Browsing by Author "Hatting, Justin Louis."

Now showing 1 - 3 of 3

Development of a new entomopathogenic nematode species, steinernema innovation : biological characterization and mass production.
(2014) Ramakuwela, Tshimangadzo.; Laing, Mark Delmege.; Hatting, Justin Louis.; Hazir, Selçuk.
Entomopathogenic nematodes belonging to the genera Steinernema and Heterorhabditis are employed as environmental friendly biopesticides to control key pests in high value agricultural crops. Their successful application in developed countries has proved their effectiveness. The production of EPN for large scale commercial application has been restricted to developed countries because high capital cost of setting up liquid fermentation, and the high running costs of such units. If EPNs are to be commercialized in developing countries, then this will probably be achieved using in vivo culturing in insect larvae, or by using in vitro solid culture on a small to medium scale. The Republic of South Africa bans the importation of exotic EPN products by Amendment 18 of Act 36 of 1983. Therefore EPN products in South Africa will need to be developed from indigenous species. This study focused on optimizing the solid culture production and biological characterization of an indigenous nematode, Steinernema innovationi, as a biocontrol agent. A good production system must achieve high yield, maintain high virulence and the product must have a reasonable shelf-life. Optimum production temperature was determined using in vivo culturing of S. innovationi inside Galleria mellonella larvae. Performance was measured by quantifying the first day of emergence of infective juveniles (IJs) from cadavers, yield of IJs and IJ length, at five temperatures ranging from 18°C to 25°C. Optimum temperature was selected as 22°C based on high yield and slow emergence. There was a yield of 92,756 ± 28,089 IJs/larva, and 84,056 ± 27,832 IJs/larva, at 22°C and 25°C, respectively, which were not significant different. However, IJ emergence was significantly slower at 22°C, which provided more time for nutrient uptake and therefore these IJs had a greater nutrient reserve. There was no correlation observed between IJ length vs. temperature and IJ length vs. yield. A medium containing a puree of the larvae of the common house fly (Musca domestica) and 3% canola oil produced the highest yield of IJs (781,678 ± 221 IJs/5g), the highest level of live IJs (>84%) and the lowest level of adults (<10%) remaining in the medium at the time of harvest, compared to five other media formulations. All media were subjected to in vitro mass production at 22°C. A liquid inoculum of the EPN gave higher yields than a solid culture inoculum, irrespective of concentration. The length of IJ did not affect virulence against last instar larvae of G. mellonella, which was >90% in all experiments. A study characterizing optimum storage temperature of S. innovationi was carried out at five temperatures ranging from 5°C to 25°C in aqueous suspension over a period of 84 days. Survival was highest and most stable at 15°C, ranging from 84% to 88% after 84 days storage. Storage of the EPN in a sponge at a concentration of 2.5 million IJs in 15ml 0.1% formalin solution was successful, with an improvement of 6% compared to aqueous storage at 15°C. Furthermore, storage of the EPN in a sponge at 25°C, after a period of low temperature (15°C) storage for 84 days, did not have a detrimental impact on IJ survival and infectivity (87% and 95%, respectively). The new EPN (S. innovationi) was characterized by studying traits related to its infectivity (infectivity under a range of temperatures [10°C to 35°C], foraging behaviour & persistence under field conditions). Time till death was shortest at 25 and 30°C (average 1 day). The highest number of established IJs was recovered at 25°C (mean = 27°C). The nematode infected Galleria mellonella larvae at all depths and was capable of covering a distance of up to 15cm in 24 hours. There was no statistical difference between in vitro (40%) and in vivo (27%) cultured IJ persistence 4 weeks post application. Furthermore, there was no statistical difference in nematode recovery after 1 and 4 weeks post application of in vitro (33% & 40%, respectively) and in vivo (60% & 27%, respectively) produced IJs. Thermal activity was optimal at 25°C, the new species was classified as a cruiser and proved to survive under field conditions for at least 4 weeks. Susceptibility of larvae and/or pupae of Eldana saccharina (Walker), Sesamia calamistis (Hampson), Chilo partellus (Swinhoe), Tenebrio molitor (Linnaeus), Galleria mellonella (Linnaeus), Cydia pomonella (Linnaeus), Plutella xylostella (Linnaeus), and Gryllidae acheta (Linnaeus) representing three orders (Coleoptera, Lepidoptera & Orthoptera) was tested at a low and high concentration of 50 and 500 IJs, respectively. The hosts G. acheta, C. partellus and P. xylostella showed least susceptibility with maximum mortalities at the 500 IJs concentration of 28%, 45% and 92%, respectively. All other hosts suffered 100% mortality. Pupal mortality ranged from 47% to 68%. An LC50 and LC70 of 3 IJs/larva and 31 IJs/larva, respectively, was calculated for the black cutworm, Agrotis ipsilon (Hufnagel). These results provided a broad guideline on the relative pathogenicity of this new species against different hosts. A cost analysis was calculated for in vitro solid culture of the EPN, including the cost of rearing an insect-based nutrient component. An estimated retail price was then compared to the costs of market products around the globe. An estimated retail price (R90.61) for S. innovationi was considerably lower than the market price for other Steinernema species, which ranged from R271.50 to R458.55, in South African rands. The production system developed in this study for S. innovationi offers a highly competitive small to medium scale production method to produce EPN products without having to invest in large scale liquid fermentation equipment, by using relatively cheap production media ingredients, and simple solid culture growing conditions.
Fungal parasitism of cereal aphids in South Africa.
(2002) Hatting, Justin Louis.; Miller, Raymond Martin.; Wraight, Stephen.; Poprawski, Tadeusz.
The Russian wheat aphid, Diuraphis noxia is one of the most destructive exotic invaders of South Africa, capable of reducing individual wheat plant yields by up to 90%. Entomopathogenic fungi are important natural mortality factors associated with this aphid in its Eurasian endemic habitats as well as in the United States and Canada; their impact often exceeding that of predators and parasitoids. The principal objectives of this study included (1) baseline characterization of the aphid-pathogenic flora associated with aphids from South Africa, with special reference to six common cereal aphids, (2) quantification of the comparative impact of the different fungal species on the cereal-aphid complex in three different wheat producing regions of South Africa, (3) field evaluation of the Hyphomycete Beauveria bassiana against D. noxia on resistant wheat, (4) screening of six fungicides for their potential usage in managing entomophthoralean epizootics within greenhouse rearings of the Russian wheat aphid, and (5) development and evaluation of a novel bioassay protocol for screening entomopathogenic Hyphomycetes against D. noxia. A total of nine species of fungi known to infect and kill aphid hosts were collected, including the six entomophthoraleans, Pandora neoaphidis, Conidiobolus thromboides, Conidiobolus obscurus, Entomophthora planchoniana, Conidiobolus coronatus and Neozygites fresenii, and three Hyphomycetes, Beauveria bassiana, Verticillium lecanii, and Paecilomyces farinosus. The former four entomophthoraleans are considered first reports from this country. For the first time, morphological characteristics of these nine South African-collected species are visually depicted and techniques for their isolation and in vitro culture discussed. Seven species of fungi were recorded from D. noxia, of which P. neoaphidis was the most important, causing up to 50% mortality during the late season under dryland conditions in the summer-rainfall region. Mycoses at epizootic levels, together with the large diversity of fungal species recorded from this host, indicated a high level of susceptibility to fungal infection. In contrast, infection of the oat aphid Rhopalosiphum padi remained < 5% despite favourable numbers of hosts and apparently suitable environmental conditions. This phenomenon strongly suggests some level of low susceptibility to fungal infection in this species. Under irrigated conditions m KwaZulu-Natal, the rose-gram aphid Metopolophium dirhodum was the predominant aphid but remained below economical injury levels. Field surveys revealed that this aphid was effectively targeted by P. neoaphidis and C. obscurus, and findings suggest that in some areas of South Africa entomophthoralean fungi effectively suppress this aphid, negating the need for insecticide applications. On average, ca. 61% control of D. noxia on resistant wheat was observed following an application of B. bassiana (5 x 10¹³ conidia per hectare) during the early flag-leaf stage of the wheat. Efficacy of B. bassiana applications on younger plants appeared to be influenced by the level of aphid activity, possibly explained by secondary pick-up of inoculum by D. noxia. These findings accentuate the importance of understanding the tritrophic relationship between host plant, pest and pathogen. The fungicides copper oxychloride, mancozeb + oxadixyl, captab + metalaxyl, bittertanol, iprodione, and mancozeb at a rate of 0.1% active ingredient moderately to strongly inhibited C. thromboides vegetative growth (mean inhibition 81.1 %). Mancozeb at concentrations of 10.0, 2.0, 1.25, 0.5, 0.08, and 0.016% was further evaluated in vitro. The fungus growth cut-off point, midway between 1.25 and 2.0% mancozeb, was calculated and a rate of 1.625% active ingredient per litre of water was used to decontaminate the fungus-infected D. noxia cultures. A novel bioassay protocol was developed, employing live host plants for rearing aphids post inoculation. Using this design against D. noxia, an average LC₅₀ estimate of 85 conidia per mm² for B. bassiana strain GHA was calculated. Control mortality was restricted to levels below 4%. The data indicated high precision due to an average coefficient of variation for slope of less than 20%, and an average chi-squared value of 5.46 ± 2.74 (n = 10 assays). The design will accommodate the use of cereal aphid species other than D. noxia, while live host plants will facilitate tritrophic studies on the effect of host-plant resistance on fungus-induced mortality of D. noxia.
Studies on the integrated control of the Russian wheat aphid, Diuraphis Noxia (Kurdjumov) (Hemiptera: Aphididae), using entomopathogenic fungi combined with sublethal doses of insecticides.
(2017) Mzimela, Nokulunga Prudence.; Laing, Mark Delmege.; Hatting, Justin Louis.
The Russian wheat aphid (RWA), Diuraphis noxia (Kurdjumov) (Hemiptera: Aphididae), is one of the most damaging aphid pests of wheat grown under dryland conditions. Host plant resistance is considered as the most, cost-effective, and ecologically attractive method of control for this cereal pest. However, it is a slow process to breed wheat varieties for resistance to RWA, and resistance-breaking biotypes continue to pose a serious threat to wheat producers in the country. Currently, there are four RWA biotypes that have been recorded in South Africa. Entomopathogenic fungi (epf), such as Beauveria bassiana, have the potential to suppress RWA as shown by Hatting et al. (2004), whereby B. bassiana together with host plant resistance managed to provide about 60% level of control of this pest. Nevertheless, that was not a satisfactory level of control. Furthermore, hyphomycetes are known to kill their target insects more slowly than chemicals. One approach to increase their efficiency, and therefore insect mortality is by combining epf with sub-lethal doses of chemical insecticides. The interaction observed from this combination can be synergistic, antagonistic or neutral. Synergistic effects would allow for reduced insecticide use, minimized environmental pollution, the preservation of natural enemies and a slowing of the development of insecticide resistance. The hypothesis for the synergistic interaction is that the insecticide acts as a stress inducer, making the insect pest more susceptible to fungal infection. Given the development of new resistance-breaking biotypes of the RWA and many reported cases of insecticide resistance, this study aimed to enhance the virulence of selected entomopathogenic fungal strains through synergism with sub-lethal doses of chemical and botanical insecticides. The first objective was to find several virulent entomopathogenic strains of B. bassiana and M. anisopliae against D. noxia. The virulence of three Beauveria bassiana and three Metarhizium anisopliae strains were evaluated against D. noxia biotype RWASA1. B. bassiana Isolate SGI921, was the most pathogenic strain, and was used for the subsequent studies. Four insecticides registered for RWA control were screened in vitro for compatibility with the selected fungal strain, SGI921, with special emphasis on germination, radial vegetative growth and sporulation intensity. All the tested insecticides (active ingredients: chlorpyriphos, dimethoate, demeton-S-methyl and acetamiprid) reduced germination, radial vegetative growth and sporulation intensity in various degrees in a concentration-dependent manner. Mospilan was found to be compatible with the selected strains and, at its highest concentration, it seemed to have stimulated vegetative growth. The last objective was to determine the effect of combining sub-lethal doses of pyrethrum-based insecticides with either a commercial formulation of Beauveria bassiana, Eco-Bb®, and Beauveria bassiana Strain R444 in both in vivo and in vitro trials. In the laboratory bioassays 10% Pyrol was able to enhance the sporulation intensity of R444. In glasshouse trials 10% Pyrol enhanced the efficacy of B. bassiana Strain R444 by increasing RWA mortality and reducing the mean time to mortality. At a concentration of 10%, Mospilan killed the aphids before either of the fungi could infect the aphids, i.e., within 48 hours post fungal inoculation. Mospilan concentrations below 10% will be further investigated to establish a minimal sub-lethal dose that does not kill the RWA but make the aphids more susceptible to epfs. The interaction of Pyrol with the unformulated strain of B. bassiana Strain R444 will be tested in other biotypes of Russian wheat aphids. Since vegetable oils are also known to enhance efficacy of entomopathogenic fungi, future research will also evaluate the interactions of the pyrethrum extract and canola oil, in order to enhance the synergistic effects of the oil in the Pyrol and B. bassiana interaction.