The potato leaf miner, Liriomyza huidobrensis (Diptera: Agromyzidae), is an important economic pest of potatoes across the world, causing substantial damage in most potato-growing areas. The use of ...local entomopathogens for the control of insect pests has been widely encouraged, coupled with several environmental advantages being contrasted to the effects achieved using chemical pesticides. The current study surveyed potato field soil to determine the presence of entomopathogenic nematode (EPN) and entomopathogenic fungi (EPF) species in the Sandveld region of the Western Cape province. Susceptibility of L. huidobrensis larvae and pupae to the isolated pathogens and other locally sourced isolates was evaluated. The results of the survey indicated the presence of an unknown free-living bacteria feeding insect-associated nematode species of the genus Acrobeloides, the EPN Heterorhabditis zealandica and two EPF species, Metarhizium robertsii and M. majus. All L. huidobrensis pupal (45-57%) and larval stages (52-72%) of the potato leaf miner showed susceptibility to the EPN species tested. Laboratory bioassays showed that the pupal and larval stages was the most susceptible (>72% infection) to Heterorhabditis baujardi. Liriomyza huidobrensis pupal stage showed high pathogenicity to isolates of M. robertsii (10C). Overall, all isolates of M. robertsii showed pupal pathogenicity ranging between 80% and 91%. Metarhizium robertsii outperformed Beauveria bassiana as a biocontrol agent against L. huidobrensis pupae in the laboratory. Results from this study indicate the potential of biologicals as future biocontrol agents in an integrated pest management system for potato production.
The control of white grub (Coleoptera: Scarabaeidae) pests of sugarcane and forest plantations is difficult due to their cryptic nature and resistance to chemicals. This study evaluated the potential ...use of entomopathogenic nematodes (EPNs) as an alternative control method. Laboratory bioassays were performed with 12 locally isolated EPN species to determine the susceptibility of third instar larvae of the white grubs
Schizonycha affinis
Boheman,
Pegylis sommeri
Burmeister,
Monochelus
sp. and
Maladera
sp. 4. Concentration trials to determine lethal dosages for three of the white grub species were performed using
Heterorhabditis zealandica
Poinar MJ2C. Bioassays were performed to determine whether nematodes could develop inside the cadavers of
S. affinis
and
P. sommeri
as these had shown the lowest susceptibility to EPNs. The mortality percentage of the white grubs, although varying significantly, was found to be low for most of the EPN species, except
H. zealandica
. The highest percentage mortality of white grubs was observed at four weeks post inoculation with the rate of mortality being highest in the first week.
Schizonycha affinis
had the lowest LD
50
of 38 Infective juveniles (IJs) per larva after 28 days, compared to
Maladera
sp. 4, with 284 IJs per larva, and
P. sommeri
, with 1035 IJs per larva. The dissection of insect cadavers revealed possible limiting factors for low susceptibility, due to the nematodes and their associated symbiotic bacteria’s inability to infect the insect haemocoel, with no EPNs being found inside some of the cadavers.
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•Heterorhabditis zealandica turned infected wax moth larvae in three distinct colors.•H. zealandica associated with three different symbionts, connected to color.•Two of the symbionts ...are new associations with H. zealandica.
Bacterial symbionts associated with entomopathogenic nematodes (EPNs) play an important role in terms of the insecticidal properties of nematodes in pest control. Galleria mellonella larvae, shortly after being infected with three different strains of Heterorhabditis zealandica, which were isolated from South African soil, changed from pale white to steel grey-blue (blue), bright red, and yellow with a green tint (green), respectively. The genetic relatedness of the bacterial symbionts that were isolated from the three strains of H. zealandica was determined by means of comparing the 16S rRNA, recA, gyrB, dnaN, gltX and infB gene sequences. Subsequently, comparing the concatenated sequences revealed the presence of three distinct Photorhabdus species. The H. zealandica strain SF41, associated with Photorhabdus heterorhabditis, produced ‘blue’ G. mellonella larvae. The H. zealandica strain MJ2C, associated with Photorhabdus thracensis, yielded ‘green’ G. mellonella larvae, while the H. zealandica strain LLM associated with Photorhabdus laumondii subsp. laumondii yielded red larvae. The colour changes in G. mellonella larvae were found to have been instigated by a particular Photorhabdus species associated with H. zealandica. The red and ‘green’ phenotypes of G. mellonella larvae were found to represent new combinations of Heterorhabditis and Photorhabdus. In future studies, the colour of infected G. mellonella larvae needs to be reported as a phenotypic character, as it indicates the different bacterial species associated with the same nematode host, as shown in the case of H. zealandica.
Steinernema nguyeni n. sp. was recovered by baiting from beneath an Olea africana tree in South Africa. The combination of morphological and molecular features suggests that S. nguyeni n. sp. is a ...member of the feltiae-kraussei-oregonense group, clustering with members of this group in Clade III. The new species is morphologically characterised by the infective juvenile body length of 737 (673-796) μm and the number of ridges in the infective juvenile lateral field is 2, 8, 2. The male of the first generation can be recognised by the spicule length of 66 (58-75) μm and a gubernaculum length of 43 (30-55) μm. The first generation female can be recognised by the vulval lips only slightly protruding and the presence of low, double-flapped epiptygmata. Analysis of the ITS and D2-D3 regions of the ribosomal DNA confirms that S. nguyeni n. sp. differs from all other known Steinernema species.
During a survey for mollusc-associated nematodes in South Africa, a new Phasmarhabditis species was isolated from the invasive slug, Deroceras reticulatum, collected from a nursery near George in the ...Western Cape province. The nematode was identified using a combination of morphological, morphometric, molecular, and phylogenetic techniques. The new species, P. safricana n. sp., is characterised by the cupola-shaped tail of the female with a spike, small, non-protruding phasmids, a fingerprint-like pattern of the cuticle covering the female tail, toothlike cephalic structures of the infective juveniles, and the distinct molecular characteristics of the species. The molecular phylogeny of the new species, as inferred from its SSU and LSU rRNA gene, places P. safricana n. sp. in close proximity to P. papillosa. Virulence tests were conducted, which demonstrated that P. safricana n. sp. caused significant mortality to the European invasive slug, D. reticulatum. The new species brings the total complement of the genus to eleven species.
Summary
Entomopathogenic nematodes (EPN) are successful biological control agents of a variety of soilborne insect pests. They have the potential to be mass-produced, using in vitro liquid culture ...technology, and can be formulated and sold as a biopesticide. To commercialise an EPN-based biopesticide successfully, the method of liquid mass production requires in-depth optimisation to reduce the cost of production and to increase yields, to make it affordable to the farming community. This study attempted to optimise the liquid culture protocol for the South African isolates, Steinernema jeffreyense and S. yirgalemense, by investigating the impact of cheaper medium ingredients on the recovery and yield of the liquid culture process. Studies were conducted by investigating alternative protein, lipid and nitrogen/yeast sources, compared to the more expensive laboratory-grade ingredients currently used. The results showed that egg yolk has no impact on the yield in the case of S. jeffreyense. However, for S. yirgalemense, egg yolk was shown to be a superior protein source to soy and insect-based protein in terms of nematode yield. Moreover, neither canola oil nor olive oil showed a significant difference in the yield of S. yirgalemense, with yeast extract being found to be the optimal nitrogen/yeast source. When comparing the yields with those in other liquid culture research on S. yirgalemense, yields have been successfully increased by 300%, with the cost of the nematode nutrient medium having decreased by 77%. Thus, it is imperative that, prior to a scale up to large bioreactors, the nutrient medium should be optimised to reduce the cost of production.
Summary
Control of white grubs, the sporadic root-feeding larvae of scarabid beetles, is difficult due to their cryptic nature and resistance to chemical insecticides. Potential exists for the use of ...entomopathogenic nematodes (EPN) for such control. The successful infection of white grubs by EPN, however, is limited by the white grubs’ innate reduced susceptibility in form of behavioural, morphological and physiological defences. This study aimed to determine the different physiological defence mechanisms of white grubs against attack by EPN. The white grub species, Pegylis sommeri and Schizonycha affinis, which were previously found to have a low level of susceptibility to EPN, and Maladera sp. 4, which had previously shown a relative high level of susceptibility, were examined. The ability of the EPN to penetrate the white grubs and of the EPN symbiotic bacteria to grow inside the insect haemolymph, the encapsulation rates, as well as the nematode growth inside the insect haemolymph, were determined. The results showed that the ability of the nematodes to penetrate white grubs, the encapsulation rates and growth in the haemolymph varied between EPN and white grub species. The different species of the EPN symbiotic bacteria were able to grow in the haemolymph of all the white grub species tested. The results indicate that the white grub species investigated tended to resist the initial penetration of most of the EPN studied, thus avoiding infection.
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.
Pseudococcus viburni (Hemiptera: Pseudococcidae), the obscure mealybug, is a serious polyphagous, cosmopolitan agricultural insect pest that affects deciduous fruit production in South Africa. The ...presence of the mealybug in orchards results in significant damage to crops, with economic implications. The potential use of two entomopathogenic fungi (EPF) and entomopathogenic nematodes (EPN), as biological control agents against P. viburni, was assessed under laboratory conditions, using screening bioassays. The EPF isolates, Metarhizium robertsii (6EIKEN) and M. pinghaense (5HEID), tested at a conidial concentration of 1 × 10
7
conidia/ml, proved to be effective against the adult stage female mealybugs, causing 96% ± 6% and 91% ± 13% mortality, respectively, 7 days post inoculation, when incubated at 25°C with humidity levels of > 95%. Heterorhabditis indica, applied at a concentration of 200 infective juveniles per insect, was found to be the most effective EPN against P. viburni, causing 79% mortality, 48 h post inoculation, while incubated at 25°C. Further bioassays showed a positive correlation between insect mortality and conidial concentration as well as exposure time. The study represents the first report on the pathogenicity of multiple local and commercial EPF isolates to control P. viburni and several local EPN isolates against P. viburni.
Entomopathogenic nematodes (EPNs) are microscopic roundworms that are found in soil worldwide. They deliver an important ecosystem service through preventing natural flares in insect reproduction by ...means of utilising the soil stages of insects as a food source and by acting as natural biocontrol agents. A survey of EPNs was conducted in the JS Marais Nature Reserve, Stellenbosch, in the Western Cape province of South Africa. Soil samples were baited with the larvae of three susceptible hosts, codling moth (Cydia pomonella), wax moth (Galleria mellonella) and mealworm (Tenebrio molitor) to determine the presence of EPN. Of the 76 soil samples collected across the reserve, 39 were found to be positive for the presence of EPN (51.32%). Among the positive samples, 87% contained Steinernema isolates, 8% contained Heterorhabditis and 5% contained the Oscheius sp. Morphological and molecular studies were performed to characterise the isolates to species level. The Steinernema species were identified as Steinernema khoisanae in 34 samples, and as Steinernema nguyeni in five samples. The only species of Heterorhabditis found was H. safricana, which was identified from three samples. An unknown Oscheius sp. was found in two samples. The reserve's population of S. khoisanae showed interesting inter-individual variation (93%) early in the internal transcribe spacer (ITS) region, leading to short single-usable sequences, which, in most cases, included only the ITS1 or ITS2 region. However, using the D2D3 confirmed their identity as S. khoisanae, with such occurring in all areas and soil types of the reserve. Conservation implications The undisturbed alluvial fynbos and renosterveld of the JS Marais Nature Reserve showed high EPN abundance and diversity in stark contrast to the agro-ecosystems present in the Cape floristic region. This finding, on a micro level, should be conserved for future bioprospecting in the fynbos for EPNs with potential as biocontrol agents.
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