No description available.

Due to the rapid development of tick resistance to synthetic chemical acaricides and the potential risk posed by these chemicals to non-target species, efforts are intensifying towards the development of entomopathogenic fungi as an environmentally benign alternative tick control strategy. The main objective of this study is the development of formulations and delivery systems for control of ticks with entomopathogenic fungi. Screening of entomopathogenic fungal isolates is the first step towards the development of mycoacaricides. Twelve isolates of Metarhizium anisopliae (Metschnik.) Sorok. and three isolates of Beauveria bassiana (Bals.) Vuill. (Ascomycota: Hypocreales) were evaluated for pathogenicity against unfed Rhipicephalus pulchellus Gerstäcker (Acari: Ixodidae) adults under laboratory conditions. Out of the 15 isolates screened, 4 isolates were virulent against R. pulchellus, inducing significantly (F value = 11.86; DF = 14, 75; P < 0.0001) higher tick mortalities; i.e. ICIPE 78 (76.1 ± 5.9%), ICIPE 69 (62.6 ± 5%), ICIPE 62 (49.8 ± 5.8%) and ICIPE 60 (49.6 ± 7.7%), four weeks post-treatment compared to the others. The results of this study demonstrate that R. pulchellus ticks were susceptible to some M. anisopliae isolates. Since the process of strain selection is an important step in the development of insect pathogens for biological control, an appropriate bioassay technique for infecting different life stages of a host is of paramount importance. Bioassays were conducted in the laboratory to evaluate the efficacy of different methods of inoculation using R. pulchellus as a model. In a preliminary experiment, an oil-based formulation of M. anisopliae titred at 109 conidia ml-1 was applied to R. pulchellus adults using a Burgerjon spray tower or a microapplicator. Inoculation by microapplicator yielded poor results (25.0% tick mortality) compared to Burgerjon’s spray tower (52.3% tick mortality) , although the mean number of fungal conidia on R. pulchellus adults was lower (1.5 x 104 ± 1.1 x 103 conidia ml-1) after spraying by Burgerjon’s spray tower compared to 1 x 106 conidia ml-1 obtained with the microapplicator. Thus, inoculation by Burgerjon’s spray tower was selected for further investigations. Different modes of inoculation were tested and included direct spray of inoculum on the tick and substrate (SS), direct spray on the substrate and tick followed by transfer of the tick to clean uncontaminated Petri dish (SP) or indirect inoculation of ticks through substrate (SW). The LC50 values following contamination of nymphs (LC50 = 1.4 x 107 conidia ml-1) and adults (LC50 = 6.7 x 107 conidia ml-1) in SS were significantly lower compared to SP; nymphs (LC50 = 5.7 x 108 conidia ml-1) and adults (LC50 = 5.3 x 109 conidia ml-1) and SW; nymphs (LC50 = 5 x 108 conidia ml-1) and adults (LC50 > 1010 conidia ml-1). Although the LC50 value in SS was the lowest, it recorded the highest tick mortality among control ticks (24.2% at two weeks post-treatment) and (23.3% at three weeks post-treatment) in nymphs and adults respectively compared to SP (2.5 and 5.8% respectively) and SW (0.0 and 0.0). Results show that among the modes of contamination tested, SP was the most appropriate for contaminating R. pulchellus adults. SW and SP were identified as appropriate techniques for infecting the R. pulchellus nymphs with conidia formulated in oil. The integrated use of semiochemicals and entomopathogenic fungus (hypocreales) may optimise the performance of a fungus as a biocontrol agent against ticks. Initially, experiments were conducted to evaluate the attraction of the hunter tick Amblyomma variegatum Fabriscius (Acari: Ixodidae) to semiochemicals. In one of the experiments, the simultaneous release of 1-octen-3-ol and AAAP together with CO2 from a trap in simulated field plots attracted up to 94 ± 6% of adult ticks from a distance of 6 m, and up to 24.0 ± 5.1% from 8 m. Formulations of M. anisopliae (dry powder, oil, and emulsifiable) applied within the trap baited with AAAP and 1-octen-3-ol resulted in high levels of contamination of the ticks attracted to the traps. However, 48 hr after autoinoculation, 89.1 and 33.3% of conidia were lost in dry powder and oil formulations, respectively. Emulsifiable formulation lost the lowest number of propagules (17.1%). Samples of ticks attracted to the baited traps were transferred to plastic basins containing grass and maintained for 5 weeks. The experiment was conducted in the rainy and dry seasons. The emulsifiable formulation gave the highest relative reduction in tick numbers in both seasons: 54.7 and 46.5% in the rainy and dry seasons, respectively, followed by the oil formulation (32 and 23.8%) and the powder formulation (38.0 and 24.4%). Following the high attraction and contamination of A. variegatum under semi-field conditions, experiments were done to evaluate the efficacy of M. anisopliae-treated semiochemical-baited traps for control of A. variegatum under field conditions. Unfed A. variegatum adults (118) were seeded in each 100-m plot. An emulsifiable formulation of M. anisopliae (consisting of 49.5% sterile distilled water, fungal conidia, 49.5% corn oil [CHEF cooking oil, Premier Oil Mills LTD] and 1% Tween 80) titrated at 109 conidia ml-1- was applied in a semiochemical-baited traps (900 cm2) which were placed at 5 spots within the plot. The control and fungal treatments were repeated after 14 and 28 days soon after rotating the traps clockwise (45o) in order to cover different sections of the plot. In the control plots, traps baited with semiochemicals only were used. Six weeks after the initiation of the experiments, five semiochemical-baited traps (untreated) were deployed in each plot for 3 successive days to trap ticks in the treated and control plots. The percentage of ticks recovered in the fungus-treated plots were significantly lower (31.1 ± 5.2%) than in the control plots (85.6% ± 3%) (P < 0.001), which represented a relative tick reduction of 63.7%. Mortality of 93.8 ± 2.3% was observed among the ticks that were recovered from the field and maintained in the laboratory for two weeks; while only 3.3 ± 0.9% died from the control plots. The results of this study open up the possibility of developing an environmentally friendly, low cost product to control these economically important ticks. / Thesis (PhD)--University of Pretoria, 2009. / Paraclinical Sciences / unrestricted

No description available.

Entomopathogenic nematodes (EPN) (Heterorhabditis bacteriophora and H. megidis) and entomopathogenic fungi (EPF) (Metarhizium anisopliae and Beauveria bassiana) were evaluated for efficacy against masked chafer white grub, Cyclocephala spp., under laboratory and greenhouse conditions, as well as their efficacy against various grub stages in the field.  Under both laboratory and greenhouse conditions, additive interactions were found between EPN and EPF in their combined application against Cyclocephala spp., except a few observations that showed antagonism or synergism.  Significantly greater control occurred from the combination of a nematode and a fungus compared with a fungus alone, but not compared with a nematode alone.  The combined effect did not differ significantly for nematode and fungi applied simultaneously or at different times.  EPF had no significant impact on EPN infection and production of infective juveniles (IJs) in grub carcasses.  Nematodes alone or in combination with fungi were comparable to the insecticide Merit 75 WP (imidacloprid) against 3rd instar Cyclocephala spp in the greenhouse. Efficacy of EPF and EPN varied dramatically between field sites and conditions; EPN and EPF applied alone or in combination were less effective than Merit 75 WP in >50% field trials, but some EPN + EPF treatments were more effective than the insecticide in reducing grub numbers.  EPN and EPF showed better potential than insecticides for providing extended control of white grubs in the subsequent generation.  In addition, the sub-lethal effects of EPF on southern masked chafer, C. lurida, were investigated.  Neither M. anisopliae nor B. bassiana had a sub-lethal effect on grub weight gain, adult longevity, oviposition, pupation and eclosion.  Finally, interaction between H. bacteriophora and M. anisopliae was examined to determine the potential of the nematode in improving fungal distribution in soil.  H. bacteriophora enhanced fungal distribution in sandy loam soil without grass thatch, but not in sandy soil with thatch.  In both soil types, soil depths significantly affected nematode and fungal distribution. In water profile, M. anisopliae conidia germinated hyphae that attached to sheath of H. bacteriophora IJs, which molted to detach from the fungus.  IJs mortality and virulence were not affected by the presence of M. anisopliae. / Ph. D.

Thesis (PhD (Paraclinical Sciences, Veterinary Science))--University of Pretoria, 2009. / Includes bibliographical references. Also available in print format.

Thesis (M.Sc.(Zoology and Entomology))--University of Pretoria, 2000. / Abstracts in Afrikaans and English. Includes bibliographical references (leaves 72-79).

Background: Steinernematid nematodes form obligate symbioses with bacteria from the genus Xenorhabdus. Together Steinernema nematodes and their bacterial symbionts successfully infect, kill, utilize, and exit their insect hosts. During this process the nematodes and bacteria disassociate requiring them to re-associate before emerging from the host. This interaction can be complicated when two different nematodes co-infect an insect host. Results: Non-cognate nematode-bacteria pairings result in reductions for multiple measures of success, including total progeny production and virulence. Additionally, nematode infective juveniles carry fewer bacterial cells when colonized by a non-cognate symbiont. Finally, we show that Steinernema nematodes can distinguish heterospecific and some conspecific non-cognate symbionts in behavioral choice assays. Conclusions: Steinernema-Xenorhabdus symbioses are tightly governed by partner recognition and fidelity. Association with non-cognates resulted in decreased fitness, virulence, and bacterial carriage of the nematode-bacterial pairings. Entomopathogenic nematodes and their bacterial symbionts are a useful, tractable, and reliable model for testing hypotheses regarding the evolution, maintenance, persistence, and fate of mutualisms.

The eastern hemlock [Tsuga canadensis (L.) Carrière] is one of the native dominant forest components of northeastern US. At present, these valuable stands face an alarming decline, in part due to the Fiorinia externa, elongate hemlock scale (EHS), (Hemiptera: Coccoidea: Diaspididae). The armored shield of F. externa provides an excellent defense against insecticides, natural enemies and adverse conditions. Chemical and classical biocontrol methods have been unable to stop the spread of this pest. Recently, the occurrence of an epizootic within the F. externa population in the Mianus River Gorge Preserve in Bedford, NY revealed a promising opportunity for control of this scale. Entomopathogenic fungi represent a valuable, although under-utilized, group of organisms with unique capabilities for self-sustaining pest management. Given the significant impact of this epizootic on F. externa, we have conducted extensive research on the biology, genetics and biological control potential of this epizootic. We molecularly identified a complex of entomopathogenic, phytopathogenic, and endophytic fungi associated with the epizootic in 36 localities within the states of New York, Pennsylvania, Connecticut and New Jersey. One fungus, Colletotrichum sp., was the most commonly isolated organism in populations of F. externa within areas of the epizootic. The host range of this Colletotrichum species comprised both insects and plants, although diverse life cycles occured in the different hosts. Endophytic growth was observed in 28 species of plants comprising 18 families (52% of the sampling), whereas in F. externa biotrophic and necotrophic growth was detected. Colletotrichum is a widely known phytopathogenic genus and reports of entomopathogenic activity are extremely rare. In order to understand the biological processes involved in the host-pathogen interactions we quantified the pathogenicity and virulence of this Colletotrichum sp. to four insect families and six plants families as well as the occurrence of sexual recombination in this Colletotrichum sp., both in vitro and in planta. We observed that this Colletotrichum sp. displays a propensy to induce rapid disease and mortality in F. externa hosts. Phylogenetic analysis comprising six of the most commonly studied nuclear genes in molecular phylogenetics (D1/D2 domain of the 28 rDNA gene, ITS region, β-Tubulin 2, GPDH gene, GS gene and HMG box at the MAT1-2 mating-type gene) and RAPDs showed this fungus is closely related to phytopathogenic strains of Colletotrichum acutatum and that it may represent a single population lineage of this species (i.e., Colletotrichum acutatum forma specialis fiorinia). Though a large body of information exists regarding the phytopathogenic genus Colletotrichum, ours is only the second reported entomopathogenic strain. It is not clear whether the colonization of an insect by this fungus is truly rare or a common but undetected event. Sexual recombination, observed in planta and in vitro, could be the means by which new genetic variants are generated leading to new biotypes with a selective advantage to colonize new hosts, which in this case is a novel host in a different kingdom.

The prevailing use of chemical pesticides has generated several problems including insecticide resistance, outbreak of secondary pests, safety risks for humans and domestic animals, contamination of ground water and decrease in biodiversity among other environmental concerns (Webster, 1982). These problems and the nonsustainability of control programs based mainly on conventional insecticides have stimulated increased interest in the development and implementation of costeffective, environmentally safe alternatives to chemical pesticides for insect pest control. One of the most promising strategies to help minimize dependence on chemical pesticides has been the recent application of entomopathogenic nematodes (EPNs) as biocontrol agents. EPNs in the families Steinernematidae and Heterorhabdidae have been shown to have considerable potential as biological control agents. As a natural process, biological control has the potential to play an important role in the suppression of field crop pests in agriculture. EPNs as biocontrol agents have the following advantages: high virulence, safety to non target organisms, ability to search for hosts, high efficacy in favourable habitats, high reproductive potential, ease of mass production, ease of application (Ferron & Deguine, 1996). To isolate the EPNs in South African soil, 200 soil samples were randomly collected from 5 locations in the agricultural research council (ARC) Pretoria, Gauteng province in April 2006; and 5 locations in Brits, North West province in March, 2006. At the different collection sites, soil samples were obtained from soils associated with various types of vegetation. The nematodes were collected from sandy soil by the insect-baiting technique and maintained successfully in vivo for 12 months on Galleria mellonella (G. mellonella), 4 months on Tenebrio molitor (T.molitor); 2 months Pupae and in vitro (lipid agar) for 2 weeks in the laboratory. Out of a total of 200 soil samples that were baited, 2 were found to be positive for EPNs.EPNs. IV In addition to completing Koch’s postulates, the colour of cadavers infected by the putative EPNs were also used as a diagnostic characteristic for categorizing the nematode isolates. Characterization and identification of the EPN isolates were based on morphological characters, as well as on a molecular marker (18S rDNA). On the basis of the morphological and molecular data that was obtained both of the EPNs isolates were placed in the family Heterorhabdidae: Heterorhabditis bacteriophora (H. bacteriophora) and Heterorhabditis zealandica (H. zealandica). Also from the phylogenetic trees generated from the 18S rDNA sequence, the indigenous putative H. bacteriophora was shown to be closely related to H. bacteriophora (accession number EF690469) and indigenous putative H. zealandica to H. zealandica (accession number AY321481). The two EPNs were found associated with Gram negative rod-shaped bacteria. The bacterial symbionts of the two isolates were isolated and a region of the 16S rDNA gene was sequenced. National Center for Biotechnology Information (NCBI-BLAST) results of the 16S rDNA sequence obtained showed the endosybiotic bacteria to be Photorhabdus luminescens laumondii (P. laumondii) (H. bacteriophora) and Photorhabdus sp (H. zealandica). Results of the tree showed that isolates from H. bacteriophora appeared to be closely related to P. luminescens subsp laumondii strain TT01 Ay 278646. The isolates from H. zealandica appeared to be most closely related to Photorhabdus sp Accession number: Q 614 Ay 216500). Bioassays were used to determine the infectivity of the two EPNs. In this experiment different infective juvenile (IJs) concentrations (5, 10, 25, 50, 100,200 400 and 500) of the two EPNs were applied per G. mellonella; T. molitor larva and pupae. The bioassay was carried out in two parts. In the first part, mortality data was collected for H. bacteriophora and H. zealandica. The results showed that the degree of susceptibility of G. mellonella, T. molitor larvae and pupae to each nematode species was different. When 24 h post-exposure mortality data for larvae exposed to the IJs of H. bacteriophora and H. zealandica were analyzed, ANOVA showed no differences V in mortality between insects exposed to different H. bacteriophora IJ doses (Fig: 8.1 ABC). However, there were significant differences in mortality between insects exposed to different IJ doses of H. zealandica such as 5 and 500 IJs/insect (Fig: 8.2 ABC) Therefore, no differences were noted when mortality data was compared between IJ doses at both 72 h and 96 h following IJ application to the insects. The highest susceptibility was observed with G. mellonella followed by T. molitor pupae and then T. molitor larvae. According to Caroli et al., (1996), the total mortality of insect such as G. mellonella and other lepidopterans, was reached within 24-72 h of exposure to nematodes at concentrations such as those tested here. In this study similar results were observed with high concentration of nematodes (100, 200 and 500). In the second part of the dose response bioassay, the number of progeny IJs emerging from EPN-infected cadavers was determined for all two EPNs. The results indicate that IJ progeny production differed among the three insect hosts used, the IJ doses they were exposed to, as well as the EPN species (Figs 8.3 & 8.4). The highest number of emerged IJs of H. zealandica was produced by G. mellonella (mean ± SEM: 220500 ± 133933 IJs), followed by T. molitor larvae (mean ± SEM: 152133 ± 45466 IJs) and the lowest then T. molitor pupae (mean ± SEM: 103366 ± 56933 IJs).

A THESIS PRESENTED TO CAPE PENINSULA UNIVERSITY OF TECHNOLOGY IN FULFILMENT OF THE REQUIREMENTS FOR THE MASTERS OF TECHNOLOGY DEGREE IN HORTICULTURE. IN THE FACULTY OF APPLIED SCIENCES 2014 / Codling moth ([Cydia pomonella (Lepidoptera: Tortricidae]) infest pomes resulting in high production losses to fruit farmers in South Africa and globally. Many farmers are adopting biocontrol methods including the use of entomopathogens as alternatives to synthetic insecticides for sustainable management of Arthropod pests. Research activities on screening and application of entomopathogenic fungi (EPF) have intensified in recent years. This study was carried out to isolate and identify virulent indigenous entomopathogenic fungal strains from soils of selected locations in the Western Cape region of South Africa that are pathogenic against codling moth (Cydia pomonella (Linn) [Lepidoptera: Totricidae]) and to evaluate the possiblility of combining semiochemicals and fungal inoculums in a simulated attract-and-fungus contamination system for codling moth control. Soil samples were collected from 10 locations in the Western Cape, South Africa for the isolation of entomopathogenic fungi (EPF) by baiting the soil samples with 5th instar larvae of codling moths. Fungal strains were identified and characterized using light microscopy and DNA analysis (internal transcribed spacer region) and elongation factor 1-α (EF 1-α) genes of fungal cultures. Thirty-nine (39) isolates belonging to six species were obtained; Purpureocillium lilacinum (8 isolates), Fusarium oxysporum (five isolates), Fusarium polyphialidicum (two isolates), Beauveria pseudo-bassiana (one isolate), Aspergillus flavus (three isolates) and Metarhizium robertii (20 isolates). Generally, M. robertsii was the most frequently encountered species representing 51% of the total number of isolates collected from the soil samples. This is the first report of the isolation of M. robertsii in southern Africa. A screening test to identify the most virulent fungal strains against codling moth was carried out on 39 isolates by immersing 5th instar larvae of C. pomonella into aqueous spore suspension 1 x 108 conidia ml-1. Mortality data from the immersion bioassay indicated that the 39 fungal isolates were pathogenic against fifth instar larvae of codling moth inducing 47-85% insect mortality at an infective concentration of 1 x108 spores’ ml-1, 5 days post-treatment. Two fungal strains MTL151 and GW461 induced over 85% larval mortality and were selected for further evaluations. The effect of MTL151 and GW461 on egg hatchability of 0-day old eggs was evaluated by exposing freshly laid eggs on wax papers that were pre-treated with fungal spores ranging from 103 -108 spores/ml. Egg hatchability reduced significantly from 93-71% (GW461) and 95-66% (MTL151) as spore concentration increases from1 x 103 to 1 x 108 spores ml-1, respectively. The highest spore concentration significantly inhibited egg hatchability resulting in lower egg hatchability compared to that obtained with the cypermethrin containing commercial insecticide (Fruitfly [registered by Kombat (PTY) Ltd] ) tested at the recommended dose of 0.25 ml/250 ml of water. The potential of the two M. robertsii strains to protect apples from infestation by codling moth neonates was assessed in an apple fruit bioassay under laboratory and field bioassay. Codling moth neonates were exposed to apples that were sprayed topically with varied conidia concentrations (103 - 108 sporesml-1). The mean number of participating apple fruits having developing/developed larvae in the core/flesh significantly reduced from 5.3 to 1 and 7.6 to 1 for MTL151 and GW461, respectively as spore concentrations increased from 1 x103 to 1x 108 spores/ml-1. A concurrent decrease in apple fruit rot as conidia concentration increased was observed. Up to 90% of apples treated with 1 x 108 spores/ml-1 had no larva present in their cores and this result compared favourably with the commercial pesticide (Fruitfly) used at a recommended dose of 0.25g/250 ml of water. No significant difference was found between the EC50 values of 1.2 x 103 (CI=1.2 x 102 -1 x 105) (MTL151) and 1.1 x 105 (CI =7 x103 – 5.6x105) (GW461) spores ml-1. However, neither the two M. robertsii strains nor the insecticide deterred the neonates from feeding on the surface of the fruits. The attraction responses of male and female adult codling moths to butyl hexanoate and codlemone blend; butyl hexanoate (0.025 mg / ml of hexane) and codlemone (0.1 mg / ml of hexane) in a ratio of 1:1 (0.5 ml: 0.5 ml from both solutions) were assessed in a one-choice wind tunnel olfactory bioassay during scoto-phase in the absence of fungal spores. The blend attracted more females (69%) than males (66.7%), however these responses were not significantly different (P > 0.5). The prospect of integrated use of entomopathogenic fungal conidia and semiochemical blend for control of C. pomonella adults was assessed in a wind tunnel containing the combo lure and M. robertsii (MTL151) spores formulated as a powder (250 mg) or aqueous spore suspension (3.1 x 108 spores m-1 ± 7 x 102 sporesml-1). Insect attraction, mean number of spores picked per insect and insect mortality was assessed. No significant difference (P > 0.5) was observed in the number of inoculi picked by unsexed adult moths in the two conidial formulations tested; spore powder (3.1 x 103 ± 1 x 103 spores ml-1) and aqueous spore suspension (3.1 x 103 ± 7 x 102 spores ml-1). No mortality was recorded among fungus or control-treated moths. Both M. robertsii (MTL151) spore formulations did not significantly (P > 0.05) affect the attraction responses of the insects to the combo lure; powder (73.3 ± 3.3%) and aqueous (68.6 ± 2.9%) compared to the control treatment (without the fungal spores) (70 ± 0.5 %). These results suggest that the fungus did not inhibit the attraction of moths by semiochemicals. Despite the successful autoinoculation of moths with fungal spores, the level of contamination was too low to induce mortality among adult moths. This study opens up opportunities for research in attract-and-fungus contamination delivery systems. This is the first report of the occurrence of M. robertsii in the southern Africa. Further, results from this study indicates that C. pomonella is susceptible to indigenous South African entomopathogenic fungi and 2 indigenous M. anisopliae strains, MTL151 and GW461 provided adequate protections against codling moth larval infestations of apple fruits. Future research endeavours to improve adherence of conidia to adult moth cuticle is recommended. Key words: Entomopathogenic fungi (EPF), codling moth, B. pseudobassiana, M. robertsii, larvae, pathogenicity and biological control, coddlemone, butyl hexanoate, attraction, semiochemicals.