harvesting nematodes only in their infective forms in relatively less contamination. ...
Entomopathogenic nematodes (EPNs) are recognized as one of the effective bio-control agents of insects that are harmful to many agricultural crops. The two EPNs genera, Steinernema and Heterorabditis, are widely used nematodes. Insect pests infesting different agricultural crops and other plants, can be controlled using these nematodes. These beneficial EPNs can be mass produced using both in vivo, baiting using insects, and in vitro techniques, using solid or liquid fermentation. For laboratory trials, greenhouse experiments, or limited agricultural field trials, in vivo production of EPNs is most appropriate. As EPNs have a crucial part in the management of numerous insect pests of field crops, stored products/grains, mushroom culture, etc., they are valuable as eco-friendly products. Our focus in this paper is to present the various procedures and components involved in the in vivo mass production of EPNs and to provide a summary of factors that affect the in vivo mass production.
... The culture of both the nematodes was maintained in the laboratory at the Department of Agriculture, University of Reading, UK and subcultured on last instar larvae of Galleria mellonella (Lepidoptera: Pyralidae) at 25 °C supplied by Livefoods Direct Ltd. Sheffield, UK following the method described Dutky (Dutky et al., 1964). Only one week old infective juveniles of S. feltiae and H. bacteriophora were selected and used in the experiments. ...
The terrestrial activities of entomopathogenic nematodes like movement, infection, development, and survival are influenced by a number of abiotic factors and among these soil moisture and temperature are regarded as the most important. Therefore, in the present studies the emergence of Steinernema feltiae from infected Galleria mellonella cadavers were evaluated in moist and dry conditions at different temperatures and the effects of cool storage on the recovery and duration of recovery of infective juveniles (IJ) from cadavers were also investigated. The results showed highly significant results regarding emergence of S. feltiae from infected G. mellonella under moist and dry regimes. A total of 154,456 IJ recovered per Galleria on moist sand as compared to 11,551 when placed on dry sand. Similarly, greater numbers of IJ recovered from the 10°C treatment than 5 °C. The total IJ recovered from cadavers at 5 °C were 292,314 and at 10°C were 381,135. The relationship between both the temperatures under wet and dry conditions was highly significant (P0.001). The number of IJ emerging from wet at 5 °C was 50,029 and from wet at 10 °C were 81,674. Under dry conditions, 40,892 IJ recovered from 5 °C and 41,260 from 10 °C. There was no significant overall effect of insect host on the numbers of active nematodes recovered from the cadavers. More IJ recovered from G. mellonella cadavers than T. molitor stored at 5 °Cbut this trend was reversed at 20 °C.
... Heterorhabditis heliothidis is a nematode species of genus Heterorhabditis described by Khan, et al., [28]. It is a parasite of insects such as the Colorado potato beetle.Heterorhabditis heliothidis, was cultured on the larvae of greater wax moth Galleria mellonella [29]. H. heliothidis is applied by conventional spray techniques and activelypenetrate both feeding and non feeding stages of susceptible insects. ...
Insect pests are always become a problem to man since they started cultivation of the crops. They cause economic losses to our commodities in different ways. But their control becomes a major problem, though it developed resistance against many insecticides. However, a variety of problem sare associated with the traditional use of non selective insecticides which include environmental issues, negative impact on nature enemies, food safety and hazards to human health. Therefore, use of other alternatives like microbesis being more conventional in the present scenario. Moreover, microbial control of insects can concern with the use of insect-specific pathogens viz., virus, bacteria, protozoa, fungi and nematodes, which are capable for killing the insect pests. Generally they are attacking to the targets and reduce the pest population below economics without disturbing the ecosystem and biodiversity. Use of microbial pesticides has number of advantages over chemical pesticides and also helps to maintain equilibrium of our natural ecosystem. The demand of microbial pesticides increased drastically since last two decades in the global market, which can become possible only due to awareness among the farmers.
... In vivo production is based on the White's trap method, which involves the natural migration of infective juveniles (IJs) from the infected host cadaver into a surrounding water layer, from where it can be harvested. White (1927) invented a method, later it was modified by several researchers (Dutky et al. 1964, Poinar, 1979, Woodring and Kaya 1988, Lindegren et al. 1993, Abdel-Razek and Abd-elgawad 2007. The selection of the insect host totally depends on the susceptibility of a particular host for the nematode infestation. ...
Free-living nematode Acrobeloides longiuterus (Rhabditida: Cephalobidae) exhibits a potential to kill some insect pests. Mass production of this species is a requirement for use it in pest management programs. Tribolium castaneum has been used as a primary host for this nematode as an alternative for Galleria mellonella. Use of artificial media is another option for mass culturing and such recipes based on soy flour are available. Production of A. longiuterus using cost effective method and easily available insect host is important in setting up of small-scale production unit. Therefore, this study has the objectives of evaluating the production feasibility of A. longiuterus on T. castaneum larvae, pupae and adults as in vivo production method. Further, feasibility of using different solid media such as soy flour, palmyra tuber flour, corn flour, black gram flour and dhal flour with other basic ingredients as in vitro conditions system was evaluated. Results revealed that pupa of T. castaneum yielded the highest number of infective juveniles (36112 IJs/ pupa) compared to other life stages tested. In vitro production of A. longiuterus on soy flour and black gram flour media yielded 21530 and 16538 IJs/20g, respectively. Pathogenicity against T. castaneum was shown up to 93% by the infective juveniles produced from the in vitro cultures. In conclusion, T. castaneum is an alternative insect that can be used as a host to produce the A. longiuterus. In addition, soy flour and black gram flour can be used as the sources for this nematode production without losing their entomopathogenicity.
The use of nematodes for biologic control of pests requires efficient and economic technology for mass production of the agent. This work deals with about the production of entomopathogenic nematodes in vitro.
KEY WORDS: Entomopathogenic nematodes; production.
Adult and nymph stages of German cockroaches Blatilla germanica were infested with two entomopathogenic nematodes, Steinernema carpocapsae and Heterorhabditis bacteriophora in vitro at 15, 20, 25o C. Results showed that S. carpocapsae was more virulent than H. bacteriophora at all temperatures over the exposure times of 24, 48 and 72 h. Infection of nymphs by S. carpocapsae at 20o C caused 100% mortality after 72h and 100% mortality of adults at 25 o C after 72h. Reproduction of S. carpocapsae significantly increased at 25o C after 72 h compared to H. bacteriophora.
Entomopathogenic nematodes (EPNs) in the genera Heterorhabditis and Steinernema are obligate parasites of insects in nature. The pathogenicity of these nematodes is facilitated by symbiotic bacteria that are carried in the nematode’s gut prior to entering an insect host. More than a dozen EPN species have been commercialized for use in biological pest suppression. The objective of this chapter is to review and analyze the production methodology for EPNs. EPNs are mass-produced using three different methods: in vivo production, in vitro culture in solid media, and in vitro culture in liquid media; each method has advantages and disadvantages. For example, in vivo production requires the least capital outlay but is lacking in the economy of scale due to costs of labor and insects. In vitro liquid culture (which accounts for the bulk of commercial production) requires the largest capital outlay but generally offers the greatest economic efficiency. In vitro solid culture is intermediate between the other two methods in most aspects. Some advancements can be made to improve production efficiency and biocontrol potential. In vivo culture can be streamlined by producing insects in-house and mechanizing the entire process to reduce labor. In vitro liquid culture can be improved through media optimization and maximizing bioreactor processing. All the production approaches will benefit from advanced strain improvement programs, and mechanisms to stabilize beneficial traits (such as utilization of selected inbred lines). As production efficiency improves the utility of EPNs in biological control will expand.
Entomopathogenic nematodes (EPN) are found in all inhabited continents except Antarctica (no report yet) and a range of ecologically diverse habitats, from cultivated fields to deserts. Steinernema and Heterorhabditis are the well studied genera which belong to the family Steinernematidae and Heterorhabditidae, associated with symbiotic bacteria Xenorhabdus and Photorhabdus respectively. As far as entomopathogenicity is concerned genus Oscheius is less studied, also having ability to kill the host insect due to the mutually associated with efficacious bacterial genera of Pseudomonas, Enterococcus and Serratia. The bacterial complex of these nematodes makes them a prominent mediator for the bio-management of many insect pests. Many studies have proven the active and main involvement of nematode’s bacterial partner by releasing secondary metabolites in causing septicemia and oenocytoids. Globally, both the genera, that is, Steinernema and Heterorhabditis, are represented by 100 and 16 species, respectively, while Oscheius is represented by 45 species, out of which 17 are from the Indian subcontinent. The information on EPN diversity is limited in India. EPN show high potential for plant protection and can play a major role in Integrated Pest Management (IPM) of insects.
India has an agrarian economy, where 1012.4 million are dependent on agricultural commodities from 124.07 million hectares cropped area cultivated by 110.7 million producers and contributed 17%–18% to country’s GDP. Over 85% of rural households depend on agriculture as their principal means of livelihood. Most of the agroproducts are damaged by pests. Farmers together with fertilizers have pesticides as a tool to control pests during the growing season of crops. In India, estimated crop losses due to the pest are worth of ₹6000 crores annually in which Insects contributing ~20%. Problem can easily be assessed viz. Helicoverpa armigera alone losses to the tune of ₹1000 crore in crops like cotton, tomato, pigeon pea, groundnut, sorghum, pearl, millet, and other crops of economic importance. It has been estimated that about ₹1200 crore worth of pesticides were used in India to control the bollworm complex of cotton. The degree of the problem is accelerating and requires a better control program in an environmentally safe manner. To reduce crop losses, tons of pesticides are being used by the farmers. The crop damage is caused highest by insects, followed by pathogens and weeds. Consequently, the use of chemical pesticides in agriculture has been an integral part of crop production in many regions, often at very high levels and unscientific pattern of application. The use-intensity has been found highest in Jammu and Kashmir, followed by Punjab and Haryana. To minimize the use of pesticides, it is required to find out such control measures that could reduce not only the application of huge quantity of the pesticides but also could be ecofriendly. Two of the prominent nemic genera, that is, Steinernema and Heterorhabditis having interaction with insects and are considered globally as highly pathogenic to insects. Such nematodes are termed as Entomopathogenic nematodes (EPN) which can be used as biopesticide against the insect pests.
EPN are everywhere, they have been found in all inhabited continents except Antarctica (no report yet) and a range of ecologically diverse habitats, from cultivated fields to deserts. Steinernema and Heterorhabditis are the most commonly studied genera that are in use to control the insect pests by causing host mortality within 48 h. The mass production and application of EPN is easy. There is a tremendous scope to train the marginal farmers for the mass production of EPN and thereby producing large quantity of EPN at their own. Thus the farmers could be able to use of EPN isolates against the specific insect pest and the use of chemical pesticides could be reduced. The expenditure incurred on pesticides would be reduced and in turn the income of the marginal farmers can automatically be increased.
In an agricultural system, to increase natural biological control, plants that attract natural enemies can be grown alongside the main crop. However, the effects of these plants on entomopathogenic nematodes (EPNs), important agents for controlling soil pests, and the action of their conservation are unknown. To assess the impact of these plants on EPNs, two experiments were carried out in a greenhouse. The first measured the effect of Crotalaria spectabilis, Crotalaria breviflora, and Tagetes erecta on the persistence and infectivity of Heterorhabditis amazonensis isolate RSC 5 for 27 days, compared to a control treatment without plants. The second trial evaluated the effect of C. breviflora and T. erecta on the displacement of the nematode. Additionally, the influence of predator Calosoma granulatum in this system was evaluated. The plants did not influence nematode behaviour in terms of persistence, infectivity, or displacement. However, C. spectabilis allowed the most significant persistence of nematodes in the substrate for a short time, and T. erecta caused the fastest suppression of the initial population of infectives juvenile. In the second experiment, neither the predator nor the plants affected the nematode’s ability to move in the soil within 5 days. These results show that prior knowledge in agricultural diversification can help to control pests by inundative application of EPNs.
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