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Control of false codling moth, Thaumatotibia leucotreta (Lepidoptera: Tortricidae), using in vitro-cultured Steinernema jeffreyense and S. yirgalemense
Abstract
False codling moth (FCM), Thaumatotibia leucotreta, is a priority pest of citrus, stone fruit and table grapes, as it causes direct crop damage. Biological control of entomopathogenic nematodes (EPNs) has not been explored for use against FCM in stone fruit and table grapes. However, EPNs have been shown to provide exceptional control against the larvae and pupae of FCM in laboratory bioassays, compared to other biocontrol agents. EPNs are particularly attractive for the control of FCM, as they attack the soil stages. In this study, the mortality, quality and age of in vitro liquid-cultured Steinernema jeffreyense was assessed in the laboratory and tested in the field. Additionally, pre- and post-application pathogenicity tests with FCM larvae were conducted to assess the EPN virulence of in vitro-cultured nematodes. Field trials were conducted by means of applying infective juveniles (IJs) to the vineyard floor. Four different Steinernema jeffreyense concentrations (0, 10, 20, and 30 IJs/cm²) were applied to 40 1 m² experimental plots that were artificially infested with FCM larvae. The insects were retrieved from the soil 48 hrs after application, to allow for the assessment of the immediate effect. The FCM-loaded cages were replaced over a period of four weeks, to determine the persistence of the original application. In a second trial, following the same procedure, S. jeffreyense and Steinernema yirgalemense were compared with regard to virulence and persistence over a four-week period. In the laboratory, the in vitro-cultured EPNs proved to be of similar quality to the in vivo-cultured S. jeffreyense, with a high percentage mortality of >80%. The semi-field study showed promising results, with the immediate effect yielding up to 77% mortality of FCM larvae, which remained >35% over the four-week period after application. The results compare favourably with those of previous field studies, using in vivo EPN, proving that EPNs would be a valuable addition to the current integrated pest management programme aimed at the control of FCM.
... Heterorhabditis bacteriophora are very effective against FCM at low concentrations of IJs (50 IJ/insect), as demonstrated in laboratory screening (Malan et al., 2011) and in semi-field trials (Malan & Moore, 2016;Steyn et al., 2019). Large-scale field trails with imported H. bacteriophora confirm their suitability for application in a systems approach to control FCM in South Africa (Moore, 2021;Moore et al., 2024). ...
... Host insect susceptibility to EPN infection might also affect infectivity. FCM are susceptible to several EPNs, including S. yirgalemense, H. zealandica, Steinernema litchi Steyn et al. and Steinernema jeffreyense Malan et al. (Steyn et al., 2017(Steyn et al., , 2019. Laboratory bioassays conducted with such EPN species resulted in high mortality of >80% (Steyn et al., 2017(Steyn et al., , 2019, with such mortality rates all being higher than what was achieved in this study with H. bacteriophora. ...
... FCM are susceptible to several EPNs, including S. yirgalemense, H. zealandica, Steinernema litchi Steyn et al. and Steinernema jeffreyense Malan et al. (Steyn et al., 2017(Steyn et al., , 2019. Laboratory bioassays conducted with such EPN species resulted in high mortality of >80% (Steyn et al., 2017(Steyn et al., , 2019, with such mortality rates all being higher than what was achieved in this study with H. bacteriophora. One explanation for such mortality rates could be that H. bacteriophora might have a lower infectivity rate compared to the other EPN species. ...
The South African table grape industry requires biological control options for pest insects to maintain export relationships. Entomopathogenic nematodes (EPNs) has shown effective biological control of the economically important table grape pest, Thaumatotibia leucotreta, the false codling moth (FCM). Critical to the success of EPNs is their ability to be mass produced using in vitro liquid mass production methods. The selection of suitable isolates for mass production is the most important step in the process of developing an EPN biocontrol product for commercial purposes. Heterorhabditis bacteriophora is the most common EPN species found in South African soils, with previous research having shown its high pathogenicity against a variety of grapevine pest insects. In this study, four local H. bacteriophora isolates were laboratory-screened to select the best candidate in terms of virulence against FCM, and differences in the symbiotic bacteria isolates were also observed. Two of the H. bacteriophora isolates, CRI_LC and LLM’s symbiotic Photorhabdus bacteria, showed slight bioluminescence, while SGI_170 and Px_SPH showed strong luminescence, indicating the biological difference between the symbiotic bacterial species that are associated with the same nematode species. Molecular analysis of the 16S gene indicate three different bacterial species, of which two were the same and two are possibly new. The H. bacteriophora isolate, SGI_170, which showed the highest virulence against late-instar FCM larvae, was chosen for the further development of in vitro liquid culture.
... Summers are marked by intense sunlight and dry heat, while winters tend to be cold and wet with potential snowfall at higher elevations. As South Africa is a water-scarce country, irrigation is essential for the maintenance of viticulture in many of these regions (Stevenson, 2005). ...
... Thus, the results obtained from field trials conducted in citrus orchards showed that local EPN species have potential for the control of the soil stages of FCM, with the added possibility of good persistence (Malan and Moore, 2016). Steyn et al. (2019) investigated the infection rate, quality and age of in vitro liquid-cultured Steinernema jeffreyense in the laboratory and in field trials. Additionally, pre-and post-application pathogenicity tests were conducted with FCM larvae to assess the EPN virulence of the in vitro-cultured nematodes. ...
... The semi-field study showed promising results, with the immediate effect yielding up to 77% mortality of the larvae, which remained at more than 35% over the 4-week period following application. These results compare favorably with those from previous field studies using in vivo EPNs, proving that EPNs can form a valuable part of an IPM program aimed at the control of FCM (Steyn et al., 2019). ...
... Additionally, measures of virulence were determined by successful infection and mortality of inoculated mealworm larvae in bioassays (Glazer and Lewis 2000). Although reproductive capacity (yield per insect) could also have been measured, the three measurements used (viability, infectivity and virulence) were considered sufficient to confirm the quality of the nematodes, particularly as T. leucotreta had already been shown to be a highly suitable host for the two EPN species used (Malan et al., 2011;Malan and Moore, 2016;Steyn et al. 2017Steyn et al. , 2019aSteyn et al. , 2019b. ...
... What can be excluded as a source of variation is the nematode species. Lepidopteran larvae and, in particular, the larvae of T. leucotreta are highly susceptible to EPNs, including H. bacteriophora and S. feltiae (Malan et al., 2011;Malan and Moore, 2016;Steyn et al. 2017Steyn et al. , 2019aSteyn et al. , 2019b. Furthermore, T. leucotreta will remain soil-borne for 21-40 days (Newton 1998), as a prepupating larva, prepupa, pupa, and the emerging moth, all of which are susceptible to EPNs, albeit less so for pupae (Malan et al., 2011). ...
... Similarly, positive results against the same pest, but on subtropical crops, are reported by Steyn et al. (2019b), who tested the effect of H. bacteriophora (in vivo-cultured, strain supplied by e-nema) in semi-field trial applications to avocado, macadamia and litchi orchards in Nelspruit, Mpumalanga province, at a concentration of 30 IJs/cm 2 . The immediate T. leucotreta infection of sentinel larvae post application was 73 %, with a persistence of >60 % after 14 days, with no difference recorded between crops. ...
Commercial-scale field studies were conducted in citrus orchards to test the efficacy of entomopathogenic nematodes (EPNs) in controlling the soil-dwelling life stages of the key pest, Thaumatotibia leucotreta. Trials were conducted in three provinces of South Africa from 2011 to 2013. Two EPN species were tested: Heterorhabditis bacteriophora and Steinernema feltiae. Both EPN species were applied underneath citrus tree canopies, either with a spray machine or through the microsprinkler irrigation system. The concentration of nematodes and frequency of application varied. Efficacy of these treatments was measured by evaluation of EPN infection of sentinel T. leucotreta larvae in the soil at various intervals after application and by T. leucotreta infestation of fruit. Successful control was recorded in several trials, with EPN infection of larvae frequently reaching 80 %. However, in a few trials, this level of infection was assisted by naturally occurring EPNs. Fruit infestation by T. leucotreta larvae was reduced by up to 88 %. Efficacy appeared to be limited by suboptimal soil moisture and temperature. The former was influenced mainly by mode and relative thoroughness of irrigation before and after EPN application. This was best obtained through microsprinkler application. Nematode species and concentration also influenced efficacy. Both EPN species survived and persisted in the soil of the treated orchards for up to 4 months. Promising results support their potential for use in an integrated pest management programme in citrus in South Africa.
... A South African isolate of Steinernema yirgalemense Nguyen, 2004 (isolate 157-C), associated with the mutualistic bacteria Xenorhabdus indica Somvanshi, 2009, was isolated from a citrus orchard in the Mpumalanga province growing region (Malan et al., 2011). This isolate has shown high virulence against key South African pests, including Phlyctinus Schönherr, 1826 (Coleoptera: Curculionidae: Entiminae), across multiple families in both laboratory and field trials (Malan et al., 2011;Le Vieux & Malan, 2015;Malan & Moore, 2016;James et al., 2018;Katumanyane et al., 2018;Dlamini et al., 2019Dlamini et al., , 2020Steyn et al., 2019). It shows reasonable soil persistence under Western Cape province orchard and vineyard conditions (at least 4 weeks, Le Vieux & Malan, 2015;Steyn et al., 2019), making it a suitable biological control candidate in these agro-ecosystems in this region. ...
... This isolate has shown high virulence against key South African pests, including Phlyctinus Schönherr, 1826 (Coleoptera: Curculionidae: Entiminae), across multiple families in both laboratory and field trials (Malan et al., 2011;Le Vieux & Malan, 2015;Malan & Moore, 2016;James et al., 2018;Katumanyane et al., 2018;Dlamini et al., 2019Dlamini et al., , 2020Steyn et al., 2019). It shows reasonable soil persistence under Western Cape province orchard and vineyard conditions (at least 4 weeks, Le Vieux & Malan, 2015;Steyn et al., 2019), making it a suitable biological control candidate in these agro-ecosystems in this region. Significant advances have been made in the in vitro mass culture of this EPN-bacterium complex, and the subsequent formulation of the produced IJ (Ferreira et al., 2016;Kagimu & Malan, 2019;Dunn et al., 2022;Nxitywa & Malan, 2022). ...
... xerophilus pupal bioassay 1 at inoculation rate of 100 IJ insect −1 ). These results highlight the importance of EPN batch quality control before use (Grunder et al., 2005;Steyn et al., 2019). A single dose infectivity test using T. molitor or Galleria mellonella larvae (Peters, 2005) before using a batch of IJ for bioassays or trials could potentially decrease variability. ...
Considerable progress has been made in the surveying, taxonomy, screening, mass production and formulation of entomopathogenic nematodes (EPN) and their associated symbiotic bacteria in South Africa. Steinernema yirgalemense isolate 157-C is one of the most promising native EPN candidates with regards to virulence, its broad insect-host spectrum, and can be readily mass-produced and formulated into a commercial product. The banded fruit weevils, Phlyctinus callosus sensu stricto and Phlyctinus xerophilus , previously grouped together under the Phlyctinus callosus sensu lato species concept, are native entimine weevils of economic importance to deciduous fruit, grapevine and berries in the Western Cape province of South Africa. This study investigated potential differences in baseline susceptibility of larvae and pupae of the two weevil species to S. yirgalemense in laboratory screenings. The test arena used was 24-well bioassay plates, with an inoculation concentration of 200 infective juveniles (IJ) insect ⁻¹ for larvae and 100 IJ insect ⁻¹ for pupae. Infection was determined 48 h and 96 h after inoculation. Field-efficacy of S. yirgalemense , applied at a concentration of 60 IJ cm ⁻² , against larvae of the two weevil species was determined in an ecologically relevant semi-field trial. In all cases in vitro mass-produced IJ of S. yirgalemense were used. No baseline differential susceptibility between P. callosus and P. xerophilus larvae was obtained in laboratory screenings. Phlyctinus pupae were approximately twice as susceptible compared to larvae, with significant differences between bioassay batches. Approximately 45% control of P. xerophilus larvae was obtained after 96 h of exposure to S. yirgalemense in the field, differing significantly from the control and P. callosus treatment. Low levels of Phlyctinus larval infection by native EPN (confirmed as Heterorhabditis bacteriophora from one P. xerophilus cadaver) occurred in both control and EPN treatment groups under field conditions.
... The diapausing larval population of codling moth overwinters in cryptic habitats, for example in old pruning wounds and cracks in the bark of apple trees, which offer an opportunity to use nematodes as a biological control agent prior to their emergence during the next growing season. The performance of EPNs has also been evaluated in laboratory and field bioassays against the false codling moth, Thaumatotibia leucotreta (Meyrick) Malan & Moore, 2016;Steyn et al., 2019) and the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Malan & Manrakhan, 2009;James et al., 2018). ...
... Steinernema jeffreyense was previously evaluated against codling moth and false codling moth in both laboratory and field environments (De Waal et al., 2011Odendaal et al., 2016a;Steyn et al., 2019), as well as against the vine mealybug (Platt et al., 2018(Platt et al., , 2019a. Methods for mass culturing this nematode species have been demonstrated by Dunn & Malan (2019). ...
... The performance of S. yirgalemense was previously evaluated against false codling moth Steyn et al., 2019), codling moth (De Waal et al., 2011) andmealybugs (Van Niekerk &Malan, 2012;Le Vieux & Malan, 2013Platt et al., 2018) in both laboratory and field environments. It has also been evaluated in terms of aboveground application for the control of codling moth (Odendaal et al., 2016b). ...
Plangia graminea, locally known as a katydids or “krompokkels”, is a minor pest of vineyards in the Western Cape province of South Africa. Is feed on leaves, and sporadically on the skin of grapevine berries. Under natural conditions, katydids are not of much agricultural importance, but pest outbreaks during favourable conditions can result in significant foliar damage. Observations indicate an increase in katydid abundance and damage intensity in recent years. Currently, no agrochemicals are registered for the control of this species, and its present natural enemies are unlikely to provide sufficient control without augmentation. In this study, 12 entomopathogenic nematode (EPN) species were evaluated against the nymphs of Plangia graminea in laboratory bioassays, and mortality by infection was investigated. Seven locally occurring nematode species achieved significant mortality, with H. zealandica, H. indica, S. jeffreyense and S. yirgalemense being found to perform the best (> 90% mortality).
... Steinernema has been previously evaluated against lepidopteran pests, under both laboratory and field conditions. Relevant EPN biocontrol research in South Africa includes that of the above-ground diapausing larval population of the codling moth, Cydia pomonella L. (Lepidoptera: Tortricidae) [11][12][13][14] and the soil stages of false codling moth Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) [15][16][17][18]. ...
... Steinernema yirgalemense Nguyen, Tesfamariam, Gozel, Gaugler & Adams and Steinernema jeffreyense Malan, Knoetze & Tiedt have been successfully mass-produced using in vitro liquid culture methods [19,20]. Promising results were obtained against false codling moth, using both in vivo-and in vitro-cultured nematodes, with no significant difference between culture types in laboratory and field trials [18]. The in vitro liquid mass production of EPNs is much more cost and labour effective than in vivo production, the latter of which is better suited for small-scale experiments and insecticidal applications [21]. ...
... Insects without a soil stage may be more susceptible to EPNs, as they may not have had the opportunity to evolve the resistance necessary to protect themselves from nematode infections. This weakness of above-ground pest defence mechanisms against microbiological pathogens can thus be exploited to provide biological control, for example, as with previous research on mealybugs, and the addition of adjuvants to nematode suspensions [18,[22][23][24][25][26] has shown. ...
Entomopathogenic nematodes (EPNs) have been successfully applied as biological control agents against above ground and soil stages of insect pests. However, for commercial application, it is crucial to mass culture these nematodes using in vitro liquid culture technology, as it is not attainable when using susceptible insects as hosts. Lobesia vanillana (Lepidoptera: Tortricidae) is regarded a sporadic pest of wine grapes in South Africa. The in vivo- and in vitro-cultured South African EPNs, Steinernema yirgalemense and Steinernema jeffreyense (Rhabditida: Steinernematidae), were evaluated against larvae and pupae of L. vanillana in laboratory bioassays. For larvae, high mortality was observed for all treatments: In vitro-cultured S. yirgalemense (98%) performed better than S. jeffreyense (73%), while within in vivo cultures, there was no difference between nematode species (both 83%). No significant difference was detected between in vivo- and in vitro cultures of the same nematode species. The LD50 of the in vitro-cultured S. yirgalemense, was 7.33 nematodes per larva. Mortality by infection was established by dissecting L. vanillana cadavers and confirming the presence of nematodes, which was > 90% for all treatments. Within in vitro cultures, both S. yirgalemense and S. jeffreyense were able to produce a new cohort of infective juveniles from L. vanillana larvae. Pupae, however, were found to be considerably less susceptible to EPN infection. This is the first study on the use of EPNs to control L. vanillana. The relative success of in vitro-cultured EPN species in laboratory assays, without any loss in pathogenicity, is encouraging for further research and development of this technology.
... heterorhabditis subsp. heterorhabditis), 'green' and red (James et al., 2018;Steyn et al., 2019a). Booysen et al. (2022) ascribed this coloration to three different symbiotic bacteria, adding two new associations for P. laumondii subsp. ...
... The lowest mortality (42%) was recorded with H. baujardi. Recently, the efficacy of four EPNs was evaluated in avocado, macadamia and litchi orchards against the larvae of false codling moth (Steyn et al., 2019a). Steinernema yirgalemense showed the highest mortality in all three orchards, ranging between 82% and 96%. ...
Extensive surveys of entomopathogenic nematodes (EPNs) have been conducted globally to identify native species. In Africa, EPN-related research has gained much attention over the past two decades, with several surveys having been undertaken in different countries, including Benin, Cameroon, Egypt, Ethiopia, Kenya, Morocco, Nigeria, Rwanda, South Africa and Tanzania. Such surveys have led to the description of 37 EPN species and 16 species of symbiotically associated bacteria being documented from Africa. Whereas South Africa and Egypt have made significant progress in the discovery and identification of EPN, in terms of their symbiotic bacteria and pathogenicity testing against pest insects, in many other African countries, the research is still in its early stages, or is else non-existent. Most research conducted so far has focused on laboratory bioassays, while research on the use of nematodes in more natural environments is scant. The present review provides a comprehensive overview of EPNs and their associated symbiotic bacteria from the African continent. The biological control potential of local EPN isolates against various economically important insect pests in Africa is also discussed.
... Previous research has highlighted several species of indigenous entomopathogenic nematodes (EPNs) (Malan et al., 2011;Malan & Moore, 2016;Steyn et al., 2019) and entomopathogenic fungi (EPF) (Coombes et al., 2016;Goble et al., 2011) as potential management tools against these soil-dwelling life stages when used alone. However, given that these microbes may be applied in the same environment, at the same time and target the same pest life stage, interactions may be apparent. ...
... Previous bioassay studies, testing only one concentration, have shown that S. yirgalemense 157-C, S. jeffreyense J194 and H. noenieputensis 158-C can induce a high level of mortality in T. leucotreta fifth instar cohorts when applied at 50 IJs/larva (Malan et al., 2011;Steyn et al., 2019). The results of this study confirm this. ...
Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is a major phytosanitary pest of citrus in South Africa. Although several management tools exist, control options registered for use against the soil-dwelling life stages are limited. Both entomopathogenic nematodes (EPNs) and entomopathogenic fungi have been investigated previously, but they have not been studied in combination against T. leucotreta. Thus, this study investigated the interaction of an indigenous entomopathogenic fungus, Metarhizium pinghaense (previously anisopliae) FCM Ar 23 B3 with three indigenous EPNs: Steinernema yirgalemense 157-C, S. jeffreyense J194 and Heterorhabditis noenieputensis 158-C for increased late instar T. leucotreta larval mortality. Before interaction experiments, lethal concentration (LC) values for each of these microbial agents were determined through dose–response bioassays. Heterorhabditis noenieputensis recorded the highest LC50 amongst the nematodes (7.11 IJs/50 µl). Using the pre-determined LC70 value of M. pinghaense and the LC50 values for each of the nematode species, interaction experiments were conducted. Combinations of the nematodes either applied simultaneously with the fungus or at 24, 48, 72 or 96 h post-fungal application showed predominantly additive interactions. Synergy between the simultaneous application of S. yirgalemense and M. pinghaense was found, whilst the interaction between H. noenieputensis and M. pinghaense applied simultaneously and S. jeffreyense applied 24 h post-fungal application, recorded antagonistic interactions. The use of these agents in combination may therefore have the potential to increase control of T. leucotreta soil-dwelling life stages in citrus orchards across South Africa and should be further investigated.
... In recent times, a Heterorhabditis bacteriophora based product was commercialized under the name Cryptonem ™ and is being produced by a South African-based Company (Malan et al. 2011). Several species of EPNs occur naturally in the soil and they have been isolated and evaluated for possible control of T. leucotreta with high levels of efficacy Steyn et al. 2019Steyn et al. , 2017. A review of EPNs evaluation and application has been given by Belien (2018) and Platt et al. (2020). ...
Fruit production in Sub-Saharan Africa is of paramount importance both socially and economically. Millions of farmers derive livelihoods from mango, avocado, citrus, cashew, and coconut farming, but native and alien invasive species constrain production The region’s capacity to contain invasives is weak due to the absence of national and institutional support systems for early detection, containment, eradication, or management of the pests. Climate change is expected to play a huge role in the influx of more alien invasive species and the shift of ecological requirements of some native species. Though a fair share of pre-and post-management pest management techniques for several insect pests has been developed, adoption and adaptation of the options are limited. Data on economic and social implications are largely lacking, making it challenging to implement informed policy decisions. The existence of the “Strategy for Managing Invasive Species in Africa 2021–2030” promises a paradigm shift in the management of invasives, from reactive thinking to coordinated proactive approaches. The uncoordinated deployment of management measures in the region and the lack of funding, play a negative role in managing the pests effectively. Prospects for enhanced future research are wide, and efforts are currently being channeled to Area-Wide-Integrated Pest Management in a bottom-up approach with stakeholders owning the process. Participatory development of technologies is also taking centre stage, paving the way for increased adoption and adaptation. Postharvest technologies promise to provide the adequate phytosanitary assurance required by countries importing fruit from Sub-Saharan Africa.
... Commercial use of entomopathogenic nematodes since the beginning of the 1980s was possible due to the dynamic development of mass cultures on artificial media. Now, biological products containing nematodes of the genera Steinernema and Heterorhabditis are commonly produced worldwide (Bedding, 1981;Wouts, 1981;Steyn et al., 2019). ...
Liquid culture is the most scalable technology for the industrial production of entomopathogenic nematodes. Variability of the recovery after inoculation into cultures of Photorhabdus luminescens remains a persistent problem in the mass production of Heterorhabditis sp. In order to enhance infective juvenile (IJ) recovery and improve nematode population management, we analysed the correlation between the nematode Heterorhabditis megidis (strain KV – 136) development in liquid cultures, the density of bacteria of P. luminescens and the culture agitation speed. Analyses focused on the impact of different agitation speeds (160 rpm and 200 rpm) on the dynamics of population growth of H. megidis in liquid cultures at constant biotic and abiotic parameters (initial dose of nematodes introduced to the culture 2300 IJs/ml, temperature 25°C, the number of bacterial colonies 0.3 × 10 ⁷ /ml). The performed experiments showed that the agitation speed of 200 rpm favourably affected the density of bacteria of P. luminescens (24.14 × 10 ⁷ /ml). High density of bacteria at this agitation speed resulted in an earlier (on the fifth day of the culture) maximum increase in the number of hermaphroditic individuals (1239.6 H/ml) than in the culture at an agitation speed of 160 rpm.
... For example, Steinernema yirgalemense Nguyen, Tesfamariam, Gozel, Gaugler & Adams was shown to be highly effective against the vine mealybug, grapevine leaf miner and the banded fruit weevil in laboratory trials (Le Vieux & Malan, 2013;Steyn et al., 2019a;Dlamini et al., 2020). Both in vivo-(Le Vieux & Malan, 2013) and in vitrocultured nematodes (Steyn et al., 2019b) have proven to be effective in laboratory trials and field trials for potentially controlling pests of grapevine. EPNs were first mentioned in South Africa in the early 1950s, when they were isolated from all the life stages of the black maize beetle, found in a maize field in the Eastern Cape province near Grahamstown (Harington, 1953). ...
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.
... All rights reserved 2013; Malan & Moore, 2016;Steyn et al., 2018) with promising results. Furthermore, Steyn et al. (2019) showed similar efficacy of in vitro-and in vivo-cultured EPNs against FCM in both laboratory and semi-field environments, indicating the feasibility of in vitro production (Dunn et al., 2020) without the loss of EPN performance, which is vital for commercial feasibility. In the current IPM systems for grapevine, deciduous fruit and citrus, no control of the soil stages of T. leucotreta is currently targeted with any kind of control measure, stressing the importance of using these biologicals against the soil-dwelling stages of FCM. ...
False codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), is a priority pest on citrus, stone fruit, and table grapes. Current control focuses on the adult stage; however, each stage of the life cycle of the insect should be targeted. The potential of local biocontrol agents against FCM immature stages were investigated. Several entomopathogenic nematode (EPN) and entomopathogenic fungi (EPF) species were identified from soil samples collected from orchards and vineyards throughout the Western Cape province in South Africa. The eggs were proven to be susceptible (30–65%) to several EPN species, at 200 infective juveniles (IJs) per 50 µl. Xenorhabdus indica, the symbiotic bacterium of Steinernema yirgalemense Nguyen et al., caused significantly more mortality than the control. The EPNs were highly virulent against the FCM larvae, with S. yirgalemense causing 100% mortality in all trials. Metarhizium robertsii (78%), M. anisopliae (50%), and Beauveria bassiana (75%) caused high mortality in FCM larvae. The fully formed pupae were the most resistant of the immature stages, with the EPNs causing low mortality at 100 IJs per insect. As the FCM larvae were highly susceptible to S. yirgalemense and M. robertsii under laboratory conditions, they should be further tested, separately and in combination, in field conditions to illustrate the benefits of including them in a FCM‐IPM programme.
... These results were confirmed with significant efficacy recorded with S. yirgalemense and S. jeffreyense against T. leucotreta in semi-field trials in other crops (vineyards, avocado, litchi, and macadamia), using a similar methodology [47,107]. ...
Thaumatotibia leucotreta, known as the false codling moth, is a pest of citrus and other crops in sub-Saharan Africa. As it is endemic to this region and as South Africa exports most of its citrus around the world, T. leucotreta has phytosanitary status for most markets. This means that there is zero tolerance for any infestation with live larvae in the market. Consequently, control measures prior to exporting must be exemplary. Certain markets require a standalone postharvest disinfestation treatment for T. leucotreta. However, the European Union accepts a systems approach, consisting of three measures and numerous components within these measures. Although effective preharvest control measures are important under all circumstances, they are most critical where a standalone postharvest disinfestation treatment is not applied, such as within a systems approach. Conventional wisdom may lead a belief that effective chemical control tools are imperative to achieve this end. However, we demonstrate that it is possible to effectively control T. leucotreta to a level acceptable for a phytosanitary market, using only biological control tools. This includes parasitoids, predators, microbial control, semiochemicals, and sterile insects. Simultaneously, on-farm and environmental safety is improved and compliance with the increasing stringency of chemical residue requirements imposed by markets is achieved.
... The results also indicate that inoculation with lower nematode concentrations should be applied in future studies, as this could indicate the difference in virulence between different EPN species. Success in using EPNs to control the soil life stages of other grapevine pests, including false codling moth, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) (Malan & Moore, 2016;Steyn et al., 2019aSteyn et al., , 2019b; fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) (James et al., 2018); and weevils, Phlyctinus callosus (Schönherr) (Coleoptera: Curculionidae) (Ferreira & Malan, 2014;Dlamini et al., 2019), has resulted in research being undertaken on mass culturing and the formulation of local entomopathogenic nematodes (Ferreira et al., 2016;Dunn et al., 2020). The pathogenicity of S. yirgalemense to A. trimenii larvae further emphasises its potential to control more than one target pest, which should enhance its acceptability by, and usefulness for, farmers. ...
Trimen's false tiger moth, Agoma trimenii (Lepidoptera: Agaristidae), recently developed as a pest of grapevine in the Northern Cape and Limpopo (Groblersdal area) provinces of South Africa. Little is known about the biology of A. trimenii and control options are lacking. The aim of this study was to test the susceptibility of A. trimenii larvae and pupae to two locally isolated entomopathogenic nematodes (EPNs), Steinernema yirgalemense and Heterorhabditis noenieputensis, and two commercially available entomopathogenic fungi (EPF), Metarhizium anisopliae and Beauveria bassiana, under laboratory conditions. Larvae and pupae were screened for pathogenicity of the two nematode species, using a concentration of 100 infective juveniles (IJs)/50 μ! of water. After 48 h, 100% mortality of the larval stage was found. However, no pupae were infected with EPNs. Larvae and pupae were screened for pathogenicity of the two EPF isolates by means of a dipping test, at a concentration of 0.2 ml/500 ml water and 0.5 g/500 ml water, respectively. Five days post-treatment, 100% larval mortality was recorded in comparison with no deaths in the controls. Overt mycosis was only observed in the case of M. anisopliae. However, in the case of pupae, no mortality was observed for both the nematode and the fungal applications. In future studies, the prepupal soil stage of A. trimenii should be screened for susceptibility to EPNs and EPF. The results of this study indicate the excellent potential of EPNs and EPF as biological control agents against the larvae of A. trimenii, especially for application to small areas with high infestation, without disrupting an integrated pest management programme.
... The bacto-helminthic relationship represents a relatively environmentally safe means of biological control of many soil-dwelling insect-pests (Lacey et al., 2015). For the past decade, research in South Africa has demonstrated the control potential of EPNs within integrated systems for key pests of grapevine, deciduous fruit and citrus (Dlamini et al., 2019;Hatting & Malan, 2017;Malan & Ferreira, 2017;Steyn et al., 2019aSteyn et al., , 2019b. However, the high cost of producing EPNs on a large scale limits their commercial application, resulting in significant efforts to improve the methods for mass production (Askary & Mahfouz, 2017). ...
Entomopathogenic nematodes (EPNs) are a safe insect biological control agent. Key to the implementation of EPNs as a biopesticide is their mass production in shake flasks and bioreactors. For commercial application, in vitro liquid culture is the predominant choice, due to the cost and scale of production, and to the ease of downstreaming. The in vitro liquid culture of EPNs begins in Erlenmeyer shake flasks to provide aeration and agitation. The initial liquid culture phase is followed by upscaling to 5-20-L desktop bioreactors and, thereafter, to 80-120-1000-L industrial-scale bioreactors. The ingredients of the liquid culture media, on which symbiotic bacteria and nematode develop, is of great importance for mass-culturing. The diet usually consists of essential nutrients that best replicate the constituency of the natural insect host, such as protein, carbohydrates and lipids. In addition, aeration and agitation are maintained, without causing shear damage due to the rotating impeller blades. Such factors, however, requires different parameters, depending on the EPN species involved, and, moreover, optimisation is required to obtain high yields and quality of infective juveniles. The objective of the current review is to assess the conditions required for optimal EPN production in liquid culture, and how the conditions might be optimised for South African EPN species.
... EPNs produced in liquid culture are equally effective, for example, in applications of S. carpocapsae (Weiser) on Popillia japonica Newman and Otiorhynchus sulcatus (F.) (Gaugler & Georgis, 1991), S. jeffreyense and S. yirgalemense on Thaumatotibia Accepted Article leucotreta (Lepidoptera: Tortricidae) (Steyn et al., 2019), of S. riobrave against Diaprepes abbreviatus (Shapiro & McCoy, 2000), of H. bacteriophora, H. megidis or S. feltiae against Diabrotica virgifera (Kurtz et al., 2009) or S. feltiae against Lucilia cuprina (Pace, Grote, Pitt, & Pitt, 1986). Even the prolonged persistence of H. bacteriophora in-vitro produced was detected 23 months after their application in bean crops, followed by rotation by wheat, with red clover as a cover crop (Susurluk & Ehlers, 2008). ...
Monoxenic liquid culture is the most suitable technology for scaling up to industrial production of entomopathogenic nematodes (EPNs); however, the variability of the yield production remains a current problem in the process. The aim of this study was to analyze the parameters and criteria for EPN production in liquid culture based on scientific and technological knowledge from the last two decades. While experimental research has permitted the yield production of Heterorhabditis bacteriophora (362 × 10³ infective juveniles [IJs]/ml) and Steinernema carpocapsae (252 × 10³ IJs/ml), simultaneously, theoretical approaches have contributed to the understanding of the culture process, based on biological parameters of the bacterium–nematode complex and hydrodynamic and rheological parameters of the complex gas–liquid–solid system. Under this interdisciplinary research approach, bioprocess and biosystem engineering can contribute to design the various control strategies of the process variables, increase the productivity, and reduce the variability that until now distinguishes the in vitro production of EPNs by the liquid culture.
... In this study, S. jeffreyense, produced in vitro outperformed in vivo produced nematodes with regard to pathogenicity. In a laboratory and field trial, Steyn et al. (2019) compared in vivo and in vitro produced IJ of S. jeffreyense to control false codling moth, with in vitro produced IJ providing better pathogenicity in both cases. A higher mortality percentage for in vitro produced IJs also provide promising prospects for upscaling to fermenters and for formulation where IJs often lose their pathogenicity due to sub-culturing and selection of genetic traits that improve mass production but decrease pathogenicity and in formulation where the IJs are subjected to a variety of stressful factors that can decrease their pathogenicity. ...
Entomopathogenic nematodes (EPNs) of the families Heterorhabditidae and Steinernematidae are efficient biological control agents against important insect pests. In vitro liquid culture production technology is a key factor in the success of implementing EPNs as a biological control agent. One of the first steps of in vitro mass culture is to use shake flasks to obtain nematode inoculum for optimising and upscaling to desktop and industrial fermenters. This study was the first attempt on the in vitro liquid mass culture of a local South African isolate, Steinernema jeffreyense, in 250 ml Erlenmeyer flasks, together with their mutualistic bacteria, Xenorhabdus khoisanae. After the successful in vitro production of S. jeffreyense-inoculum, different parameters for optimizing infective juvenile (IJ) recovery (developmental step when the IJ moult to initiate the life cycle) and yield, were investigated. This includes the effect of the volume of liquid medium in the flasks, two different orbital shakers setups and the initial IJ inoculum density. With 30 ml of liquid medium the mean percentage recovery of IJ after six days was 86%, with a yield of 121,833 IJ ml−1 after 14 days, in comparison to 75% and 99,875 IJs ml−1 respectively when 50 ml of liquid medium was used. No significant difference was found between IJ recovery and yield, using different orbital shakers setups. Among the three inoculum concentrations tested (1000, 2000 and 3000 IJ ml−1), the lowest concentration gave the highest IJ recovery and yield. Pathogenicity of IJs cultured in vitro was higher than those cultured in vivo.
Background
The bollworm complex consisting of Helicoverpa armigera and Earias vittella is a major threat in cotton production globally. The habit of developing resistance to many insecticides including Bt transgenic cotton necessitates the exploration of an alternate strategy to manage bollworms. The entomopathogenic nematodes (EPN) Steinernema carpocapsae strain APKS2 and Heterorhabditis bacteriophora strains KKMH1 and TRYH1 at different concentrations of 1 × 10 ⁹ infective juveniles (IJs)·hm ⁻² , 2 × 10 ⁹ IJs·hm ⁻² , and 3 × 10 ⁹ IJs·hm ⁻² in 500 L of water were evaluated as a foliar spray in fields naturally infested with H. armigera and E. vittella located at Eastern Block and and Cotton Research Farm of Tamil Nadu Agricultural University, Coimbaotre, India during October 2010–February 2011 and October 2011–February 2012, respectively.
Results
In general, all three tested EPN strains reduced the larval population of H. armigera and E. vittella ; reduced square and boll damage; and subsequently increased cotton yield compared with the untreated control. The S. carpocapsae APKS2 is most effective against H. armigera whereas both S. carpocapsae APKS2 and H. bacteriophora KKMH1 were equally effective against E. vittella. The higher dose of 3 × 10 ⁹ IJs·hm ⁻² was highly significant in the reduction of H. armigera larvae. However, the doses 2 × 10 ⁹ IJs·hm ⁻² and 3 × 10 ⁹ IJs·hm ⁻² were equally effective for E. vittella control. The S. carpocapsae APKS2 at 3 × 10 ⁹ IJs·hm ⁻² caused a 62.2% reduction of H. armigera larvae, 34% reduction of square damage, 58.5% reduction of boll damage, and yielded 45.5% more seed cotton than the untreated control plots. In E. vittella infested field, S. carpocapsae strain APKS2 and H. bacteriophora strain KKMH1 at 2 × 10 ⁹ IJs·hm ⁻² resulted in 60.6%~62.4% larva reduction, 68.4%~70.7% square damage reduction, 66.6%~69.9% boll damage reduction and 45.9% yield increase over the untreated control. The effective EPN treatments were comparable to the chemical insecticide chlorpyriphos 20% emulsifiable concentrate spraying at 2 mL·L ⁻¹ .
Conclusions
This study has shown that EPN have great potential in the management of the bollworm complex in cotton. Foliar spraying EPN strain S. carpocapsae (APKS2) at 3 × 10 ⁹ IJs·hm ⁻² and S. carpocapsae (APKS2) or H. bacteriophora (KKMH1) at 2 × 10 ⁹ IJs·hm ⁻² five times at10 days intervals are the best for the management of H. armigera and E. vittella , respectively.
Entomopathogenic nematodes (EPNs) of the family Steinernematidae are effective biological control agents against important pest insects. The in vitro liquid culture method of mass production is used to produce EPNs of high quantity, quality and with reduced cost by upscaling. The first step in liquid mass production is the use of shake flasks to obtain monoxenic infective juvenile (IJ) inoculum for optimisation purposes, followed by upscaling to a desktop fermenter. This study was undertaken to assess the impact of the addition of agar and glucose to the culture medium, as well as assessing the impact of bacterial cell density inoculum on IJ recovery and yield. Steinernema jeffreyense was cultured in 250 ml Erlenmeyer flasks, with the mutualistic bacterium Xenorhabdus khoisanae. In this study the impact of four different agar and glucose concentrations showed negligible impact on nematode recovery and yield. Different initial bacterial inoculum densities inoculated to the culture medium showed a low inoculum density of 2 % (6 × 106 cells) of the bacteria culture to be the optimal inoculum concentration. A bacterial growth curve for X. khoisanae in tryptic soy broth, showed 40–44 h to be the optimum time for introduction of the initial nematode inoculum into the complex medium.
The in vivo - and in vitro -cultured South African entomopathogenic nematodes (EPNs), Steinernema yirgalemense and Steinernema jeffreyense (Rhabditida: Steinernematidae), were evaluated against larvae and pupae of Lobesia vanillana in laboratory bioassays. For larvae, high mortality was observed for all treatments: In vitro -cultured S. yirgalemense (98%) performed better than S. jeffreyense (73%), while within in vivo cultures, there was no difference between nematode species (both 83%). No significant difference was detected between in vivo - and in vitro cultures of the same nematode species. The LD 50 of the in vitro -cultured S. yirgalemense , the best performing species, was 7.33 nematodes per larva. Mortality by infection was established by dissecting cadavers and confirming the presence of nematodes, which was > 90% for all treatments. Within in vitro cultures, both S. yirgalemense and S. jeffreyense were able to produce a new cohort of infective juveniles from L. vanillana larvae. Pupae, however, were found to be considerably less susceptible to EPN infection. The relative success of local in vivo - and in vitro -cultured EPN species against a tortricid species in laboratory assays is encouraging for further research and development of this technology.
Biological control of insect pests using entomopathogenic nematodes is fraught by costly and scarce formulation and application techniques. For conceivable solutions, we evaluated Steinernema yirgalemense using different formulation techniques. Encapsulation of the infective juveniles (IJs) in alginate beads, as well as the use of diatomaceous earth with reduced water activity (aw-value at 0.97) to induce quiescence, and to reduce the metabolism of the IJs were undertaken. Survival of the IJs in the formulations was determined at 6 0 C, 14 0 C and 25 0 C over 4 weeks. Nematodes readily survived the encapsulation process, with 10-20% being able to break through the bead, at varying temperatures. At 6 0 C the desiccation effect of diatomaceous earth had the greatest affect on the survival of IJs. In both formulations, survival significantly differed at 6 0 C compared to 14 0 C and 25 0 C, with a drastic decrease over time. Steinernema yirgalemense did not survive at low temperatures in either formulation methods. The beads successfully retained most of the IJs, and can therefore be stored for a longer time. Of the two methods, diatomaceous earth is the formulation that requires further investigation, since it can easily be optimised, dissolved in water and applied in the field.
False codling moth (FCM), Thaumatotibia leucotreta (Lepidoptera: Tortricidae) is an important pest of various fruit crops in South Africa. Current FCM control strategies include the use of chemical insecticides. However, FCM has developed resistance to some of the insecticides, and stringent chemical residue restrictions have been imposed by some foreign markets. Thus, the demand for high-quality fruit has translated into a need for new, efficient and effective integrated pest management (IPM) strategies. One such strategy is the control of the soil-dwelling life stages of FCM, using entomopathogenic nematodes (EPNs) and entomopathogenic fungi (EPF). Both of the biocontrol agents concerned have individually been shown to be effective against FCM. However, it is possible that, if they are applied simultaneously, a synergistic relationship might be observed between EPNs and EPF that could serve to enhance their efficacy against the target pest. In addition to reviewing previous and current control options against FCM in South African fruit crops, this study investigates the potential for using EPNs and EPF individually, and in combination, as biological control agents against FCM within an IPM system.
A survey was undertaken to determine the diversity and frequency of endemic entomopathogenic nematodes (EPN) in subtropical regions in the Mpumalanga, Limpopo and KwaZulu-Natal provinces of South Africa. A total of 136 soil samples were randomly taken from cultivated and uncultivated habitats, including subtropical fruit tree orchards (avocado, litchi, macadamia, mango and guava) and adjoining natural vegetation. EPNs were isolated from 14 samples (10.3%) by means of baiting with Tenebrio molitor larvae. Heterorhabditis was the most common genus isolated from 12 samples, whereas only two Steinernema species were isolated. The most common Heterorhabditis isolated were Heterorhabditis noenieputensis and H. zealandica, which were both isolated from four samples each. The present report is the first occurrence of H. taysearae and H. baujardi in South Africa. The two Steinernema species are both unknown species. Laboratory bioassays were conducted to determine the potential of seven native EPN isolates to control the false codling moth (Thaumatotibia leucotreta) (FCM). Last instar FCM larvae were found to be most susceptible to S. yirgalemense (157-C), H. zealandica (WS 23) and Steinernema litchii (WS9), when exposed to 50 IJs/50 µℓ water for 48 h, causing 100%, 94.2% and 93.5% mortality, respectively.
False codling moth (FCM), Thaumatotibia leucotreta, is one of the most important insect pests of citrus in South Africa. No treatment is currently directed towards the soil stages of FCM, with entomopathogenic nematodes (EPNs) having the potential to fill the niche. Laboratory bioassays in orchard soil, using Heterorhabditis bacteriophora, showed the LD50 to be ≈3 and the LD90 to be ≈58 infective juveniles (IJs) per FCM larva. In a field trial, after application of three concentrations (20, 40 and 80 IJs/cm2) of H. bacteriophora, mortalities of >90 %were obtained for FCM larvae, with significant difference between the lower concentrations, but not with higher concentrations. Twenty-one days after application, there was no further FCMcontrol.With the field application of H. zealandica after 6 days, no significant differences were found in FCM mortality of >80 % between three nematode concentrations (5, 10 and 20 IJs/cm²). After 21 and 35 days no significantly different FCM mortality was found for all three treatments compared to the untreated trees. In a field trial using three nematode species, treatment with H. zealandica resulted in significant control for each evaluation day, up to day 49. Results from field trials showed local EPN species to have great potential for control of the soil stages of FCM, with the added possibility of good persistence.
Wax moth larvae (Galleria mellonella L.) and yellow mealworm larvae (Tenebrio molitor L.) were reared on five different diets respectively, to determine which diet resulted in the highest increase in insect larval weight. Wax moth eggs were placed on each diet and larvae that developed therefrom were weighed after 51 days. A diet containing 118 g wheat flour, 206 g wheat bran, 118 g milk powder, 88 g brewer's yeast, 24 g wax powder, 175 ml honey and 175 ml glycerol, produced the heaviest larvae, with an average weight of 0.19 g per larva. Mealworms were weighed 62 days after adult beetles were placed on the various diets. The heaviest larvae were produced on a diet consisting of wheat bran only, with each larva weighing, on average, 0.0154 g. Nematode production of Heterorhabditis zealandica and H. bacteriophora using wax moth, mealworm, codling moth (Cydia pomonella) and false codling moth (Thaumatotibia leucotreta) larvae were determined, as well as was the correlation between the weight of the host larvae and that of the nematode progeny produced. Wax moth larvae produced the highest number of H. zealandica and H. bacteriophora per g of host. The mean number of H. zealandica and H. bacteriophora infective juveniles produced were 1 459 205 and 1 898 512, respectively, per g of host. The mean number of H. zealandica produced per g of codling moth and false codling moth larvae was 57 582 and 192 867, respectively. Lower mean numbers were produced for codling moth and false codling moth larvae when using H. bacteriophora, with 39 653 and 97 652 nematodes produced per g of the respective hosts. A significant positive linear relationship existed between weight of wax moth larvae and that of mealworm and the number of nematodes produced. Using frozen hosts of wax moth larvae and mealworm, comparable reproduction of nematodes was only found with mealworm.
The development and use of entomopathogens as classical, conservation and augmentative biological control agents have included a number of successes and some setbacks in the past 15 years. In this forum paper we present current information on development, use and future directions of insect-specific viruses, bacteria, fungi and nematodes as components of integrated pest management strategies for control of arthropod pests of crops, forests, urban habitats, and insects of medical and veterinary importance.
During a non-targeted survey for entomopathogenic nematodes in South Africa, a new species of Steinernema was isolated from a soil sample collected from underneath a guava tree, close to the shore at Jeffrey's Bay. The nematode was isolated by means of the insect-baiting technique using last-instar larvae of Galleria mellonella. It is described herein as Steinernema jeffreyense n. sp. The nematode can be separated from other described, closely related species in terms of the morphological and morphometric characteristics of the different life stages, and in terms of the characterization and phylogeny of DNA sequences of the internal transcribed spacer (ITS) rDNA of the 18S gene, and of the D2D3 region of the 28S rDNA gene. The new species is placed molecularly in the arenarium-glaseri-karii-longicaudatum group characterized by the following morphological characters: infective third-stage juvenile with a body length of 926 (784-1043) μm, distance from head to excretory pore of 87 (78-107) μm, tail length of 81 (50-96) μm, with an E% of 109 (86-169), and eight evenly spaced ridges (i.e. nine lines) in the middle of the body. First-generation males have a spicule length of 88 (79-95) μm and gubernaculum length of 57 (51-61) μm. Male mucron is absent in both generations. First-generation females have an asymmetrical protuberance and a short, double-flapped epiptygmata, with both flaps directed to the front. The tail of the first-generation female is shorter than the anal body width, with a mucron on the dorsal tail tip, with D% = 78 (59-99). Cross-hybridization with S. khoisanae, S. tophus and S. innovationi showed the new species to isolate reproductively from the others. The analyses of ITS rDNA and D2D3 sequence of the 18S and 28S rDNA genes support the studied nematode isolate to be a valid new species belonging to the ‘glaseri’ group (Clade V).
Lipids represent the main source of energy in entomopathogenic nematodes. In the infective juvenile (IJ) phase, the level of such reserves can be influenced by storage, and this may affect their infectivity. The aim of this study was to evaluate the percentage of lipids and the associated infectivity in IJs of Steinernema carpocapsae, S. riobrave, Heterorhabditis sp. JPM4, Heterorhabditis sp. CCA and Heterorhabditis sp. PI that had been stored under different temperatures (8-28°C) for various times (0 to 180 days). The amounts of lipids present in IJs were evaluated histologically using a colorimetric method, while infectivity was assayed against Galleria mellonella larvae. Lipid levels diminished with increasing storage time for all nematodes, but the rates of decrease varied according to storage temperature and species. Lipid reserves were conserved for longer storage periods at 8, 16 and 20°C, while at 24 and 28°C the percentage of lipids decreased rapidly. The infectivities of IJs of Heterorhabditis spp. were less tolerant than those of Steinernema spp. to temperatures of 8, 24 and 28°C. Thus, while storage at 8°C was optimal for conserving lipid reserves, infectivity was best preserved at temperatures of 16 and 20°C gave rise to the least reduction in infectivities after 180 days of storage. In this way, lipids and infectivity are influenced by different storage temperatures for the species tested. These data are useful for greater success in using entomopathogenic nematodes as bio control agents.
Commercial use of entomopathogenic nematodes against key insect pests of deciduous fruit, grapes and citrus in South Africa requires massive numbers of nematodes for inundative field application. High-technology in vitro liquid culture requires development to mass culture and to formulate these nematodes for commercial purposes. Heterorhabditis zealandica was identified as a species with potential as a biological control agent against a wide range of key insect pests. The first step towards the development of in vitro mass culture
of H. zealandica was to establish monoxenic cultures of both the nematode and its Photorhabdus sp. symbiont, using in vitro liquid culture technology. The body length of various H. zealandica life stages during in vitro development was measured to determine the growth characteristics of H. zealandica in liquid culture. The growth curve of the symbiotic bacteria during the process time was measured to determine when the stationary phase was reached, as this would indicate the optimum time required for inoculating infective juveniles (IJs) and for aiding in maximum IJ recovery.Onday 15, the IJs reached a maximum density of 41 100/ml, while the hermaphrodites and females reached their highest density on day 16 at 9800/ml and 7700/ml, respectively, after which the experiment was terminated. Bioassays using Galleria mellonella were performed to compare the virulence between in vitro- and in vivo-produced nematodes, which indicated in vitro-produced nematodes to be significantly less virulent. This study illustrates that H. zealandica and its Photorhabdus sp. symbiont can be successfully cultured in liquid. However, two generations occurred during the process time, instead of the desirable one generation. Future research goals would include developing methods to increase the percentage recovery in the liquid culture, as doing so would increase the number of nematodes produced per ml and it would also reduce the processing time significantly.
Production and application technology is critical for the success of entomopathogenic nematodes (EPNs) in biological control. Production approaches include in vivo, and in vitro methods (solid or liquid fermentation). For laboratory use and small scale field experiments, in vivo production of EPNs appears to be the appropriate method. In vivo production is also appropriate for niche markets and small growers where a lack of capital, scientific expertise or infrastructure cannot justify large investments into in vitro culture technology. In vitro technology is used when large scale production is needed at reasonable quality and cost. Infective juveniles of entomopathogenic nematodes are usually applied using various spray equipment and standard irrigation systems. Enhanced efficacy in EPN applications can be facilitated through improved delivery mechanisms (e.g., cadaver application) or optimization of spray equipment. Substantial progress has been made in recent years in developing EPN formulations, particularly for above ground applications, e.g., mixing EPNs with surfactants or polymers or with sprayable gels. Bait formulations and insect host cadavers can enhance EPN persistence and reduce the quantity of nematodes required per unit area. This review provides a summary and analysis of factors that affect production and application of EPNs and offers insights for their future in biological insect suppression.
The effect of culture method on the efficacy of entomopathogenic nematodes was assessed by analysis of 511 greenhouse and field trials. In tests against larvae of the Japanese beetle, Popillia japonica Newman, and black vine weevil, Otiorhynchus sulcatus (F.), infective juveniles of Steinernema carpocapsae (Weiser) reared by in vivo, in vitro solid, and in vitro liquid culture methods provided equivalent larval reductions. Heterorhabditis bacteriophora Poinar infective juveniles produced in vivo and on solid media provided equivalent results in trials against Japanese beetle larvae and northern masked chafer, Cyclocephala borealis (Arrow). By contrast, H. bacteriophora produced by liquid culture achieved significantly lower Japanese beetle reductions than those reared in vivo or on solid media. Against the northern masked chafer, liquid culture H. bacteriophora provided significantly less host mortality than solid culture nematodes. Field persistence studies provided further evidence that liquid culture H. bacteriophora were inferior to infective juveniles produced on solid media. The poor performance of liquid culture H. bacteriophora was attributed to low lipid assimilation.
A new species of entomopathogenic nematode, Steinernema khoisanae n. sp. is described from South Africa. The new species is characterised by morphometrics of the infective juvenile with body length 1076 μm, narrow body diameter of 33 μm, excretory pore 94 μm from anterior end, tail 85 μm long, a = 33, D% = 68, H% = 57, and E% = 111. The lateral field pattern of the new species is 2, 7, 8, 6, 4 and 2. The male of the first generation can be recognised by the spicule and the gubernaculum shape, excretory pore located posteriorly near the end of the pharynx, D% = 88, and SW% = 199. The first generation female can be recognised by the non-protruding vulva and tail bearing a prominent mucron. Steinernema khoisanae n. sp. is characterised genetically by sequences of the internal transcribed spacers and D2/D3 regions of 28S ribosomal DNA, by composition of their sequences and by numerous unique, derived, nucleotide character states. Phylogenetic trees show that S. khoisanae n. sp. and other members of the S. glaseri-group form a monophyletic assemblage.
The stability of traits important for biological control was studied in the entornopathogenic nematode-bacteria complexes Heterorhabditis bacteriophora and Steinernema carpocapsae. Five experimental lines of each species were subcultured for 20 serial passages in Galleria mellonella larvae to assess trait stability. Subculturing impaired performance of both H. bacteriophora and S. carpocapsae. Virulence, heat tolerance and fecundity deteriorated in all H. bacteriophora experimental lines, and four out of five experimental lines deteriorated in host-finding ability. All S. carpocapsae experimental lines deteriorated in heat tolerance and nictation, and four out of five experimental lines declined for reproductive capacity, whereas virulence declined in two experimental lines. Determination of whether trait deterioration was due to changes in nematode, bacteria, or both symbiotic partners was tested by exchanging nematodes or bacteria from control populations with nematodes or bacteria from the most deteriorated experimental lines and assessing trait recovery. The source of deterioration varied according to trait, but only the bacterial partner played a role in trait reductions for every trait and species, whereas the nematode was the main source only for S. carpocapsae nictation. These results emphasise the important role each symbiotic partner plays in the stability and expression of beneficial traits.
Entomopathogenic nematodes (genera Steinernema and Heterorhabditis) kill insects with the aid of mutualistic bacteria. The nematode-bacteria complex is mass produced for use as biopesticides using in vivo or in vitro methods, i.e., solid or liquid fermentation. In vivo production (culture in live insect hosts) is low technology, has low startup costs, and resulting nematode quality is high, yet cost efficiency is low. In vitro solid culture, i.e., growing the nematodes and bacteria on crumbled polyurethane foam, offers an intermediate level of technology and costs. In vivo production and solid culture may be improved through innovations in mechanization and streamlining. In vitro liquid culture is the most cost-efficient production method but requires the largest startup capital and nematode quality may be reduced. Liquid culture may be improved through progress in media development, nematode recovery, and bioreactor design. A variety of formulations is available to facilitate nematode storage and application.
Cane toads (Bufo marinus) are large anurans (weighing up to 2 kg) that were introduced to Australia 70 years ago to control insect pests in sugar-cane fields. But the result has been disastrous because the toads are toxic and highly invasive. Here we show that the annual rate of progress of the toad invasion front has increased about fivefold since the toads first arrived; we find that toads with longer legs can not only move faster and are the first to arrive in new areas, but also that those at the front have longer legs than toads in older (long-established) populations. The disaster looks set to turn into an ecological nightmare because of the negative effects invasive species can have on native ecosystems; over many generations, rates of invasion will be accelerated owing to rapid adaptive change in the invader, with continual 'spatial selection' at the expanding front favouring traits that increase the toads' dispersal.
This book provides a comprehensive review of entomopathogenic nematology. It discusses the fundamental biology and taxonomic foundation for nematodes and their bacterial symbionts. The functional processes involved in parasitism and nematode ecology are also discussed. Technological advances and control methodologies are described.
False codling moth, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), is an important pest of fruit trees in South Africa. The subtropical fruit tree crop industries use a combination of techniques for T. leucotreta suppression. Semi-field trials were conducted in avocado, litchi and macadamia orchards, using four entomopathogenic nematode (EPN) species. The effect on T. leucotreta mortality, directly after application (two days), as well as on persistence for 7, 14, 21, 28, 35, and 42 days post-application, was determined. Results from the field trials, using 30 IJs cm² of Steinernema yirgalemense, showed the highest mortality of 86% directly after application, with Steinernema litchii being the least effective of the four EPN species, with 63% mortality. High persistence was found until day 14, with a steep decline thereafter until day 28. Local South African EPN species showed great potential for the future control of the late instar T. leucotreta, with the added advantage of persistence.
Invertebrate pests pose a significant threat to food security on the African continent. In response, South Africa has become one of the largest importers of chemical pesticides in sub-Saharan Africa, with several hundred active ingredients registered. To address the over-reliance on such chemicals, the South African Department of Agriculture, Forestry and Fisheries (DAFF) has eliminated or restricted several pesticides since the late 1970s. The recent launch of the South African National Bio-Economy Strategy and establishment of the South African Bioproducts Organisation (SABO), together with new guidelines for registration of biopesticides in 2015, also support this endeavour. Concurrently, entomopathogen-related research and bioproduct development has increased over the past decade. Currently, 31 products (seven manufactured locally) are registered under the Fertilizers, Farm Feeds, Agricultural Remedies and Stock Remedies Act 36 of 1947. Commercially important microbes include Beauveria bassiana (Cordycipitaceae), Metarhizium anisopliae (Clavicipitaceae), Cydia pomonella granulovirus, Cryptophlebia leucotreta granulovirus, Helicoverpa armigera nucleopolyhedrovirus (Baculoviridae) and Bacillus thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai (Bacillaceae). Both parasitic and entomopathogenic nematodes (EPNs) show potential for development as bioinsecticides with one commercial EPN product, based on Heterorhabditis bacteriophora (Heterorhabditidae), registered under the Act. Rapid scientific progression, supported by a favourable legislative environment, should facilitate further advances in microbial control of phytophagous invertebrate pests in South Africa.
Entomopathogenic nematodes (EPNs) of the genera Steinernema and Heterorhabditis and their associated symbiotic bacteria of the genera Xenorhabdus and Photorhabdus, are efficient biological control agents, due to their ease of culture, their high fatality against key insect pests and their safety in use. However, their commercial utilisation is limited by their finite shelf life, both in storage and in formulations. Thus, efficient storage in the formulation of EPNs is essential so as to attain success in integrated insect pest management strategies. This paper reviews the latest information that is available on EPN storage, formulation, quality and application methods, coupled with improvement strategies for the effective control of insects. Nematode survival mechanisms investigated were heat and cold tolerance, desiccation, osmotic stress, hypoxia and energy reserves, among others, in storage, field or formulations. Their influence on the formulation of EPNs is also discussed.
Low-cost mass production of entomopathogenic nematodes (EPNs) is an important prerequisite towards their successful commercialisation. This study evaluated six low-cost solid substrate media for in vitro mass production of Steinernema innovationi. Cost analysis was undertaken and an estimated retail price was calculated. This was then compared to the costs of commercial EPN products currently on the market. The highest yield of infective juveniles (IJs) was obtained from a medium containing a puree of house fly, Musca domestica, larvae + 0.15g canola oil, (781,678 ± 221 IJs/5g medium). This medium also had the lowest number of adults remaining in the medium and dead IJs (<10%) at the time of harvest (Day 28). The estimated retail price (R243.27 per 50 million IJs) for S. innovationi produced with our solid culture system was considerably lower than the market price for other Steinernema species products sold by E∼nema, BASF corporation, Koppert, BioBest and Natural Insect Control. The production system developed in this study offers a competitive technology to produce EPN products without having to invest in large scale liquid fermentation equipment, by using a relatively cheap production medium and simple solid culture growing conditions using Erlenmeyer flasks.
Entomopathogenic nematodes have become a valuable addition to the range of biological control agents available for insect control. An endemic nematode, Steinernema yirgalemense, has been found to be effective against a wide range of key insect pests. The next step would be the mass production this nematode for commercial application. This requires the establishment of monoxenic cultures of both the nematode and the symbiotic bacterium Xenorhabdus indica. First-stage juveniles of S. yirgalemense were obtained from eggs, while X. indica was isolated from nematode-infected wax moth larvae. The population density of the various life stages of S. yirgalemense during the developmental phase in liquid culture was determined. The recovery of infective juveniles (IJs) to the third-stage feeding juveniles, was 67 ± 10%, reaching a maximum population density of 75,000 IJs ml- 1 on day 13 after inoculation. Adult density increased after 8 days, with the maximum female density being 4600 ml- 1 on day 15, whereas the maximum male density was 4300 ml- 1 on day 12. Growth curves for X. indica showed that the exponential phase was reached 15 h after inoculation to the liquid medium. The stationary phase was reached after 42 h, with an average of 51 × 107 colony-forming units ml- 1. Virulence tests showed a significant difference in insect mortality between in vitro- and in vivo-produced nematodes. The success obtained with the production of S. yirgalemense in liquid culture can serve as the first step in the optimizing and upscaling of the commercial production of nematodes in fermenters.
Written by 39 leading entomologists, mostly from South Africa, this book is the most comprehensive account of the phytophagous insects of cultivated plants and natural pastures in southern Africa published to date.
Although the scope of the book covers Botswana, Lesotho, southern, Mozambique, Namibia, South Africa, Swaziland and Zimbabwe, it should be equally relevant to most other countries in sub-Saharan Africa, since many of the insects and their associated host plants included in this book also occur elsewhere on the African continent, and further afield.
The book deals with plant-feeding insects that damage field crops, deciduous, subtropical and other fruits, vegetables, plantation trees and ornamental plants, in addition to natural pastures. In all, 75 cultivated plants, or groups of related plants, are dealt with.
To make the information about the insects and their host plants more readily accessible to readers, the arrangement of the text is plant-commodity based; each pest is dealt with according to the plants with which it is associated.
• Full accounts of the 416 most important pests are given, with an additional 277 species of lesser importance being listed, with an indication of the parts of the plant on which they feed or damage.
• Each pest species account includes details on its scientific and common names, origin and distribution, identification, host plant range, damage, natural enemies and management, while key references to the literature are also provided.
• To facilitate identification, 860 high quality full-colour images, depicting adults and immature stages of the pests and their damage symptoms, are provided.
• An extensive glossary of entomological terms is provided to facilitate the use of the book.
With a foreword by V.C. Moran
Publisher Summary
This chapter focuses on the techniques used for identifying, isolating, propagating, assaying, and preserving nematodes that are parasitic in or pathogenic to insects. Nematodes are nonsegmented animals with excretory, nervous, digestive, reproductive, and muscular systems but lacking circulatory and respiratory systems. The stage of entomogenous and entomopathogenic nematodes that is infective varies depending on the group. A good stereomicroscope is essential for nematode identification and should have a range of magnification between 10 and 100X, a fairly fiat field, and good resolution. The gonads and other structures of fixed nematodes may be obscured by the granular appearance of the intestine. Specimens can be cleared by processing to lactophenol or glycerin. The cephalic structures and the number of longitudinal chords are diagnostic characters for genetic or specific determination of certain groups of nematodes. Extraction methods for insect nematodes are derived from techniques developed with plant-parasitic nematodes. It is found that the most common methods are the Baermann funnel, sieving, elutriation, and centrifugal flotation.
The characteristics of morphometric (body length and width), biochemical (fatty acid content), motility, and penetration rate of infective juveniles ofSteinernema carpocapsaeBeijing strain reared on four different culture media [e.g., plant protein medium (I), animal protein medium (II), plant and animal protein medium (III), andin vivoculture (IV) were systematically compared in this research. The results showed that the average lengths of infective juveniles were 497.4 ± 0.09, 514.3 ± 0.08, 525.7 ± 0.09, and 556.6 ± 0.09 μm, the average widths were 24.9 ± 0.006, 25.6 ± 0.005, 26.1 ± 0.006, and 27.9 ± 0.004 μm, and the average dry weights per million infective juveniles were 49.2 ± 2.2, 58.6 ± 2.4, 59.6 ± 1.8, and 80.7 ± 1.7 mg cultured by media I, II, III, and IV, respectively. The highest relative content of fatty acid of infective juveniles was obtained from medium IV at 15.4 ± 1.2 × 105μV/s, and the lowest one was 6.76 ± 0.3 × 105μV/s from medium I and 11.8 ± 0.2 × 105and 13.7 ± 0.3 × 105μV/s from media II and III, respectively. The numbers of nematodes that moved a vertical distance of 5 cm in sand column within 48 h were 24 ± 3.6, 75 ± 11.6, 69 ± 9.7, and 92 ± 13.2 and the penetration rates into theGallerialarva within 24 h were 2.8 ± 0.45, 6.0 ± 1.14, 6.4 ± 0.74, and 6.0 ± 0.7% from media I to IV, respectively. The results indicated that the quality of entomopathogenic nematode was influenced by the cultural medium component. The animal protein, especially from insects which were presented in media II, III, and IV, has a strong positive effect on nematode quality.
The biocontrol potential of South African isolates of Heterorhabditis zealandica, Steinernema citrae, S. khoisanae, S. yirgalemense, and Steinernema sp., was evaluated against codling moth, Cydia pomonella. Codling moth was susceptible to all six nematode isolates at a concentration of 50 infective juveniles/insect (78–100% mortality). Low temperatures (10 h at 17°C; 14 h at 12°C) negatively affected larvicidal activity (≤3%) for all isolates. All tested isolates were most effective at higher levels of water activity (aw=1). The average aw50-values for all isolates tested was 0.94 (0.93–0.95), except S. khoisanae 0.97 (0.97–0.98). Regarding host-seeking ability, no positive attraction to host cues could be detected amongst isolates, except for H. zealandica. Three of the isolates, H. zealandica, S. khoisanae, and the undescribed Steinernema sp., were selected for field-testing and proven to be effective (mortality >50%). Insect containment methods used during field experimentation was shown to influence larvacidal activity, as different levels of mortality were obtained using various containment methods (wooden planks vs. pear tree logs vs. mesh cages). Pear tree logs were impractical. Predictive equations were subsequently developed, enabling future trials to be conducted using either planks or cages, enabling the prediction of the expected level of control on tree logs. All tested isolates therefore showed a certain degree of biological control potential, however, none of the experiments showed clear efficacy-differences amongst isolates. The study highlighted the importance of environmental factors to ensure the successful application of these nematodes for the control of diapausing codling moth larvae in temperate regions.
A survey was conducted to determine the diversity and frequency of endemic entomopathogenic nematodes (EPN) in citrus orchards in the Western Cape, Eastern Cape and Mpumalanga provinces of South Africa. The main aim of the survey was to obtain nematodes as biological control agents against false codling moth (FCM), Thaumatotibia leucotreta, a key pest of citrus in South Africa. From a total of 202 samples, 35 (17%) tested positive for the presence of EPN. Of these, four isolates (11%) were found to be steinernematids, while 31 (89%) were heterorhabditids. Sequencing and characterisation of the internal transcribed spacer (ITS) region was used to identify all nematode isolates to species level. Morphometrics, morphology and biology of the infective juvenile (IJ) and the first-generation male were used to support molecular identification and characterisation. The Steinernema spp. identified were Steinernema khoisanae, Steinernema yirgalemense and Steinernema citrae. This is the first report of S. yirgalemense in South Africa, while for S. citrae it is the second new steinernematid to be identified from South Africa. Heterorhabditis species identified include Heterorhabditis bacteriophora, Heterorhabditis zealandica and an unknown species of Heterorhabditis. Laboratory bioassays, using 24-well bioassay disks, have shown isolates of all six species found during the survey, to be highly virulent against the last instar of FCM larvae. S. yirgalemense, at a concentration of 50IJs/FCM larva caused 100% mortality and 74% at a concentration of 200IJs/pupa. Using a sand bioassay, S. yirgalemense gave 93% control of cocooned pupae and emerging moths at a concentration of 20IJs/cm(2). This is the first report on the potential use of EPN to control the soil-borne life stages of FCM, which includes larvae, pupae and emerging moths. It was shown that emerging moths were infected with nematodes, which may aid in control and dispersal.
Recent studies on butterflies have documented apparent evolutionary changes in dispersal rate in response to climate change and habitat change. These studies often assume a trade‐off between dispersal rate (or flight capacity) and reproduction, which is the rule in wing‐dimorphic species but might not occur equally in wing‐monomorphic species such as butterflies.
To investigate the relationship between dispersal rate and fecundity in the Glanville fritillary butterfly Melitaea cinxia we recorded lifetime individual movements, matings, ovipositions, and maximal life span in a large (32 × 26 m) population cage in the field. Experimental material was obtained from 20 newly established and 20 old local populations within a large metapopulation in the Åland Islands in Finland.
Females of the Glanville fritillary from newly established populations are known to be more dispersive in the field, and in the cage they showed significantly greater mobility, mated earlier, and laid more egg clutches than females from old populations. The dispersive females from new populations exhibited no reduced lifetime fecundity in the cage, but they had a shorter maximal life span than old‐population females.
These results challenge the dispersal–fecundity trade‐off for nonmigratory butterflies but instead suggest a physiological trade‐off between high metabolic performance and reduced maximal life span. High metabolic performance may explain high rates of dispersal and oviposition in early life.
In fragmented landscapes, an ecological trade‐off exists between being more dispersive and hence spending more time in the landscape matrix vs. having more time for reproduction in the habitat. We estimate with a dispersal model parameterized for the Glanville fritillary that the lifetime egg production is 4% smaller on average in the more dispersive butterflies in a representative landscape, with much variation depending on landscape structure in the neighbourhood of the natal patch, from −26 to 45% in the landscape analysed in this paper.
Sustainability in Plant and Crop Protection: Ecology and Applied Technologies for Sustainable Plant and Crop Protection
- R Campos-Hererra
Campos-Hererra, R., 2015. Sustainability in Plant and Crop Protection: Ecology and
Applied Technologies for Sustainable Plant and Crop Protection. Springer, Cham.
Production technology
- Gaugler
Gaugler, R., Han, R., 2002. Production technology. In: Gaugler, R. (Ed.),
Entomopathogenic Nematology. CABI, Wallingford, UK, pp. 289-310.
Formulation of Steinernema yirgalemense, S. jeffreyense and Heterorhabditis bacteriophora in alginate beads and diatomaceous earth
- N Kagimu
- A P Malan
Kagimu, N., Malan, A.P., 2019. Formulation of Steinernema yirgalemense, S. jeffreyense and
Heterorhabditis bacteriophora in alginate beads and diatomaceous earth. Biocontrol 64,
413-422. https://doi.org/10.1007/s10526-019-09945-1.
Dispersal-related life-history trade-offs in a butterfly metapopulation
- Hanski
Field control of the false codling moth, Thaumatotibia leucotreta (Meyrick) in avocado, litchi and macadamia in South Africa, using entomopathogenic nematodes
- Steyn
Development and population dynamics of Steinernema yirgalemense and growth characteristics of its associated Xenorhabdus symbiont in liquid culture
- Ferreira