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Capsules containing entomopathogenic nematodes as a Trojan horse approach to control the western corn rootworm

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Aims The use of entomopathogenic nematodes in the biological control of soil insect pests is hampered by the costly and inadequate application techniques. As a possible solution we evaluated a nematode encapsulation approach that offers effective application and may possibly attract the pest by adding attractants to the capsule shell. Methods Heterorhabditis bacteriophora nematodes, which show high virulence against the maize root pest Diabrotica virgifera virgifera, were encapsulated in a polysaccharide shell derived from the algae Laminaria ssp. Shells of varying thickness and composition were evaluated. Results Nematodes readily survived the encapsulation process and were able, varying with shell thickness and temperature, to break through the shell and subsequently infect hosts. The added attractants and feeding stimulants to the shell attracted the pest larvae as much as maize roots. In field trials, encapsulated H. bacteriophora nematodes were more effective in controlling D. v. virgifera than those sprayed in water over the soil surface, but in these trials the addition of stimulants did not increase the control efficiency. Conclusions The study demonstrates that nematodes can be successfully applied in capsules in the field. Further improvements are needed to make the capsules a cost effective alternative to conventional field application of nematodes.
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... Kaya and Nelsen (1985) were the first to encapsulate Heterorhabditis bacteriophora and Steinernema feltiae in calcium alginate beads. Progressively, several studies reported on the fight against the western corn rootworm Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) with EPNs formulations (Hiltpold et al., 2012;Kim et al., 2014;Jaffuel et al., 2020;Kim et al., 2021). The treatments protected maize plants from D. v. virgifera damage, when applied at right time of the season. ...
... The protocol of the alginate beads was adapted from Hiltpold et al. (2012). We previously determined that a maximum of 10% of the potato extract could be added without significantly changing bead shape and structure. ...
... The longer lasting EPNs activity may also be explained by the properties of the bead system itself. For instance, tests on the use of capsules containing H. bacteriophora in the field against rootworms have shown that the IJs had a high survival rate in the capsules and that this formulation was more effective than water sprays (Hiltpold et al., 2012;Kim et al., 2021). ...
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Wireworms are polyphagous soil-dwelling pests that are hard to control. Attract-and-kill strategies, combining attractive semiochemicals with biocontrol agents, have great promise to control insect pests. We hypothesized that the combination of plant semiochemicals and entomopathogenic nematodes (EPNs) in an attract-and-kill system could greatly enhance the nematodes’ efficiency against wireworms. We evaluated the potential of alginate beads loaded with plant extracts and EPNs to control Agriotes obscurus. We tested the efficiency to kill wireworms or to reduce their feeding activity when combining potato tuber extracts as attractants with any of seven different EPN populations. While a direct application of EPNs on wireworms did not reduce the feeding activity nor increased their mortality, the combination of attractants and EPNs encapsulated in alginate beads resulted in attraction and consumption of the EPNs and caused up to 50% wireworm mortality with the EPN species Steinernema carpocapsae. Beads with EPNs caused a significant reduction of the wireworms’ feeding activity. This study shows that wireworms feeding on EPN-containing beads have their feeding activity and survival negatively affected. Considering their long developmental time and the survival capability of EPNs in the soil, implementing this attract-and-kill system in the field might be a suitable strategy for the long-term management of wireworms.
... EPN formulations are usually prepared using either solid or semi-liquid substrate (Jaffuel et al. 2020). These formulations are made based on the addition of antidesiccants to keep the gel high in moisture level (Jaffuel et al. 2020;Hiltpold et al. 2012). In formulation, addition of yeast can be useful in attraction of mosquitoes; the yeast consumes sugar and releases CO 2 which plays a significant role in hostseeking process of mosquitoes (Smallegange et al. 2010). ...
... Studies carpocapsae with calcium alginate gel showed survival value of higher than 50%, up to 40 days (Hussein and Abdel-Aty, 2012). Addition of xanthan gum increased the viscosity of the solution in the alginate bead formulation (Kapranas et al. 2020;Hiltpold et al. 2012). In the present study, by using Arabic gum with sodium alginate gel formulation, S. carpocapsae and R. blumi showed longevity of up to 12 weeks, while 96% survival was observed up to 3 weeks at different concentrations of IJs. ...
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Entomopathogenic nematodes (EPNs) are effective biopesticides used in insect control. There are several formulations of entomopathogenic nematodes which are commercially available, but their popularity is low because the requirement of periodic release of stored entomopathogenic nematodes in field has reduced infectivity and is a constraint in large-scale repeated application of entomopathogenic nematodes in field. So, in order to overcome this impediment, the current study focuses on storage of entomopathogenic nematodes for a longer period of time using encapsulating beads. In this study, three EPNs, namely, Steinernema carpocapsae, Steinernema monticolum, and Rhabditis blumi, were encapsulated with Arabic gum and sodium alginate gel beads at different concentrations. Bio-efficacy of stored EPNs at different concentrations (50, 100, 200, 500, and 1000 infective juveniles (IJs)) in varying time intervals of 3, 6, 9, and 12 weeks were tested against Culex quinquefasciatus larvae. The results show that, after 3 weeks of storage, these entomopathogenic nematode species showed survival up to 96%, and in 12-week storage, survival rate was 50%. The virulence of the progeny that emerged from these stored entomopathogenic nematodes was similar to the fresh infective juveniles. The results of the present study show that Arabic gum–sodium alginate combination is a promising approach toward storing entomopathogenic nematodes. Furthermore, semi-field trials showed 70–90% larval mortality at a concentration of 3000 IJs (encapsulated and stored entomopathogenic nematodes in S. monticolum, S. carpocapsae, and R. blumi) on C. quinquefasciatus larvae. Key points • Increased survival of stored EPNs (S. carpocapsae, S. monticolum, and R. blumi) in encapsulated gel using Arabic gum, sodium alginate, and yeast cells was assessed. • The efficacy was greater in stored EPNs at different concentrations from 3 to 12 weeks. • Semi-field trials of stored encapsulated EPNs are efficient in mosquito larval control.
... The first attempt at EPN encapsulation was made by Kaya and Nelsen (1985), whereby the alginate EPN solution was dropped into the CaCl 2 solution, resulting in the immediate formation of capsules of about 1.5 mm diameter, with the nematodes being immobilised on the inside of the solid core. However, the nematodes reportedly can escape from the beads when they are too soft (Hiltpold et al., 2012;Kim et al., 2015). In an experiment by Kim et al. (2015), the IJs escaped from the soft beads within a few days, especially when the formulation was not refrigerated. ...
... Ideally, the viability of the IJs should be maintained for a couple of months, with the EPNs entrapped inside the capsule or bead until required for use (Kim et al., 2015). Similarly, Hiltpold et al. (2012) reported that the IJs escaped from the soft capsules a few days after formulation, which proved that the capsule properties needed adjustment to improve the entrapment and to achieve extended shelf life for the EPNs. Kagimu and Malan (2019) also reported an increase in the number of IJs escaping from the beads as the storage temperature increased. ...
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Entomopathogenic nematodes (EPNs) are insect parasites that are used successfully as biological controlagents against key pest insects of grapevine. To achieve low chemical residues and the sustainableproduction of grapes, it is important that biological control agents such as entomopathogenic nematodesfor the control of grapevine insect pests be incorporated in an integrated pest management system forgrape production. However, the commercialisation and large-scale use of EPNs is limited by their shortshelf life in formulations and in storage, thus leading to poor quality and reduced efficacy against insectsin the field. In South Africa, interest in the use of EPNs within an integrated pest management system hasgrown over the past two decades, therefore developing a formulation technique with an acceptable storagesurvival period, while maintaining infectivity, is essential. Moreover, the successful control of insects usingEPNs is only achievable when the formulated product reaches the end user in good condition. This reviewis focused on the different types of formulations required for storage and ease of transport, together withthe application formulation for above-ground pests and the factors affecting them. The quality assessment,storage and handling of formulated EPNs are also discussed.
... Several reports described Ca 2+ alginate-based EPN formulations (Kaya & Nelsen, 1985;Kaya et al., 1987;Navon et al., 1998Navon et al., , 2002Chen and Glazer 2005;Hiltpold et al., 2012;Kim et al., 2015Kim et al., , 2021Ruiz-Vega et al., 2018;Kagimu & Malan, 2019). Chen and Glazer (2005) formulated Steinernema feltiae IJs in alginate-based capsules, which were exposed to osmotic treatment before capsule formation, resulting in 99% survival during six months at 23°C and 100% RH. ...
Article
Entomopathogenic nematodes (EPNs) are effective biocontrol agents. Extending the shelf-life and storage at room temperature, along with maintaining their pathogenicity, are the most important goals of EPNs formulation. Alginate is a natural anionic polysaccharide utilised in manufacturing plant protection products. In the present study, two types of alginate-based capsules (containing 0.5% sodium alginate and 2% CaCl2·2H2O (w/v), and containing 2% sodium alginate and 0.5% CaCl2·2H2O w/v) were used for the formulation of infective juveniles (IJs) of Steinernema carpocapsae IRMoghan1. Additionally, the efficacy of the manufactured capsules against Mythimna loreyi Duponchel was evaluated. Based on the results, the survival rate of the nematodes was not significantly different among the treatments (including the two capsule types and water-suspended IJs) at 60 days after capsule formation; however, water-suspended IJs showed a significantly lower survival rate than the IJs extracted from both capsule types. The IJs extracted from the capsules containing 0.5% sodium alginate (w/v) and 2% CaCl2·2H2O (w/v) exhibited the highest and the water-suspended IJs showed the lowest pathogenicity against larvae of Galleria mellonella. Application of the alginate-based capsules manufactured with 0.5% sodium alginate (w/v) and 2% CaCl2·2H2O (w/v) against M. loreyi prepupae in the soil (at a dose of 800 IJs per insect) caused 75% insect mortality. Appropriate survival rate (74% for 120 days post-formulation at 24°C) and maintenance of pathogenicity of the nematodes over 120 days-period and effective application of the alginate-based capsules against M. loreyi is a promising successful prospect for the use of this formulation against soil pests.
... In order to protect nematodes from biotic and abiotic stress, significant improvements have been achieved. For that purpose, different encapsulation methods, i.e., alginate-based entomopathogenic nematode capsules, provide an improvement of their efficacy [73,74]. ...
... EPNs kept in the calcium alginate granules described by Chen and Glazer (2005) could still cause a 100% host insect mortality after storage for a 6 month period. Additionally, EPNs in alginate gel formulations were reported for having high infectivity against Corcyra cephalonica (Umamaheswari et al. 2006) and Diabrotica virgifera virgifera (Hiltpold et al. 2012). Moreover, Kim et al. (2015) produced hard capsules by adjusting the capsule properties in order to increase the release time of the nematodes. ...
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Background Entomopathogenic nematodes (EPNs) have long been used for controlling soil-dwelling insects. Steinernema carpocapsae HB310, previously showed a high virulence against many pests including Agrotis ipsilon Hufnagel (Lepidoptera: Noctuidae). Due to the lack of durable formulations, up until now, S. carpocapsae HB310 has thus far been prevented from use in large-scale farming. The present study aimed to get a better EPNs capsule formulation suitable for long-term storage and effective application. Results An improved EPNs capsule formulation, herein named: Capsule-C was prepared by the following composition: Solution I: 18% glycerol, 0.075% formaldehyde, 1% sodium alginate, 0.2% xanthan gum, 0.5% potassium sorbate, 9% glucose, 2% fructose, 2% sucrose, and the remainder was distilled water. The nematodes suspension was added to the alginate mixture in 2 × 10 ⁴ IJs/mL; Solution II: 18% glycerol, 0.075% formaldehyde, 0.5% calcium chloride, 0.5% potassium sorbate, with the remainder being distilled water. After storage for 180 days at 16 °C and 100% RH, the survival rate of nematodes in Capsule-C was 75.68 ± 0.48% and the nematodes caused 82.33 ± 1.45% mortality in the 5th instar larvae of Galleria mellonella. A. ipsilon larvae preferred to chew and ingest Capsule-C due to the addition of the glucose compound. The feeding rate of A. ipsilon larvae on Capsule-C reached to 100% within 24 h and the larval mortality of A. ipsilon was 90.48 ± 6.35%. Conclusion EPNs-containing capsules were as effective as sprayed EPNs in water solution at killing A. ipsilon. These results will provide ideas to acquire a stable and efficient EPNs capsule formulation and further promote the application of environmental friendly biological pesticides.
... The above finding is reassuring in terms of the implications that it holds for the much-desired room temperature storage of IJs in DE formulation. The improvement in the number of nematodes in DE formulation has undoubtedly influenced the survival of EPNs in terms of the current research (Silver et al. 1995;Hiltpold et al. 2012;Matadamas-Ortiz et al. 2014). ...
Article
The commercial use of entomopathogenic nematodes after mass production requires the development of formulation techniques that extend nematode survival and prevent virulence loss during storage. In this study, the room temperature viability of Steinernema yirgalemense, in diatomaceous earth (DE), was investigated. The shelf life was maintained above 70% mean survival rate by week 4. To avoid microbial contamination, the direct effect of antifungal agents, peroxyacetic acid (PAA), trans-cinnamic acid (TCA) and nipagin on the efficacy of the infective juveniles (IJs) was preliminarily investigated. Short-term exposure of PAA lowered the efficacy of IJs, yet low concentrations of TCA and nipagin had no effect. Finally, the combined effect of DE and nipagin on IJ efficacy was assessed. High concentrations of nipagin in DE killed all the IJs in the first week of storage. Lower concentrations of nipagin in DE have potential to be incorporated in EPN products.
... In citrus, S. riobrave is applied to the soil using tractor-mounted sprayers or through drip irrigation (Duncan et al. 1999), but more targeted application methods used in other cropping systems for other entomopathogenic nematode species may be better suited to or adjusted for small applications in strawberry (Shapiro-Ilan & Dolinski 2015). Here we tested aqueous preparations of S. riobrave applied as a soil drench around the base of individual strawberry plants, but formulation of entomopathogenic nematodes may affect efficacy and ease-of-application, with pelletized baits showing promise in some scenarios (e.g., Hiltpold et al. 2012). ...
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Diaprepes abbreviatus L. (Coleoptera: Curculionidae) is an occasional root pest of plasticulture strawberry in central Florida, USA. There are few chemical insecticide options for larval D. abbreviatus in strawberry. Therefore, we tested soil-applied aqueous Steinernema riobrave Cabanillas, Raulston and Poinar (Rhabditida: Steinernematidae), which is used for D. abbreviatus control in citrus. When S. riobrave was applied (100 infective juveniles per cm2) to the root zones of plants in a D. abbreviatus-affected area of a commercial strawberry field, less than 12% of plants were severely wilted or dead 17 d after treatment, whereas 23% of plants in control plots were wilted or dead. In research plots, peripheral plants and a central plant in each plot were infested with 4 late-instar D. abbreviatus and treated with 1 or 2 applications of S. riobrave (25 infective juveniles per cm2), 1 application of imidacloprid or water (control). Dead S. riobrave-infected D. abbreviatus larvae were recovered from plots to which S. riobrave was applied, but there was no effect of treatment on numbers of live larvae recovered 1, 2, and 3 wk post-application in the peripheral plants. At the end of the experiment, no live D. abbreviatus larvae were recovered from the central plants or the plants proximal to the central plants in plots treated once or twice with S. riobrave. Plant wilting and death due to larval D. abbreviatus root feeding was minimal (averages of 11.4 on a 15 rating scale) in plants proximal and distal to the central D. abbreviatus-infested plant. Treatment did not affect plant wilting and death rates in the proximal plants, but death of distal plants occurred only in control plots. Our results showed S. riobrave infected and killed late-instar D. abbreviatus in plasticulture Florida strawberry, and further research should be conducted to optimize S. riobrave applications and develop it into a management strategy for D. abbreviatus.
... Thus, we confirmed that the Paraconiothyrium strains are able to migrate from Brachiaria plants after desiccation with herbicide, colonize sclerotia and reduce carpogenic germination of S. sclerotiorum. We referred to this mode of action as a Trojan horse approach, as previously reported for other plant-microbe interactions (Hiltpold et al, 2012). In addition, B. ruziziensis may benefit from endophytic colonization of P. estuarinum and P. cyclothyrioides since growth promotion of plants inoculated with these strains was observed. ...
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Endophytic Paraconiothyrium strains associated with Brachiaria spp. and Panicum maximum were screened in vitro and in vivo for antagonism against Sclerotinia sclerotiorum and grass growth promotion. Based on multilocus phylogenetic analyses, three Paraconiothyrium estuarinum strains and two Paraconiothyrium cyclothyrioides strains were identified. In vitro, P. estuarinum strain CML 3699 was the most efficient in S. sclerotiorum mycelial growth inhibition. Volatile organic compounds (VOCs) produced by Paraconiothyrium strains reduced the sclerotia number, sclerotia weight and apothecia number in comparison to the control. The VOCs of P. cyclothyrioides strain CML 3698 promoted higher inhibitory effects on apothecium production. P. estuarinum CML 3695, when inoculated into Brachiaria ruziziensis seeds, promoted increased biomass in comparison to those in non-inoculated seeds. All strains were sensitive to higher glyphosate doses, which reduced their mycelial development. However, when the mycelia of the endophytic fungi were removed and transferred to new PDA medium, the fungi recovered growth. After desiccation of Paraconiothyrium-inoculated B. ruziziensis with glyphosate, Paraconiothyrium strains recovered the ability to colonize the sclerotia of S. sclerotiorum and reduced carpogenic germination via a Trojan-horse-like mode of action. Considering that grass can be deployed by farmers as a physical barrier to S. sclerotiorum ascospore release, the growth promotion and parasitism of sclerotia exerted by Paraconiothyrium strains towards B. ruziziensis reinforce this disease control strategy.
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